World association for the advancement of veterinary parasitology (WAAVP) guideline for testing the efficacy of ectoparasiticides for fish.

This guideline is intended to assist in the planning and execution of studies designed to assess the efficacy of ectoparasiticides for fish. It is the first ectoparasite-specific guideline to deal with studies set in the aquatic environment and therefore provides details for the maintenance of environmental standards for finfish. Information is included on a range of pre-clinical study designs as well as clinical studies in commercial/production sites, set within a regulatory framework. It provides information on the study animals, their welfare, husbandry and environmental requirements during the study. The most commonly pathogenic ectoparasites are presented with relevant points regarding life history, host challenge and numeric evaluation. Preparation and presentation of both topical and oral test treatments is provided, together with guidance on data collection and analysis. The guideline provides a quality standard or efficacy studies on finfish, which will assist researchers and regulatory authorities worldwide and contribute to the wider objective of harmonisation of procedures.

The fate of Lernaeocera branchialis (L.) (Crustacea; Copepoda) in Atlantic cod, Gadus morhua L.

Lernaeocera branchialis, a copepod crustacean parasite of gadoids, represents a potential threat to both wild and farmed cod, Gadus morhua. The pathological changes associated with the early stages of experimental infection have previously been reported in detail, and this article describes the lesions associated with later chronic stages of experimental infection. Chronic infection is characterised by extravascular granuloma formation and proliferation of fibrovascular tissue around intact and fragmented, degenerate parasites within both the gill arch and cardiac region. The majority of parasite granulomas are located within connective tissues of the gill arch or pericardium; however, low numbers are present within the wall of large vessels. The intraluminal parasites and thrombi of early stage infection are largely absent in these later lesions. We propose that organisation and incorporation of the parasite thrombus into the vessel wall with subsequent granuloma formation and extrusion into the surrounding connective tissue leads to the elimination of the parasite from the vascular system. Thus, rather than being a negative consequence of infection thrombosis is protective, allowing the host to survive the substantial initial vascular insult.

Infection dynamics of two renal myxozoans in hatchery reared fry and juvenile Atlantic cod Gadus morhua L.

In order to study the infection dynamics of 2 renal myxozoans, Zschokkella hildae Auerbach, 1910 and Gadimyxa atlantica Køie, Karlsbakk and Nylund, 2007 in cultured Atlantic cod, Gadus morhua L. aged 3-19 months, a specific single-round PCR assay and a double-label in situ hybridization protocol were developed. The results demonstrated that the 2 myxozoans show spatial separation of their development with regard to spore formation inside the renal tubules versus the collecting ducts and ureters, as well as temporal separation with Z. hildae proliferating and developing spores only once the G. atlantica infection decreases, despite the presence of both myxozoans in the smallest fry studied. These results strongly suggest within-host competition of the 2 myxozoans with potential suppression of Z. hildae by G. atlantica until G. morhua acquires immunity against G. atlantica. The quantification of the G. atlantica infection inside the renal tubules before and after a 29-day experimental growth performance study using fry from hatcheries with differing filtration systems showed that the intensity of infection with G. atlantica seems to be controlled if prolonged exposure to the myxozoan transmission stages takes place from hatching onwards. Surprisingly, growth rates in the trial were inversely affected suggesting that G. atlantica does not negatively influence cod fry growth performance.

Survival analysis after pancreatic resection for ampullary and pancreatic head carcinoma: an analysis of clinicopathological factors.

