Oral vaccination of Nile tilapia (Oreochromis niloticus) against francisellosis elevates specific antibody titres in serum and mucus.

Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed.

A novel use of social media to evaluate the occurrence of skin lesions affecting wild dusky grouper, Epinephelus marginatus (Lowe, 1834), in Libyan coastal waters.

The social media network Facebook™ was used to gather information on the occurrence and geographical distribution of dusky grouper dermatitis, a skin lesion affecting the dusky grouper, Epinephelus marginatus. Dusky grouper are common targets for spear fishermen in the Mediterranean and by monitoring spearfishing activity in Libyan waters, it was possible to document skin lesions from their entries on Facebook. Thirty-two Facebook accounts and 8 Facebook groups posting from 23 Libyan coastal cities provided a retrospective observational data set comprising a total of 382 images of dusky grouper caught by spearfishing between December 2011 and December 2015. Skin lesions were observable on 57/362 fish, for which images were of sufficient quality for analysis, giving a minimal prevalence for lesions of 15.75%. Only dusky grouper exceeding an estimated 40 cm total length exhibited lesions. The ability to collect useful data about the occurrence and geographical distribution of pathological conditions affecting wild fish using social media networks demonstrates their potential utility as a tool to support epidemiological studies and monitor the health of populations of aquatic animals. To our knowledge, this represents the first time that such an approach has been applied for assessing health in a wild population of fish.

Ulcerative dermatitis in wild dusky grouper Epinephelus marginatus (Lowe) from Libyan waters.

In the period 2013-2015, wild dusky grouper, Epinephelus marginatus (Lowe), caught in Libyan coastal waters and ranging in size from 42 to 92 cm in total length, were observed to have distinctive skin lesions of unknown aetiology. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digenean trematode parasites carrying similar eggs were also seen within the blood vessels of the deep and superficial dermis. The cause of this distinctive condition, termed dusky grouper dermatitis (DGD), and its potential impact upon already threatened Mediterranean wild dusky grouper populations and upon cultured grouper more widely have yet to be established.

Economic costs of protistan and metazoan parasites to global mariculture.

Parasites have a major impact on global finfish and shellfish aquaculture, having significant effects on farm production, sustainability and economic viability. Parasite infections and impacts can, according to pathogen and context, be considered to be either unpredictable/sporadic or predictable/regular. Although both types of infection may result in the loss of stock and incur costs associated with the control and management of infection, predictable infections can also lead to costs associated with prophylaxis and related activities. The estimation of the economic cost of a parasite event is frequently complicated by the complex interplay of numerous factors associated with a specific incident, which may range from direct production losses to downstream socio-economic impacts on livelihoods and satellite industries associated with the primary producer. In this study, we examine the world's major marine and brackish water aquaculture production industries and provide estimates of the potential economic costs attributable to a range of key parasite pathogens using 498 specific events for the purposes of illustration and estimation of costs. This study provides a baseline resource for risk assessment and the development of more robust biosecurity practices, which can in turn help mitigate against and/or minimise the potential impacts of parasite-mediated disease in aquaculture.

Role of kairomones in host location of the pennellid copepod parasite, Lernaeocera branchialis (L. 1767).

The life cycle of the parasitic copepod Lernaeocera branchialis involves 2 hosts, typically a pleuronectiform host upon which development of larvae and mating of adults occurs and a subsequent gadoid host, upon which the adult female feeds and reproduces. Both the copepodid and adult female stages must therefore locate and identify a suitable host to continue the life cycle. Several mechanisms are potentially involved in locating a host and ensuring its suitability for infection. These may include mechano-reception to detect host movement and chemo-reception to recognize host-associated chemical cues, or kairomones. The aim of this study was to identify the role of kairomones in host location by adult L. branchialis, by analysing their behaviour in response to fish-derived chemicals. Experiments demonstrated that water conditioned by immersion of whiting, Merlangius merlangus, elicited host-seeking behaviour in L. branchialis, whereas cod- (Gadus morhua) conditioned water did not. Lernaeocera branchialis are considered a genetically homogeneous population infecting a range of gadoids. However, their differential response to whiting- and cod-derived chemicals in this study suggests that either there are genetically determined subspecies of L. branchialis or there is some form of environmental pre-conditioning that allows the parasite to preferentially recognize the host species from which it originated.

