Pbb Crosbie

In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch’s postulates

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Kochs postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Kochs postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.

Preliminary success using hydrogen peroxide to treat Atlantic salmon Salmo salar L. affected with experimentally induced amoebic gill disease (AGD)

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H₂O₂), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H₂O₂ in sea water (500, 1000 and 1500 mg L⁻¹) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post-exposure and after 24 h. A concentration/duration combination of 1000 mg L⁻¹ for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H₂O₂ at 12 and 18 °C for 15 min at 1250 mg L⁻¹ and their re-infection rate was compared to freshwater-treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post-bath. However, the results were largely equivocal in terms of disease resolution over a 3-week period following treatment. These data suggest that H₂O₂ treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.

Amoebic gill disease in hatchery-reared ayu, Plecoglossus altivelis (Temminck & Schlegel), in Japan is caused by Neoparamoeba perurans

Amoebic gill disease (AGD) is a potentially fatal infection in fish farmed in marine environments and has been reported throughout the world in numerous species, including salmonids (Kent, Sawyer & Hedrick 1988; Munday, Foster, Roubal & Lester 1990) and turbot (Dyková, Figueras & Novoa 1995). Gill pathology, including epithelial hyperplasia resulting in lamellar fusion, is usually initiated by colonisation and proliferation of a marine amoeba harbouring an endosymbiont, on the gills of susceptible fish (Adams & Nowak 2003, 2004). Neoparamoeba perurans has been shown to be the AGD-causing agent in Atlantic salmon from Australia, the USA, Scotland and Ireland; rainbow trout from Tasmania; chinook salmon from New Zealand, and turbot from Spain (Young, Dyková, Snekvik, Nowak & Morrison 2008a). The first reported outbreak of AGD in farmed Atlantic salmon in Norway was attributed to N. perurans infection based on sequence analyses of 18S cDNA derived from AGD-affected gill tissue (Steinum, Kvellestad, Rønneberg, Nilsen, Asheim, Fjell, Nygård, Olsen & Dale 2008). Therefore, when mortalities were seen in sea water-reared juvenile ayu, Plecoglossus altivelis (Temminck & Schlegel), which displayed gross gill pathology typical of AGD, we applied the available molecular tools to determine whether N. perurans caused this infection. This article reports on that investigation and is the first reported incidence of AGD in tank-reared ayu. Ayu is an important commercial and recreational species in Japan. It is a diadromous fish, which naturally spawns in downstream stretches of rivers in autumn and larvae enter the sea where they stay until spring (Masuda, Amaoka, Araga, Uyeno & Yoshino 1984). Young ayu swim upstream and grow in the middle stretches of rivers. The life span of ayu is 1 year. Ayu is farmed for stocking local rivers and the rearing includes both freshwater and marine phases. Thus, spawners are reared in fresh water, where fertilised eggs hatch. Larvae spend their early days in sea water and then are reared in fresh water for the final stage of seed production. A total of 1.89 million eggs of ayu were collected from spawners and fertilised at the Fukui Prefectural Inland Fisheries Centre, Fukui City, in October 2007. Eyed eggs were transported to the Fukui Prefectural Centre for Fish Stock Enhancement, Obama City. Hatched larvae were fed rotifers and subsequently formulated feed. In February 2008, ayu juveniles were transported to facilities Journal of Fish Diseases 2010, 33, 455–458 doi:10.1111/j.1365-2761.2009.01137.x

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort(2005)) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE=1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U microl(-1) and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U microl(-1) (SE=6.26 U microl(-1)) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort(2005) were compared to a 2006 cohort (Cohort(2006)) in March 2006 and August 2006 to determine if prior infection in Cohort(2005) elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort(2005) had significantly lower abundances and prevalences of blood fluke infection than Cohort(2006). Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort(2006) was significantly greater than that of Cohort(2005) in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.

Production of polyclonal antisera against barramundi (Lates calcarifer Bloch) serum immunoglobulin derived from affinity columns containing mannan-binding protein or staphylococcal protein A

Barramundi (Lates calcarifer Bloch) immunoglobulin (Ig) was purified by affinity chromatography using staphylococcal protein A (SpA) or mannan-binding protein (MBP) as capture ligands. Both ligands realised a pure product, although the SpA column resulted in a higher yield. In common with other teleosts, barramundi Ig appears to be a tetramer made up of two heavy chain (HC) and two light chain (LC) moieties with individual molecular weights (MW), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), of 24 and 86 kDa, respectively. Gel filtration indicated that the native Ig molecule had a molecular weight of approximately 929 kDa. Western blot analyses demonstrated that rabbit antiserum raised against the whole MBP fraction was specific for HC and LC Ig components, whereas antisera directed against the HC or LC constituents of the SpA fraction were specific for HC and LC components, respectively. Analyses by flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed reactivity of all antisera with varying percentages of barramundi lymphocytes and SpA-purified barramundi Ig, respectively.

