Sleeping disease and pancreas disease: comparative histopathology and acquired cross-protection

Sleeping disease (SD), a disease of freshwater-reared rainbow trout, Oncorhynchus mykiss (Walbaum), was histologically compared to pancreas disease (PD), a disease of sea water-reared Atlantic salmon, Salmo salar L. Some weeks after injection of kidney homogenates from SD- or PD-affected fish in rainbow trout, the fish successively developed pancreatic, heart and muscular lesions in both populations, although the SD-injected population was the most affected. An acquired protection was observed against PD and SD after an initial injection with PD- or SD-infective material. This acquired cross-protection and the similar histopathologies in PD and SD suggest the two diseases could be caused by a similar or identical agent.     

Quality of raw and smoked fillets from clinically healthy Atlantic salmon,Salmo salar L., following an outbreak of pancreas disease (PD)

Pancreas disease (PD) is a viral disease of farmed salmonid fish, which causes huge economic losses. Pathological changes in skeletal muscle, pancreas and heart are hallmarks of PD. Stakeholders in the fish‐smoking industry have claimed that fillets from PD‐affected Atlantic salmon, Salmo salar L., are of poor quality. We therefore examined harvest‐ready, clinically healthy Atlantic salmon from a population of fish previously affected by PD. Histopathological changes in skeletal muscle tissues ranged from minor to severe. Fillet quality measurements showed that fish with severe skeletal muscle changes provided a paler raw fillet and a yellowish and harder cold‐smoked fillet than normal. PD had no significant effect on fillet gaping, bacteriological quality or off‐odour development during storage. An unexpected finding was a significant subendocardial fibrosis in 23% of the PD‐affected fish. The latter may indicate susceptibility to stress‐related heart failure.     

Pancreas disease (PD) in sea‐reared Atlantic salmon,Salmo salar L., in Norway; a prospective,longitudinal study of disease development and agreement between diagnostic test results

A prospective longitudinal study was performed on three cages at each of three Norwegian Atlantic salmon seawater sites that experienced outbreaks of pancreas disease (PD). Once salmonid alphavirus (SAV) ribonucleic acid (RNA) was detected by real‐time RT‐PCR (Rt RT‐PCR) at a site, it became detected in all studied cages and was persistently found until the end of the study period up to 19 months after first detection. SAV‐specific antibodies were detected at all sites until the end of the study period and were also found at a high prevalence in broodfish at the time of stripping. No evidence of increased viral activity was detected in these broodfish. One site tested negative over several months prior to the first detection of SAV by Rt RT‐PCR and SAV‐specific antibody, which occurred 1 month prior to clinical manifestations of PD. Moribund fish or thin fish/runts that were sampled after the first PD diagnosis had almost twice the risk of testing positive by one or more diagnostic tests compared to that of randomly selected apparently healthy individuals. This paper describes the first detailed investigation of the disease development of PD at site and cage level in Norway, as well as an assessment of the performance and agreement of the commonly used diagnostic tests.     

Mortality and weight loss of Atlantic salmon,Salmon salar L., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from Norway

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD‐endemic zone. This subtype has spread rapidly among fish farms in mid‐Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific‐based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post‐smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3‐infected fish showed higher mortality than SAV 2‐infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV‐infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection.     

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co‐varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.     

Heritability of mortality in response to a natural pancreas disease (SPDV) challenge in Atlantic salmon,Salmo salar L., post‐smolts on a West of Ireland sea site

Pancreas disease (PD) is an economically important disease of European farmed Atlantic salmon. It can cause significant losses because of morbidity, mortality and reduced production. The disease is caused by an alphavirus, known as salmon PD virus (SPDV) or salmonid alphavirus subtype 1 in Ireland. To examine whether it is possible to improve the natural resistance of Atlantic salmon to SPDV by selective breeding, 6000 genotyped, tagged, pedigreed fish from 150 full‐sib families were exposed to a natural challenge during 2005 in a sea cage on a commercial salmon farm in the West of Ireland. Histopathological and serological examination was performed weekly on a proportion of all moribund fish to determine the onset of the infection and the likely cause of death. Heritabilities and genetic correlations are presented for resistance to a natural PD challenge and smolt input weight. The results indicate that the susceptibility of salmon to SPDV could be reduced by selective breeding based on the survival in a natural challenge to the virus.     