Gill pathology in Scottish farmed Atlantic salmon, Salmo salar L., associated with the microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009.

Gill disorders have emerged in recent years as a significant problem in the production of marine-stage Atlantic salmon Salmo salar L. The multi-aetiological condition 'proliferative gill inflammation' (PGI) has been reported to cause heavy losses in western Norway, yet reports of Scottish cases of the disease have remained anecdotal. In the present study, histopathological material from a marine production site in the Scottish Highlands experiencing mortalities due to a seasonal gill disease with proliferative-type pathology was examined using light microscopy, special staining techniques and transmission electron microscopy (TEM). The microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009 (syn. Paranucleospora theridion) was identified by staining using a Gram Twort method and TEM associated with distinctive proliferative and necrotic pathology confined to the interlamellar Malpighian cell areas of the primary filaments. Epitheliocystis was not a feature of the gill pathology observed. It is believed this is the first report of D. lepeophtherii being identified associated with pathology in a Scottish gill disease case, and supports anecdotal reports that a disease at least partly synonymous with PGI as described by Norwegian researchers is present in Scottish aquaculture.

Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomus regius Asso (Perciformes: Sciaenidae).

Marine cultured meagre, Argyrosomus regius Asso, in central and western Greece were affected by an outbreak of systemic granulomatous disease subsequently demonstrated to be nocardiosis. The fish were originally imported as juveniles from hatcheries in France and Italy and on-grown in Greece, the latter also providing broodstock for a small number of local Greek hatcheries for the production of second-generation juveniles. The disease in cage reared fish had been present throughout the year, particularly in the 1+ and 2+ year old fish with a low to variable morbidity and 1-4% total mortality. Multiple lesions were visible externally on the skin of affected fish, with severe ulcerations and necrosis. Internally, multifocal yellowish-white nodules, 0.1-0.5 cm in diameter, were visible on the surface of several internal organs. Histopathology revealed systemic granulomatous inflammation. Fite-Faraco staining clearly demonstrated the presence of Nocardia-like organisms which were Gram-positive, long, rod to beaded filamentous bacteria. Nocardia genus-specific 16s RNA primers NG1 and NG2 were used to generate a 600 bp fragment recovered from affected tissue, confirming the diagnosis of Nocardia spp. To our knowledge, this is the first report of nocardiosis in meagre.

Cell types and structures involved in tench, Tinca tinca (L.), defence mechanisms against a systemic digenean infection.

Histopathological and ultrastructural investigations were conducted on 36 tench, Tinca tinca (L.), from Lake Trasimeno (Italy). The gills, intestine, liver, spleen, kidney and heart of 21 individuals were found to harbour an extensive infection of larvae of an unidentified digenean trematode. The eyes, gonads, swim bladder and muscles were uninfected. The parasites in each tissue type were embedded in a granulomatous proliferation of tissue, forming a reactive fibroconnective capsule around each larva. Most of the encysted larvae were metacercariae, in a degenerative state, but on occasion some cercariae were found. Many of the granulomas were either necrotic or had a calcified core. Within the granuloma of each, the occurrence of granulocytes, macrophages, rodlet cells and pigment-bearing macrophage aggregates was observed. Hearts bore the highest parasitic infection. Whilst the presence of metacercariae within the intestine was found positioned between the submucosa and muscle layers, metacercariae in the liver were commonly found encysted on its surface where the hepatocytes in close contact with the granuloma were observed to have electron-lucent vesicles within their cytoplasm. Metacercariae encysting adjacent to the cartilaginous rods of gill filaments were seen to elicit a proliferation of the cartilage from the perichondrium. Rodlet cells, neutrophils and mast cells were frequently observed in close proximity to, and within, infected gill capillaries. Given the degenerated state of most granulomas, a morphology-based identification of the enclosed digeneans was not possible.

Innate immune defence mechanisms of tench, Tinca tinca (L.), naturally infected with the tapeworm Monobothrium wageneri.