BACKGROUND AND OBJECTIVES: Surgery remains the only curative option for the treatment of pancreatic and ampullary carcinomas. To examine the survival differences between ampullary and pancreatic head carcinomas after pancreaticoduodenectomy. METHODS: A retrospective review of patients with ampullary or pancreatic head adenocarcinoma undergoing curative resection during a 6-year period prior to 2000. RESULTS: A total of 104 patients underwent pancreaticoduodenectomy for pancreatic head and ampullary carcinomas (n = 65 and n = 39, respectively). Histologically, pancreatic cancer was worse, with more lymph node involvement and more positive resection margins and vascular and perineural invasions than found in ampullary carcinoma. The median disease-free and overall survival rates were significantly better for ampullary cancer when compared with pancreatic cancer (17 vs. 9 months [P = 0.001] and 35 vs. 24 months [P = 0.006], respectively). The actuarial 5-year disease-free and overall survival rates were 4.4% and 10.5%, respectively, for pancreatic carcinoma and 27.9% and 31.8%, respectively, for ampullary carcinoma. Multivariate analysis showed that microscopic resection margin involvement (P = 0.02) and involvement of over three nodes (P < 0.001) were significant factors affecting the overall survival for pancreatic and ampullary carcinomas, respectively. CONCLUSIONS: In this study, patients with ampullary carcinoma have a better prognosis and survival than those with pancreatic carcinoma.

Using length-frequency data to elucidate the population dynamics of Argulus foliaceus (Crustacea: Branchiura).

This study uses a novel method for discriminating cohorts and investigating the population dynamics of the parasitic crustacean, Argulus foliaceus. Analysis of parasite length-frequency data was carried out in order to elucidate the timings and drivers behind the parasite's life cycle. Up to 6 cohorts of the parasite emerge through the course of 1 year in still-water trout fisheries in England. Recruitment ceases over the winter months; however, 3 cohorts of the parasite over-winter, 2 as eggs and 1 as a hatched stage. The technique, when used in conjunction with temperature data, also allowed for the reliable prediction of growth rates and provided estimates of egg incubation times and the length of hatching periods. These data showed that growth rates increased exponentially between the observed temperatures of 4 to 22 degrees C. The method allowed for the time taken from hatching to egg laying under field conditions to be predicted and produced estimates that were validated against independent laboratory studies on the growth of the parasite.

The influence of risk factors on the abundance, egg laying habits and impact of Argulus foliaceus in stillwater trout fisheries.

Fishery managers perceive the ectoparasitic crustacean, Argulus foliaceus to be a cause of significant economic loss through reduced fish capture rates. This study investigates the influence of previously identified risk factors on the abundance, egg laying habits and impact of this parasite through a longitudinal study of five trout fisheries of varying management intensity. Low water clarity, slow stock turnover and high temperatures showed a significant association with a high abundance of A. foliaceus. High infection levels, low water clarity and low temperature were also associated with reduced rates of fish capture, suggesting abundance of A. foliaceus alone may not affect the catch rates. Depth of egg laying varied in each site throughout the study, increasing in depth as temperature and water clarity increased. Eggs were found to be most abundant in natural fish holding areas and it is hypothesized that the location of egg laying is determined by the habitat usage of host fish.

Laparoscopic Habib 4X: a bipolar radiofrequency device for bloodless laparoscopic liver resection.

BACKGROUND: In recent years the progress of laparoscopic procedures and the development of new and dedicated technologies have made laparoscopic hepatic surgery feasible and safe. In spite of this laparoscopic liver resection remains a surgical procedure of great challenge because of the risk of massive bleeding during liver transection and the complicated biliary and vascular anatomy in the liver. A new laparoscopic device is reported here to assist liver resection laparoscopically. METHODS: The laparoscopic Habib 4X is a bipolar radiofrequency device consisting of a 2 x 2 array of needles arranged in a rectangle. It is introduced perpendicularly into the liver, along the intended transection line. It produces coagulative necrosis of the liver parenchyma sealing biliary radicals and blood vessels and enables bloodless transection of the liver parenchyma. RESULTS: Twenty-four Laparoscopic liver resections were performed with LH4X out of a total of 28 attempted resections over 12 months. Pringle manoeuvre was not used in any of the patients. None of the patients required intraoperative transfusion of red cells or blood products. CONCLUSION: Laparoscopic liver resection can be safely performed with laparoscopic Habib 4X with a significantly low risk of intraoperative bleeding or postoperative complications.

The pathology of the early stages of the crustacean parasite, Lernaeocera branchialis (L.), on Atlantic cod, Gadus morhua L.