Epidemiological observations of pancreas disease of farmed Atlantic salmon, Salmo salar L., in Ireland

Epidemiological investigations into the pancreas disease (PD) of farmed salmon were conducted on populations of Atlantic salmon reared in Ireland during 2003 and 2004. The investigations surveyed all marine salmon farms operating in Ireland through a detailed questionnaire with follow-up farm visits. Information was gathered on 21 populations of fish in 2003 and 14 populations in 2004. Thirteen of the 21 populations suffered PD in 2003 and 12 of the 14 in 2004. The mean mortality due to PD on affected farms was 18.8% in 2003 and 14.8% in 2004 and the loss of growth due to PD was estimated at 11.4% over the 2-year period. The highest risk periods for outbreaks of PD were early summer and early autumn and the farms most seriously affected by PD mortality were in the western counties of Ireland. Factors which showed an indication of association with a PD outbreak or high mortality during a PD outbreak were: livestock movement to another sea site, high feeding rate prior to any PD outbreak, the presence of another PD positive farm in the same water body, greater than 250000 fish on a site, a previous history of PD on a site, a high sea lice burden, and sites located in the western regions of Ireland which reared a specific strain of salmon.     

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea‐caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real‐time RT‐PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.     

服用多烯鱼油对母乳中DHA含量的动态研究

为研究国产鱼油制品对母乳中二十二碳六烯酸（ＤＨＡ）含量的影响,随机抽取１２名哺乳母亲为受试者,以气相色谱法测定母乳样本。测试结果表明,有８３．３％的受试者服用鱼油后的第２天乳汁中ＤＨＡ含量开始升高,所有受试者在服用鱼油的第８天乳汁中ＤＨＡ含量均高于服用鱼油前。证明哺乳期妇女服用高含量ＤＨＡ鱼油制品是提高母乳中ＤＨＡ含量的最直接有效的途径。     

Increased dietary protein‐to‐lipid ratio improves survival during naturally occurring pancreas disease in Atlantic salmon,Salmo salar L.

This study demonstrated that increased dietary protein‐to‐lipid ratio (P/L‐ratio) improved survival of farmed Atlantic salmon naturally affected by pancreas disease (PD). In addition to diet, body weight (BW) and delousing mortality prior to the PD outbreak also contributed significantly (p < 0.05) to explain the observed variation in PD‐associated mortality. Subsequent to the PD outbreak, large amount of fish failed to grow and caused thin fish with poor condition (runts). At the end of the trial, significantly (p < 0.05) lower amounts of runt fish and increased amount of superior graded fish where detected among fish fed increased P/L‐ratio and within the fish with the largest BWs prior to PD. Diet, BW and delousing mortality contributed significantly (p < 0.05) to explain the variation in the amount of superior graded fish, whereas BW and diet explained the variation in the amount of runt fish. A significant (p < 0.01) negative linear relationship was observed between the amount of superior graded fish and the total mortality, whereas a positive linear relationship was detected between percentage of fillets with melanin and the total mortality. Thus, increased dietary P/L‐ratio seem to reduce the mortality and impaired slaughter quality associated with PD.     

Immune response of DNA vaccine against lymphocystis disease virus and expression analysis of immune‐related genes after vaccination

In this study, we found that an intramuscular injection of Japanese flounder (Paralichthys olivaceus, 60–80 g in weight and 15–20 mL in length) with 5 μg of a DNA vaccine (pEGFP‐N2‐LCDV‐cn‐MCP 0.6 kb, containing lymphocystis disease virus major capsid protein gene) induced a strong immune response. Subsequent real‐time polymerase chain reaction showed that the expression of immune‐related genes [e.g., major histocompatibility complex (MHC) class I α, MHC II α, T‐cell receptor (TCR), tumour necrosis factor (TNF), tumour necrosis factor receptor (TNFR), Mx, interleukin (IL)‐1β, CXC and IL‐8R] was significantly changed after DNA vaccination. The most remarkable alternation was the expression of MHC I α and MHC II α genes: MHC II α reached the maximum on day 8 in different tissues, and MHC I α on day 2 in the intestine and gills. The expression of TCR increased and reached a plateau in 2 days in the spleen, gills, kidney and liver after vaccination and then decreased after day 8. In contrast, the expression of TCR in the intestine increased and reached a plateau in 8 days. The expression of IL‐8R reached the maximum on day 2 in different tissues and then decreased on day 8. Mx increased in the gills, kidney, spleen and liver on days 2, 8, 2 and 2, but decreased in the intestine, gills, spleen and liver on days 2, 8, 8 and 8 respectively. The TNFR expression increased in the spleen, kidney and gills on days 2, 8 and 8, but decreased in intestine, liver and gills on days 2, 8 and 8 respectively. The expression of TNF, CXC and IL‐1β increased 2 and 8 days after the injection of DNA vaccine. However, the expression of TNF, CXC and IL‐1β altered on days 2 and 8 with different patterns in different tissues respectively. The fish responded to the DNA vaccine by yielding a specific immunoglobulin against lymphocystis disease virus (LCDV) as observed with indirect ELISA. The DNA vaccine induced a unique humoral response, suggesting that the DNA vaccine activated both cellular and humoral defences of the specific immune system of Japanese flounder.     

