Sarah J Haig

Robert L Davies

Timothy J Welch

R Allan Reese

David W Verner- Jeffreys
email: david.verner-jeffreys@cefas.co.uk

Dr David W Verner -Jeffreys

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

Keywords:

A c c e p t e d M a n u s c r i p t 2 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 150-250g 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 o C (74 and 33%, respectively) than at 12ºC (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.

Introduction

The Gram negative enterobacterium Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease, a chronic to acute haemorrhagic septicaemia of salmonid fish species, in particular rainbow trout (Oncorhynchus mykiss Walbaum) and Atlantic salmon (Salmo salar L) (Horne and Barnes 1999). A single serogroup (serovar 1 or serotype O1) is responsible for most outbreaks in (rainbow) trout farmed worldwide (Stevenson andAirdrie 1984, Davies 1990;1991a, Wheeler et al. 2009)). Wheeler et al. (2009) revealed that isolates recovered from UK salmon were genetically and serologically distinct from trout isolates, suggesting that they may have emerged or been introduced separately. Interestingly, the emergence of these novel Atlantic salmon isolates has necessitated the modification of the vaccines used to immunise salmon fry by the inclusion of bacterins prepared from these novel isolates (L. A. Laidler, Marine Harvest (Scotland) Ltd, personal communication). This further suggests that these isolates are divergent to typical rainbow trout isolates. While these isolates were reportedly recovered from fish displaying clinical signs of ERM, their virulence to Atlantic salmon has not been verified.

The complement system plays an important role in the killing and neutralisation of microorganisms in teleost fish, as in other vertebrate phyla, with effects in teleosts mediated by activation of both classical and alternative pathways [START_REF] Claire | The complement system in teleosts[END_REF]. Davies (1991b) demonstrated that virulent trout isolates possessed serum-resistance factors, which likely allowed them to evade complement-mediated killing in the absence of antibody. To date, no comparable studies have been performed on salmon isolates with salmon sera.

The current study aimed to compare the virulence of five isolates from salmon and trout against salmon and rainbow trout under experimental conditions. We also investigated whether salmon isolates possess serum resistance factors that might explain differences in the virulence of these organisms towards both species.

2.0Materials and methods

Bacterial strains

The Yersinia ruckeri isolates used in this study are described in Table 1.These included nine salmon isolates that had been recovered from Scotland, Norway and Tasmania over a 30 year period and four isolates from rainbow trout. Two further isolates were recovered from eel and whitefish. The isolates represented a wide range of biotypes, OMP types, O serotypes and pulsotypes recovered from other salmonids (Wheeler et al. 2009).

Fish stocks

Four stocks of disease-free trout and salmon were used for the challenge studies. These included larger trout (150-250g), rainbow trout fry (0.35g), salmon parr (5-10g) and salmon fry (0.4g).

Larger trout were challenged in 300L tanks, whereas the rainbow trout fry, salmon fry and salmon parr were challenged in half full 30L tanks. The tanks were supplied with dechlorinated fresh water (0.2-0.4 L min -1 for the 30L tanks and 3-4L min -1 for the300L tanks), from the Weymouth potable drinking water supply (sourced from local chalk and limestone boreholes). A 12h-day length, 30min dusk/dawn was provided with day light illumination set to provide approximate 200 Lux light at the water surface. Tank water temperatures were maintained to within 0.1 ºC test temperature(16ºC and 12ºC, see below) by central computer control throughout the studies. All fish were reared from surface-disinfected eggs in the experimental facility on separate flow-through systems, under conditions of strict biosecurity. All fish were acclimated in their test tanks to test temperature for a minimum of seven days prior to treatment.

Preparation of challenge doses

For the first intraperitoneal infection experiments, isolates were recovered from cryopreserved stocks by inoculation onto Tryptone Soya Agar (TSA, Oxoid) and incubated at 22°C for 48h.