Lernaeocera branchialis is a penellid copepod, the adults of which are commonly found on gadoids in the North Atlantic and North Sea. L. branchialis has a severe negative impact on wild gadoids and is a potentially serious pathogen of farmed cod, Gadus morhua. This paper describes an experimental infection by cohabiting the intermediate host, the flounder, Platichthys flesus, heavily infected with juvenile stages of L. branchialis, with hatchery-reared cod in order to study the pathology of the early stages of infection of the final host. Early stages of L. branchialis induce extensive local gill hyperplasia, large intravascular thrombus formation and a moderate cellular immune response in the cardiac and branchial tissues of G. morhua. The parasite appears to migrate within the lumen of the afferent branchial artery, ventral aorta and in turn, that of the bulbus arteriosus. These findings are consistent with the presence of a haematophagous, voluminous and rapidly invasive parasite.

The epidemiology of Argulus spp. (Crustacea: Branchiura) infections in stillwater trout fisheries.

Problem infections caused by species of the crustacean ectoparasite, Argulus, in UK stillwater trout fisheries appear to have increased in recent years. A cross-sectional study of 77 such fisheries was conducted to establish the perceived problem and the extent and severity of this problem, and to identify associated risk factors. An interview-based study was conducted in 2001 using a standardized questionnaire based on the management and infection status of each fishery in the previous year. Logistic regression was used to identify potential risk factors. Argulus spp. were perceived to cause economic losses in infected fisheries through a reduction in the number of anglers due to reduced aesthetic appeal and catchability of fish. Of the sites studied, 29% experienced such a problem infection in 2000. Argulus foliaceus was identified in all but one case and was found to be widely distributed throughout the UK. The remaining case was identified as Argulus coregoni. Three risk factors were associated with problem infections: the presence of an algal bloom, slow rates of stock turnover and whether water level dropped by <1 m during the summer months.

Molecular studies on the seasonal occurrence and development of five myxozoans in farmed Salmo trutta L.

Five myxozoan species, Tetracapsuloides bryosalmonae, Sphaerospora truttae, Chloromyxum schurovi, Chloromyxum truttae and a Myxobolus species were detected in farmed brown trout, Salmo trutta L. from Central Scotland. Using PCR and in situ hybridization, this study investigated the seasonal occurrence and tissue location of these species in young of the year brown trout. C. schurovi, C. truttae and Myxobolus sp. were first detected in brown trout in April, 2 months before T. bryosalmonae and S. truttae. T. bryosalmonae and S. truttae showed proliferation in the blood with intravascular stages of T. bryosalmonae accumulating in the heart. In contrast, only small amounts of PCR products of C. schurovi and C. truttae were obtained from the blood, suggesting that these species use the vascular system for transport but proliferate only in their target tissues from which large amounts of PCR product were obtained and where parasites were visible in histological sections. Large amounts of PCR product were obtained for T. bryosalmonae, S. truttae and both Chloromyxum species from the gills of brown trout, suggesting the gills as entry locus for these species. The neurotropic Myxobolus species formed plasmodia predominantly in the peripheral nerves, possibly indicating an entry route through the skin. Presporogonic stages of all other species had disappeared by September and mature spores were present from August onwards.

A molecular study of Eubothrium rugosum (Batsch, 1786) (Cestoda: Pseudophyllidea) using ITS rDNA sequences, with notes on the distribution and intraspecific sequence variation of Eubothrium crassum (Bloch, 1779).

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) of fish cestodes of the genus Eubothrium were sequenced. The ITS sequences of Eubothrium rugosum were determined and compared with previously analysed congeneric species, Eubothrium crassum and Eubothrium salvelini. The ITS-1 sequences of E. rugosum and E. crassum were 535 bp long, the length of E. salvelini ITS-1 was 536 bp. The ITS-2 region was found to be 403 bp in E. rugosum and E. crassum, and 401-402 bp in E. salvelini. The ITS-1 region of E. rugosum was closer to E. salvelini (identity 98.9-99.1%) than to E. crassum(97.9-98.1%), while the sequence similarity within the ITS-2 region was almost identical (97.5-98.0% for E. crassum; 97.7-98.3% for E. salvelini). Several restriction enzymes were found to be suitable for the differentiation of the three Eubothrium species by PCR-RFLP. The intraspecific sequence variation of E. crassum from different fish hosts (freshwater and marine) and European regions was very low, 0.2% for ITS-1 and 0.5% for ITS-2. Analysis of the ITS sequences of specimens from rainbow trout from three localities in Scotland revealed that both E. crassum and E. salvelini are present in this fish host.

Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique.

Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5' biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules.

A hyperparasitic microsporidian infecting the salmon louse, Lepeophtheirus salmonis: an rDNA-based molecular phylogenetic study.

The sea louse, Lepeophtheirus salmonis, is an obligate ectoparasitic copepod that lives on the external surface of salmonid fish. It is the most common ectoparasite of marine cage-reared salmonids, causing major economic loss to the aquaculture industry. During a sea louse monitoring programme, samples of L. salmonis were found to harbour an unreported microsporidian parasite. The microsporidian was observed in pre-adult and adult stages of both male and female copepods, with a prevalence of up to 5%. Unfixed spores were slightly pyriform in shape measuring 2.34 microm by 1.83 microm (+/- 0.01 microm) and were not observed to be enclosed by a sporophorous vesicle. The microsporidian infection was observed in all areas of the copepods' body, xenoma-like cysts forming directly under the cuticle in the epidermal tissue layer. In the present study, rDNA (530f-580r) sequence data gathered from the unidentified microsporidian parasite isolated from infected sea lice were compared with equivalents available in the databases in an attempt to identify its systematic position. The microsporidian was found to group within the phylogenetic clade containing the family Enterocytozoonidae, being most similar to members of the intranuclear genus Nucleospora. This is the first report of a hyperparasitic microsporidian infecting a caligid copepod.

Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes.

The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.

Does size really matter? Effects of fish surface area on the settlement and initial survival of Lepeophtheirus salmonis, an ectoparasite of Atlantic salmon Salmo salar.

The effect of the size of the surface area of a fish host on settlement and initial survival of Lepeophtheirus salmonis (Krøyer, 1837) was determined. Atlantic salmon Salmo salar L. of various sizes-small (43 +/- 4 g), medium (173 +/- 32 g) and large (644 +/- 62 g)-were initially examined to ascertain their respective body surface area, excluding gill surface. The initial examination showed a size-dependent body to fin area ratio, with the fin area of small fish representing 34% of the total body area and the fin area of medium and large fish representing 26 and 23%, respectively. Regression analysis of the body weight and standard length against total body surface area gave a good correlation and high R2 values. Two simultaneous experimental infections with approximately 7,000 copepodids of L. salmonis were carried out on fish populations of mixed sizes consisting of 30 small fish, 10 medium-sized fish and 5 large fish, with an approximate total surface area per size group of 2700 cm2. Higher numbers of parasites were found on the small size group, which also had the highest parasite density, with 0.25 and 0.45 parasites cm(-2). Comparison of samples of 5 fish per experimental group revealed that the larger fish had the highest mean numbers of parasites, but the smaller fish still retained the highest density. There was a statistically significantly higher settlement on the fins than on the remainder of the body surface in all size groups. Highest numbers of parasites were particularly found on the dorsal and pectoral fins. Examination of the surface surface area revealed that the dorsal fin had the greater parasite density, with >2 parasites cm(-2) in all fish size groups. These data provide insight into the effects of the amount of host surface area available on parasite settlement and survival, and highlight the potentially increased susceptibility of farmed salmon smolts to infection of this ectoparasite.

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae).

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.

Commercial trials using emamectin benzoate to control sea lice Lepeophtheirus salmonis infestations in Atlantic salmon Salmo salar.