密斯特黄彩鲶仔、稚、幼鱼的生长与发育研究

在水温22～24℃,pH7.0左右的人工培育条件下,对密斯特黄彩鲶(Mystus sp.)仔、稚、幼鱼的形态特征和生长发育进行了观察与研究。密斯特黄彩鲶的鱼苗发育分为卵黄囊期仔鱼期(初孵至第5天)、晚期仔鱼期(第6～10天)、稚鱼期(第11～20天)和幼鱼期(第20天以后)。初孵仔鱼平均全长5.12 mm,卵黄囊体积1.63 mm3;仔鱼第3天开始摄食,第5天卵黄囊被完全吸收,鱼苗主要外部器官主要集中在晚期仔鱼阶期和稚鱼期内形成。仔鱼在饥饿状态下最高初次摄食率出现在出膜后第6天,为84.0%,其饥饿的不可逆点则为出膜后第8天,第8天以后即进入不可逆PNR期,至第11天绝大部分饥饿仔鱼死亡。     

Effects of fish oil replacement by vegetable oil blend on digestive enzymes and tissue histomorphology of European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) juveniles

The impact of replacing circa 70 % fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca—PC, AI, PI, distal intestine—DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70 % FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology.     

Optimal periods of night‐time lighting in the sea cage culture of Pacific bluefin tuna,Thunnus orientalis,juvenile (Temminck and Schlegel)

We have previously reported that night‐time lighting prevents the mass death of Pacific bluefin tuna (PBT) juvenile due to collision and/or contact with the walls of sea cages, immediately after transfer to the cages, and that night‐time lighting does not negatively impact fish stress levels. However, the period of night‐time lighting should be limited to minimize negative impacts on the surrounding environment and aid management. Therefore, we investigated the optimal period of night‐time lighting by evaluating the whole‐body cortisol and glucose levels as stress parameters, growth performance and survival of PBT juvenile in four cages with different periods of night‐time lighting (i.e. unlit, 4‐day, 8‐day and 12‐day lighting). The results showed that almost all fish were died 1 day after transfer to the unlit cage. In comparison, the other groups (4‐day, 8‐day and 12‐day lighting) had high survival rates (92.5–96.0%) without significant difference. However, in the 4‐day‐lighting group, an obvious stress response was recorded on day 5, and growth performance was significantly lower. In the 8‐day‐lighting group, whole‐body cortisol levels were slightly elevated on day 9; however, significantly elevation was not recorded on day 12. These results indicate that the recommended lighting period of night‐time lighting in sea cages is 8–12 days.     

Patchiness Formation and Development of Schooling Behavior in Pacific Threadfin Polydactylus sexfilis Reared with Different Dietary Highly Unsaturated Fatty Acid Contents

Abstract.— Nutritional condition in larval fish culture has generally been evaluated based on growth and survival, and stress tolerance by air exposure. Here we made direct observations of fish in rearing tanks to estimate the quality of the fish. Pacific threadfin Polydactylu.s sexfilis larvae were reared with rotifers and Artemia nauplii enriched with three levels of highly unsaturated fatty acids (HUFA). The three levels were defined as no-, medium-, or high-HUFA groups, in which the fatty acids emulsions contained 0, 30, or 50%. respectively. of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The formation of patchiness (dense aggregations of fish on the surface) and development of schooling behavior were observed in four replicate rearing tanks for each feed treatment. Patchiness was formed in every tank with the highest densities of 60–250 fish/L on day 7–8 posthatch. Both medium-HUFA and high-HUFA groups showed schooling behavior at 19 d after hatching, whereas fish in the no-HUFA group did not form schools even at day 25. The body lengths of fish from each group were not significantly different, although the no-HUFA group suffered high mortality after day 20. Our results suggest that HUFA deficiency has a serious negative impact on P. sexfilis during metamorphosis rather than during the early larval stage. We also consider that direct observation rather than stress tests by air exposure may be an effective tool to measure the quality of juvenile fish.     