A histochemical and ultrastructural investigation of the cellular inflammatory response within the intestines of tench Tinca tinca L. naturally infected with the caryophyllidean cestode Monobothrium wageneri was conducted and the data obtained compared to those in uninfected counterparts. Cestode infections within the intestines were evident through the appearance of raised inflammatory swellings induced by the deep penetration of their scolices into the intestinal wall. Cestodes typically attached in tight clusters, inducing a massive hyperplastic granulocyte response of mast cells and neutrophils, which were significantly more numerous (P < 0·01) in the intestines of infected (n = 14) than of uninfected (n = 9) tench. Neutrophils were more abundant than mast cells (P < 0·01) in host tissues in close proximity to the parasite tegument. In transmission electron microscopy sections, mast cells and neutrophils were frequently observed in contact with or inside capillaries, and in close proximity to the cestode. Degranulation of both cell types was seen in the submucosa and lamina muscularis, notably in the immediate tissues surrounding the scolex of M. wageneri. No tegumental secretions were seen at the host-parasite interface. Occasional rodlet cells were encountered in the submucosa of infected fish.

An assessment of the use of drug and non-drug interventions in the treatment of Ichthyophthirius multifiliis Fouquet, 1876, a protozoan parasite of freshwater fish.

Infection by the ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876 causes significant economic losses in freshwater aquaculture worldwide. Following the ban on the use of malachite green for treating food fish, there has been extensive research aimed at identifying suitable replacements. In this paper we critically assess drug and non-drug interventions, which have been tested for use or have been employed against this parasite and evaluate possibilities for their application in farm systems. Current treatments include the administration of formaldehyde, sodium chloride (salt), copper sulphate and potassium permanganate. However, purportedly more environmentally friendly drugs such as humic acid, potassium ferrate (VI), bronopol and the peracetic acid-based products have recently been tested and represent promising alternatives. Further investigation, is required to optimize the treatments and to establish precise protocols in order to minimize the quantity of drug employed whilst ensuring the most efficacious performance. At the same time, there needs to be a greater emphasis placed on the non-drug aspects of management strategies, including the use of non-chemical interventions focusing on the removal of free-swimming stages and tomocysts of I. multifiliis from farm culture systems. Use of such strategies provides the hope of more environmentally friendly alternatives for the control of I. multifiliis infections.

Development of a light microscopy stain for the sclerites of Gyrodactylus von Nordmann, 1832 (Monogenea) and related genera.

Recent developments in semi-automated identification techniques and the increasing ability to rapidly access digital images and taxonomic descriptions offer to increase the range of individuals capable of performing taxonomic identifications. The present study details methodological approaches undertaken in developing a dedicated stain for the visualisation of monogenean haptoral skeletal elements and reproductive sclerites. The histochemical protocols centre around the use of fluorescent dyes and standard light and laser scanning confocal microscopy to support studies of the functional morphology of these hard structures in small, relatively uncompressed specimens, making these structures more amenable to semi-automated analysis and identification techniques. Staining of the sclerites was achieved using a tissue digestion step to remove the tegument and tissues enclosing the sclerites and then staining them in situ with 40 mM chromothrope 2R (C2R) containing 3 mM phosphotungstic acid (PTA) and 0.5% acetic acid (AA) at room temperature for up to 2 days. Visualisation of the armature of the male copulatory organ of warm water Gyrodactylus species was achieved using 40 mM C2R containing 3 mM PTA for 3 days, whilst cold water species were best stained in 6.4 mM C2R for 1 day without an NaOH pre-treatment. The developed techniques allow for good visualisation of the skeletal elements in a number of monogenean groups and promise to assist the preparation and identification/description of specimens. The 2D/3D digital images of specimens prepared in this manner should provide a useful resource for taxonomists and others needing material to assist specimen identification.

Confirmation of the hosts involved in the life cycle of an acanthocephalan parasite of Anguilla anguilla (L.) from Lake Piediluco and its effect on the reproductive potential of its amphipod intermediate host.