Two trials were conducted at commercial salmon farms to evaluate the efficacy of emamectin benzoate (Slice, 0.2% aquaculture pre-mix, Schering-Plough Animal Health) as a treatment for sea lice Lepeophtheirus salmonis (Krøyer) and Caligus elongatus Nordmann infestations in Atlantic salmon Salmo salar L. Trials were carried out in 15 m2 commercial sea pens, at temperatures of 5.5 to 7.5 degrees C and 10.8 to 13.8 degrees C. Each pen was stocked with 14,000 to 17,500 fish with mean weights of 0.44 to 0.74 and 1.33 to 1.83 kg. Fish were naturally infested with sea lice at the start of each trial. At Day -1, samples of 10 or 15 fish were taken from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in feed, to 4 replicate pens, at a dose of 50 micrograms kg-1 biomass d-1 for 7 consecutive days (Days 0 to 6). Sea lice were counted again, between Days 7 and 77, and comparisons made with untreated control fish. Despite adverse weather conditions, wide variations in fish weights and exposure to new infestations, treatment was effective against chalimus and motile stages of L. salmonis. In the autumn trial, efficacy at Day 27 was 89%, and lice numbers remained lower on treated fish than on control fish 64 d from the start of treatment. In the winter trial, reductions in lice numbers at low temperatures were slower but good efficacy was achieved by Day 35. Although control fish had to be treated with hydrogen peroxide at Day 21, fish treated only with emamectin benzoate on Days 0 to 6 still had 89% fewer lice than control fish at Day 35. There were very few C. elongatus present, but at the end of both trials numbers were lower on treated fish. No adverse effects were associated with treatment of fish with emamectin benzoate.

The use of statistical classifiers for the discrimination of species of the genus Gyrodactylus (Monogenea) parasitizing salmonids.

This study applies flexible statistical methods to morphometric measurements obtained via light and scanning electron microscopy (SEM) to discriminate closely related species of Gyrodactylus parasitic on salmonids. For the first analysis, morphometric measurements taken from the opisthaptoral hooks and bars of 5 species of gyrodactylid were derived from images obtained by SEM and used to assess the prediction performance of 4 statistical methods (nearest neighbours; feed-forward neural network; projection pursuit regression and linear discriminant analysis). The performance of 2 methods, nearest neighbours and a feed-forward neural network provided perfect discrimination of G. salaris from 4 other species of Gyrodactylus when using measurements taken from only a single structure, the marginal hook. Data derived from images using light microscopy taken from the full complement of opisthaptoral hooks and bars were also tested and nearest neighbours and linear discriminant analysis gave perfect discrimination of G. salaris from G. derjavini Mikailov, 1975 and G. truttae Gläser, 1974. The nearest neighbours method had the least misclassifications and was therefore assessed further for the analysis of individual hooks. Five morphometric parameters from the marginal hook subset (total length, shaft length, sickle length, sickle proximal width and sickle distal width) gave near perfect discrimination of G. salaris. For perfect discrimination therefore, larger numbers of parameters are required at the light level than at the SEM level.

Cercarial emergence of Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Rudolphi, 1819) (Digenea: Strigeidae): contrasting responses to light:dark cycling.

Many digenean cercariae have been shown to emerge from their molluscan hosts with distinct shedding patterns that have enabled the discrimination of morphologically similar species, or even strains. In this study the cercarial emission patterns of three strigeid species, Ichthyocotylurus erraticus, I. variegatus and Apatemon gracilis, from experimentally infected natural hosts were found to exhibit rhythms that correlated with the light:dark cycle. Both Ichthyocotylurus spp. exhibited a diurnal pattern of release in which cercariae emerged during the light period. Each demonstrated a latent period before the liberation of large numbers of cercariae and yielded similar numbers of cercariae daily. These rhythms offered no means for the discrimination of these two morphologically similar species. A. gracilis cercariae demonstrated a very different circadian rhythm in which the majority emerged at the onset of darkness with no latent period, whereas the cercarial numbers released daily were far greater. Differences could be related to piscine host behaviour.

Towards an automated system for the identification of notifiable pathogens: using as an example.

Simple and rapid identification of pathogen species is crucial to the control of many diseases. Here, James Kay, Andrew Shinn and Christina Sommerville demonstrate that statistical classifiers discriminate a notifiable pathogen Gyrodactylus salaris Malmberg, 1957, a lethal ectoparasite of Atlantic salmon, Salmo salar L., from its benign close relatives.