饲料脂肪对黄颡鱼卵巢脂类代谢以及PI3KCa甲基化和转录水平的影响

为了探究饲料不同脂肪水平对黄颡鱼卵巢脂肪代谢潜在的影响,本研究设计了活体与离体两个实验。活体实验中,分别投喂3组不同脂肪水平的饲料(脂肪含量分别为6.98%、11.34%和15.41%)8周。离体实验中,分离原代黄颡鱼卵母细胞,采用(0、0.2和0.5 mmol/L)3组不同脂肪酸浓度孵育48 h。活体实验结果显示:与6.98%和15.41%脂肪水平组相比,11.34%脂肪水平显著增加黄颡鱼卵巢甘油三酯水平(TG),并上调FAS、G6PD、6PGD和ME的酶活性水平,以及LPL和CD36基因表达水平。与6.98%和11.34%脂肪水平组相比,15.41%脂肪水平组显著升高黄颡鱼的性腺指数(GSI),并上调CPTIA和DNMT3b的基因表达水平,以及PI3KCa启动子-64和-52 CpG位点的甲基化水平,而显著降低PI3KCa的基因表达水平。体外细胞实验显示:与对照组(0)相比,0.5 mmol/L脂肪酸孵育显著增加卵母细胞TG含量,并上调FAS、G6PD和ME酶活水平,以及G6PD和PI3KCa的基因表达水平。同时,与对照组相比,脂肪酸孵育组显著降低CPTIA、ACCb、LPL、CD36、DNMT1以及DNMT3b基因的表达水平。然而,脂肪酸孵育对卵母细胞PI3KCa的启动子甲基化水平没有影响。研究表明,饲料不同脂肪水平影响卵巢TG的合成,可能主要是通过脂肪转运相关基因,并且高脂肪很可能是通过影响黄颡鱼卵巢PI3KCa启动子甲基化水平来影响PI3KCa的表达。而离体条件下,脂肪酸促进卵母细胞TG的合成很可能是通过升高脂肪合成相关基因、降低脂肪分解和转运相关基因来实现,但脂肪酸孵育不通过影响黄颡鱼卵母细胞PI3KCa甲基化水平来影响PI3KCa基因表达。     

Salmonid alphavirus (SAV) and pancreas disease (PD) in Atlantic salmon,Salmo salar L., in freshwater and seawater sites in Norway from 2006 to 2008

A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD‐affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real‐time RT‐PCR from samples collected in the freshwater phase, nor were any SAV‐neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (±7.06) (range 0.7–26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (±1.11) (range 1–6).     

Comparison of passive and active immunization of fish against streptococcosis (enterococcosis)

Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), was carried out to determine the persistence of anti-Streptococcus sp. antibodies (ASA) raised in sheep, rabbits or rainbow trout. The protection afforded by passive immunization was compared with the protection obtained from active immunization by immersion in or intraperitoneal (i.p.) injection with formalin-killed cells. Assessments were undertaken concurrently for up to 3 months post-immunization (PI) to evaluate the practical potential of passive immunization. Passively administered sheep and rabbit antibodies were detected in fish sera by enzyme-linked immunosorbcnt assay for more than 60 days after i.p. injection. Fish responded immunologically to these antibodies and the highest humoral responses to sheep and rabbit ASA occurred at 2 months PI. The relative per cent survival (RPS) of rainbow trout challenged with virulent Streptococcus sp. after an i.p. injection (0.1 ml 100 g^?1 fish body weight) of sheep, rabbit or fish ASA was: 88.8, 50 and 0% after 1 month; 33.3, 6.8 and 6.8% after 2 months; and 13.3, 0 and 6.6% after 3 months PI, respectively. Fish immunized actively had an RPS of 88.8 and 11.1% after 1 month, 38.1 and 4.7% after 2 months, and 36 and 0% after 3 months PI for the i.p. injection and immersion routes, respectively.     

Reduction of transport stress of ayu by obligated schooling

We investigated stress responses of ayu Plecoglossus altivelis under the hypothesis that overcrowding during transport can be a stressor and the stress predisposes fish to disease by decline in immunocompetence. The extent of stress response was determined under different fish densities. As inferred from the serum concentration of cortisol 1 day after the treatment, the density at more than 23 kg/m³, which was within the range of normal transporting procedures, acutely evoked intense stress responses. To examine a possible way to reduce the stress response of fish under crowding conditions, we compared two fish groups established in water tanks with circulating (whirling) and turbulent currents at the same density. The cortisol levels 1 day after the treatment were 2.5-fold higher in turbulent current than in whirling current. Fish in whirling current formed a school, while those in turbulent current aggregated with each other. Schooling of fish induced by water flow may reduce their stress responses during transport. A transport system with a directional water flow should keep fish in good condition without reducing the number of fish contained.     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.