A total of 37 European eels, Anguilla anguilla, collected from Lake Piediluco, Central Italy, and measuring 35 to 75.5 cm in total length (mean±1 SD, 56.41 ± 10.89 cm) were examined, and their acanthocephalan infections assessed. Thirty-two (86.49%) eels were infected with Acanthocephalus rhinensis (mean±1 SD, 67.38 ± 65.16; range, 1-350), a species that, purportedly, can be discriminated on the basis of a characteristic band of orange-brown pigmentation encircling the anterior end of the trunk. This feature, however, was not seen on any of the A. rhinensis specimens that were removed, either attached to the gut wall or free within the gut lumen, from infected eels. Approximately 40% of the eels were coinfected with the dracunculid swimbladder nematode Anguillicoloides crassus, while a single eel was also coinfected with eight specimens of a second acanthocephalan, Dentitruncus truttae. From the stomachs of two eels, 109 intact and partially digested specimens of amphipod Echinogammarus tibaldii (Pinkster & Stock 1970) were recovered, 16 (14.6%) of these were infected with one to two cystacanths of A. rhinensis per host. From a sample of 850 E. tibaldii taken from the peripheral lakeside vegetation, 102 (12%; sex ratio, 1:1) gammarids were infected with one to two A. rhinensis cystacanths. Unparasitised ovigerous female E. tibaldii specimens had significantly higher numbers of eggs in their brood pouches compared with their infected counterparts (t-test, P < 0.01).

Larval gryporhynchid tapeworms (Cestoda: Cyclophyllidea) of British freshwater fish, with a description of the pathology caused by Paradilepis scolecina.

Larvae of the cyclophyllidean tapeworms Paradilepis scolecina (Rudolphi, 1819), Neogryporhynchus cheilancristrotus (Wedl, 1855) and Valipora campylancristrota (Wedl, 1855), are described from British freshwater fish. The morphometrics of the rostellar hooks, infection characteristics and host ranges of these parasites from fisheries in England and Wales are presented. Difficulties in the detection, handling and identification of these tapeworms are highlighted, and may in part explain the paucity of records from Britain. Tissue digestion was shown to be a useful technique for the examination of these parasites, providing clear and consistent preparations of the rostellar hooks for measurement. The pathological changes caused by P. scolecina to the liver of wild tench, Tinca tinca, are detailed for the first time. Tapeworms located in the hepatic parenchyma and pancreatic tissues caused little pathological damage and invoked only mild inflammatory responses. The small size of these tapeworms and their encapsulation within host tissues appear to limit the severity of pathology, compared with parasites that insert their rostellum during attachment.

Introduction and spread of non-native parasites with Silurus glanis L. (Teleostei: Siluridae) in UK fisheries.

Despite growing concern of the ecological risks posed by the European catfish Siluris glanis L. in freshwater fisheries, little information exists on the parasite fauna of this silurid catfish in Britain. Parasitological examinations of released S. glanis from four still-water fisheries in England revealed the presence of Thaparocleidus vistulensis (Siwak, 1932) and Ergasilus sieboldi (Nordmann, 1832), both non-native parasites, the latter known to be an important fish pathogen. This represents the first record of T. vistulensis from British freshwater fish. The human-assisted movement of S. glanis between UK recreational still-water fisheries provides a clear avenue for the introduction and spread of non-native parasites.

The response of intestinal mucous cells to the presence of enteric helminths: their distribution, histochemistry and fine structure.

Histochemical and ultrastructural investigations were conducted on the mucous cells of the intestine of brown trout, Salmo trutta L., naturally infected with the cestode Cyathocephalus truncatus (Pallas, 1781) and the acanthocephalan Echinorhynchus truttae Shrank, 1788. A subpopulation of 45 S. trutta were examined of which 15 specimens harboured E. truttae, 15 of which were infected with C. truncatus and 15 fish, the control group, were uninfected. In histological sections, hyperplasia and hypertrophy of the mucous cells were evident at the site of parasite infection. Enhanced mucus secretion was also recorded in infected fish. The number of mucous cells close to the site of parasite attachment within the intestine was significantly higher than the number detected in uninfected individuals and in infected individuals at sites 1 cm or greater from the point of parasite attachment. There were no significant differences between the number of mucous cells found at the latter two sites. Alcian blue and periodic acid-Schiff's staining of representative histological sections revealed a significant increase in the number of mucous cells staining positively for acid glycoconjugates compared to the number of cells found in the intestines of uninfected S. trutta. In transmission electron microscopy sections, each mucous cell typically possessed an elongated, basally positioned nucleus. The cytoplasm was observed to possess numerous electron dense and lucent vesicles, in addition to well-developed rough endoplasmic reticulum, Golgi apparatus and a few round mitochondria.

Morphometric analysis of four species of Eubothrium (Cestoda: Pseudophyllidea) parasites of salmonid fish: an interspecific and intraspecific comparison.

