Immunoassays are not immune to errors: Examples from two studies of steroid output from freshwater trout farms.

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L-1) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L-1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed.

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.).

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.

Model for ranking freshwater fish farms according to their risk of infection and illustration for viral haemorrhagic septicaemia.

We developed a model to calculate a quantitative risk score for individual aquaculture sites. The score indicates the risk of the site being infected with a specific fish pathogen (viral haemorrhagic septicaemia virus (VHSV); infectious haematopoietic necrosis virus, Koi herpes virus), and is intended to be used for risk ranking sites to support surveillance for demonstration of zone or member state freedom from these pathogens. The inputs to the model include a range of quantitative and qualitative estimates of risk factors organised into five risk themes (1) Live fish and egg movements; (2) Exposure via water; (3) On-site processing; (4) Short-distance mechanical transmission; (5) Distance-independent mechanical transmission. The calculated risk score for an individual aquaculture site is a value between zero and one and is intended to indicate the risk of a site relative to the risk of other sites (thereby allowing ranking). The model was applied to evaluate 76 rainbow trout farms in 3 countries (42 from England, 32 from Italy and 2 from Switzerland) with the aim to establish their risk of being infected with VHSV. Risk scores for farms in England and Italy showed great variation, clearly enabling ranking. Scores ranged from 0.002 to 0.254 (mean score 0.080) in England and 0.011 to 0.778 (mean of 0.130) for Italy, reflecting the diversity of infection status of farms in these countries. Requirements for broader application of the model are discussed. Cost efficient farm data collection is important to realise the benefits from a risk-based approach.

Expert consultation on risk factors for introduction of infectious pathogens into fish farms.

An expert consultation was conducted to provide quantitative parameters required to inform risk-based surveillance of aquaculture holdings for selected infectious hazards. The hazards were four fish diseases endemic in some or several European countries: infectious salmon anaemia (ISA), viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), and koi herpes virus disease (KHD). Experts were asked to provide estimates for the relative importance of 5 risk themes for the hazard to be introduced into and infect susceptible fish at the destination. The 5 risk themes were: (1) live fish and egg movements; (2) exposure via water; (3) on-site processing; (4) short distance mechanical transmission and (5) distance independent mechanical transmission. The experts also provided parameter estimates for hazard transmission pathways within the themes. The expert consultation was undertaken in a 2 step approach: an online survey followed by an expert consultation meeting. The expert opinion indicated that live fish movements and exposure via water were the major relevant risk themes. Experts were recruited from several European countries and thus covered a range of farming systems. Therefore, the outputs from the expert consultation have relevance for the European context.

Investigation of mortality in Pacific oysters associated with Ostreid herpesvirus-1 µVar in the Republic of Ireland in 2009.

High levels of mortality in Pacific oysters Crassostrea gigas in the Republic of Ireland were recorded during the summer of 2009. The new variant of Ostreid herpes 1 (OsHV-1 µVar) which first emerged in France in 2008 was identified from affected stocks. Retrospective data was collected from 70 oyster farmers through an interviewer-administered questionnaire to investigate the distribution and determinants of the mortality. Based on farmer recall, data were recorded at the batch level for cumulative mortality during 2009, start dates and duration of the mortality event and the age of animals affected. Observable mortalities were recorded in 109 out of 346 batches at 47 sites; 104 of the 109 batches were located in bays where OsHV-1 µVar had been detected. The records from bays where OsHV-1 µVar had been detected were analysed to characterize the pattern of mortality and potential risk factors. Batch mortality averaged 37% (18-65% quartiles) but showed a bimodal distribution (half the batches had mortality less than 45%). Mortalities started at the end of May and continued until early August, peaking in early July. On average oysters died over a period of 18 days. Mortality varied considerably both between and within bays. Mortality started in recently introduced batches and occurred later in the summer in established oysters, which is consistent with the introduction of an infectious agent. Mortality was significantly lower in adults compared with other age groups, which supports observations from France. Three variables were significantly (P<0.05) associated, in both bivariate screening and a logistic regression, with high batch-level mortality (>40%): oysters (i) introduced as juveniles, (ii) during or since the winter of 2008/9 and (iii) which spent less than 8h out of water (in a tidal cycle) (compared with oysters introduced as adults before the winter of 2008/9 and spending more than 8h out of water). Twenty-one percent of triploid batches experienced "high" (>40%) mortality compared with 10% for diploid batches which was significant (P<0.05) in the initial bivariate screening but not in the final logistic regression model. Future studies should develop improved methods to assess oyster mortality and follow stocks over time to better determine the influence of management and environmental factors on mortality.

Comparative susceptibility of Atlantic salmon and rainbow trout to Yersinia ruckeri: relationship to O antigen serotype and resistance to serum killing.

A study was undertaken to compare the virulence and serum killing resistance properties of Atlantic salmon and rainbow trout Yersinia ruckeri isolates. Five isolates, covering heat-stable O-antigen O1, O2 and O5 serotypes, were tested for virulence towards fry and juveniles of both species by experimental bath challenge. The sensitivity of 15 diverse isolates to non-immune salmon and rainbow trout serum was also examined. All five isolates caused significant mortality in salmon fry. Serotype O1 isolate 06059 caused the highest mortality in salmon (74% and 70% in fry and juveniles, respectively). Isolate 06041, a typical ERM-causing serotype O1 UK rainbow trout strain, caused mortalities in both rainbow trout and salmon. None of the salmon isolates caused any mortalities in 150-250 g rainbow trout, and only serotype O2 isolate 06060 caused any significant mortality (10%) in rainbow trout fry. Disease progression and severity was affected by water temperature. Mortality in salmon caused by the isolates 06059 and 05094 was much higher at 16 °C (74% and 33%, respectively) than at 12 °C (30 and 4% respectively). Virulent rainbow trout isolates were generally resistant to sera from both species, whereas salmon isolates varied in their serum sensitivity. Convalescent serum from salmon and rainbow trout that had been infected by serotype O1 isolates mediated effective classical pathway complement killing of serotype O1 and O5 isolates that were resistant to normal sera. Overall, strains recovered from infected salmon possess a wider range of phenotypic properties (relative virulence, O serotype and possession of serum-resistance factors), compared to ERM-causing rainbow trout isolates.