Immunity to Amoeba

Amoebic infections in fish are most likely underestimated and sometimes overlooked due to the challenges associated with their diagnosis. Amoebic diseases reported in fish affect either gills or internal organs or may be systemic. Host response ranges from hyperplastic response in gill infections to inflammation (including granuloma formation) in internal organs. This review focuses on the immune response of Atlantic salmon to Neoparamoeba perurans, the causative agent of Amoebic Gill Disease (AGD).

First record of amoebic gill disease caused by Neoparamoeba perurans in South Africa

Amoebic gill disease (AGD) has been described from cultured marine fish worldwide since first reported in farmed coho salmon, Oncorhynchus kisutch (Walbaum) (see Kent, Sawyer & Hedrick 1988). AGD was initially ascribed to Paramoeba pemaquidensis (see Kent et al. 1988), later reclassified as Neoparamoeba pemaquidensis (see Dykov a, Figueras & Peric 2000). With the aid of molecular techniques, Neoparamoeba perurans was shown to be responsible for AGD in farmed Tasmanian salmon, Salmo salar L. (see Young et al. 2007). It was subsequently found in Chile, Ireland, Japan, New Zealand, Norway, Scotland, Spain and the USA (Steinum et al. 2008; Young et al. 2008; Crosbie et al. 2010; Bustos et al. 2011). Neoparamoeba perurans affects salmonids, turbot, Scophthalmus maximus (L.), ayu, Plecoglossus altivelis (Temminck & Schlegel), European seabass, Dicentrarchus labrax (L.), and sharpsnout seabream, Diplodus puntazzo (Walbaum) (see Crosbie et al. 2010; for review see Munday, Zilberg & Findlay 2001) with Africa and Antarctica remaining the only continents where AGD has not been reported. Koch’s postulates have been fulfilled for the role of N. perurans as a causative pathogen of AGD (Crosbie et al. 2012). Here, we describe two cases of AGD from South Africa from two different hosts and two different geographical locations, constituting the first report of N. perurans from Africa. The first case was of turbot ongrown in a pilot scale land-based system, located on an abalone farm in Jacobsbaai, on the West coast of South Africa. The system consisted of sixteen circular tanks, each with a volume of approximately 50 m. The tanks were on seawater flow through with no recirculation and a turnover time of about 4.5 h. The fish received a pelleted ration. Turbot were imported as 5 g juveniles from Tongoy in Chile. Fish were first stocked in 1998. Chronic lowgrade mortality started in the summer of 2001. Most fish exhibited no external signs of disease. On necropsy, multifocal pale nodules, about 1 mm diameter, were present in various organs, including heart, liver, spleen and kidney. The gills were macroscopically normal. Wet preparations of the gills revealed large numbers of sessile peritrichous ciliates and an unknown species of Trichodina. Samples for histopathology were fixed in 10% neutral buffered formalin and processed using standard methods for paraffin wax embedding (Austin & Austin 1989). Sections were stained using Harris’ haematoxylin and eosin. Histological examination showed the nodules to be granulomas of unknown cause. The gills contained multiple areas of epithelial hyperplasia and fusion of the secondary lamellae. Fusion was seen to occur at the tips of the lamellae, leading to formation of a proximal cavity. These changes were highly suggestive of AGD (Kent et al. 1988; Correspondence A Mouton, Amanzi Biosecurity, Private Bag X15, Suite 190, Hermanus 7200, South Africa (e-mail: anna. mouton@amanzivet.co.za)

Neoparamoeba perurans loses virulence during clonal culture.

Amoebic Gill Disease affects farmed salmonids and is caused by Neoparamoeba perurans. Clonal cultures of this amoeba have been used for challenge experiments, however the effect of long-term culture on virulence has not been investigated. Here we show, using in vitro and in vivo methods, that a clone of N. perurans which was virulent 70 days after clonal culture lost virulence after 3 years in clonal culture. We propose that this is related either to the lack of attachment to the gills or the absence of an extracellular product, as shown by the lack of cytopathic effect on Chinook salmon embryo cells. The avirulent clonal culture of N. perurans allowed us to propose two potential virulence mechanisms/factors involved in Amoebic Gill Disease and is an invaluable tool for host-pathogen studies of Amoebic Gill Disease.

Support for the coevolution of Neoparamoeba and their endosymbionts, Perkinsela amoebae-like organisms.

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.