Four species of the genus Eubothrium (E. crassum, E. fragile, E. rugosum and E. salvelini) were subjected to morphometric comparison. Discriminant analysis was conducted utilising 17 characters measured on the scolex and strobila of 101 specimens. Univariate statistics were first used to detect features that were useful for separating individual Eubothrium species and two different host populations of E. salvelini. Subsequent multivariate discriminant analysis, combining all the measured variables, made it possible to separate all four species. A comparison of the four taxa revealed that (1) E. fragile is the most distinct species, possessing a much smaller scolex than the other congeners, and its similarity with the other marine species E. crassum is not proven; (2) the two freshwater taxa, E. rugosum and E. salvelini are the most similar; (3) the characters most suitable for species differentiation are the length of the scolex, the width of the apical disc, the width of the neck and its area, the width of eggs and the number of testes; (4) the width of the apical disc was confirmed to be the most stable character at the intraspecific level (within E. salvelini host populations) and is therefore considered to be a trait of the highest discriminative power in the subset of four Eubothrium species.

Fine structure and cellular responses at the host-parasite interface in a range of fish-helminth systems.

A series of ultrastructural-based studies were conducted on the interface region in different fish-helminth systems: (a) an intestinal infection of the cestode Monobothrium wageneri in tench, Tinca tinca; (b) an extensive intestinal submucosa and mucosal infection in tench by metacercariae of an unidentified digenean trematode; (c) an intestinal infection in brown trout, Salmo trutta, by the acanthocephalan Dentitruncus truttae; (d) an extraintestinal infection by larvae of the acanthocephalan, Pomphorhynchus laevis in three-spined sticklebacks, Gasterosteus aculeatus; and (e) an infection in the livers of Eurasian minnow, Phoxinus phoxinus, by larvae of the nematode Raphidascaris acus. Endoparasitic helminths frequently cause inflammation of the digestive tract and associated organs, inducing the recruitment of various immune cells to the site of infection. In each of the fish-helminth systems that were studied, a massive hyperplastic granulocyte response involving mast cells (MCs) and neutrophils in close proximity to the helminths was documented. The current study presents data on the interface region in each fish-helminth system and documents the penetration of mast cells granules within the tegument of P. laevis larvae. No extracellular vesicles containing tegumental secretions from any of the four different taxa of endoparasitic helminths species at the host-parasite interface region were seen.

Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia.

Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.

The application of chaetotaxy in the discrimination of Gyrodactylus salaris Malmberg, 1957 (Gyrodactylidae: Monogenea) from species of the genus parasitising British salmonids.

The chaetotaxy of argentophilic structures on three species of the monogenean genus Gyrodactylus was investigated in an attempt to distinguish species of this genus. Maps were prepared for Gyrodactylus salaris from Scandinavia and compared with two native species of Gyrodactylus parasitising salmonids in Britain, namely Gyrodactylus derjavini and Gyrodactylus truttae. The maps were subsequently refined and analysed for zones of homology and differentiation. The results demonstrate that G. salaris can be readily distinguished by this technique, which is, therefore, of great potential value in the identification of this notifiable pathogen. The key aggregations of sensilla discriminating G. salaris are, ventrally, the antero-ventral set, the medio-lateral set and the postero-lateral set, and, dorsally, the postero-dorsal set.

A study of the composition of the sclerites of Gyrodactylus Nordmann, 1832 (Monogenea) using X-ray elemental analysis.

The elemental compositions of the hamuli, marginal hooks and ventral bars were examined following their release from the opisthaptor of the monogenean parasites Gyrodactylus salaris, G. caledoniensis and G. colemanensis. Their compositions were determined using X-ray elemental analysis on an analytical scanning electron microscope, and the variation between each species investigated. Hamuli and marginal hooks exhibited a major peak for sulphur, whilst the ventral bar had high peaks of both sulphur and calcium. The high sulphur content and structure of the hamuli suggest they are composed of a keratin-like protein, while the nature of the ventral bar is less clear. The hamuli of G. colemanensis were differentiated from those of G. caledoniensis by virtue of their calcium fraction. Low levels of several other elements, including vanadium, were also detected in some sclerites. The composition of the hooks of Gyrodactylus and their relationship to the sclerites of monopisthocotyleans and polyopisthocotyleans is commented upon.