A c c e p t e d M a n u s c r i p t 5 Isolated pure colonies were inoculated into Tryptone Soya Broth (TSB; Oxoid, Basingstoke, UK) and incubated for 18-24 h at 22ºC with shaking. The cultures were harvested and washed three times by centrifuging (300g for 15 min) and resuspension in phosphate-buffered saline (PBS). The final suspension was adjusted with PBS to an optical density to give the required challenge dose, based on an A 550 of 1.0 being equivalent to approximately 1.0 x 10 9 c.f.u. ml -1 .

Serial ten-fold dilutions (0.1 ml) of each suspension were inoculated onto TSA plates, incubated at 22 o C for 48h, and resultant colony forming units (c.f.u.) counted to confirm the challenge doses.

For the bath challenge experiments (see below), similar procedures were followed to produce challenge doses, except that fresh subcultures of isolates recovered from the head kidneys of i.p.-challenged fish in the first experiments were used. These in vivo-passaged isolates were maintained on solid media (TSA) at 4ºC and used as challenge inocula source for all four experimental bath challenges.

Challenge by intra-peritoneal (i.p.) injection

Nine groups of ten salmon parr (5-10g) were each challenged by intraperitoneal injection with each of nine different salmon isolates, (Table 1). Ten salmon were also challenged with the rainbow trout isolate 06041 (Table 1). This virulent ERM-causing UK trout isolate was used as a positive-control strain in a previous study (Davies 1991b). All fish were fasted for 24h prior to treatment, then taken from their tanks and placed in a bucket containing tricaine methane sulphonate (MS222) at strength sufficient to induce light anaesthesia within 2-3 minutes of introduction (approximately 90 mg L -1 ). Fish were injected into the peritoneal cavity with a 0.1ml dose containing 2.3x10 7 +1.4x10 7 c.f.u., then returned to their original tank and observed to confirm that they had safely recovered from anaesthesia. Supply of feed was resumed 24 h post challenge. Test temperature was 16 ºC for the i.p. challenge experiments.

A c c e p t e d M a n u s c r i p t 6

Challenge by bath exposure

For salmon and rainbow trout fry and salmon parr in 30 L tanks, fish were challenged in situ, flows were stopped and the tank volume was adjusted to 5L. One hundred ml of bacterial suspensions were then added to the tank water to give final concentrations between 1.19 x 10 7 -1.3 x 10 8 c.f.u. ml -1 for the salmon parr challenge, and 1.2 -4.8 x 10 7 c.f.u. ml -1 for both fry challenges. For challenge of 250-300g rainbow trout (bath challenge experiment 4; Table 2), rainbow trout were transferred into an aerated bucket containing 40L clean tank water.

Bacterial suspensions were then added to the bucket to give final concentrations between 8.7x 10 6 -2.3 x 10 8 c.f.u. ml -1 . In all cases, fish were kept in the bacterial suspension for 4h with constant aeration. After 4h, flow was resumed to the tanks where fish had been challenged in the holding tanks and the larger trout were returned to their respective challenge tanks. All fish were challenged at 16 ºC, except for two groups of salmon fry that were challenged at 12ºC in parallel with two of the isolates (06059 and 05094). Experiment duration for the salmon parr and larger rainbow trout challenges (experiment 2 and 4; Table 2) was 33-35 days. The fry challenges were continued for 17 days (this was more than three days after the last mortality in any challenge tank, and approximately ten days after the main peaks of mortalities had subsided). In experiments 1 & 3, two tanks of twenty-five fish were exposed to each isolate. In experiments 2 & 4, two tanks of twelve fish were exposed to each isolate. For experiments 1, 3 and 4, the two tanks of fish challenged with each isolate were each exposed to separately prepared bacterial suspensions on different days. Isolates recovered in pure culture from a minimum of two fish per challenge tank were confirmed as Y. ruckeri, based on colony appearance, primary test results and Mono-Yr latex agglutination testing (Bionor Laboratories, Norway).

Serum killing assays

Non-immune serum killing assay

Non-immune sera were obtained by caudal venipuncture from 350g trout and 400g salmon from disease-free stock. These fish had been reared from eggs in the Cefas Weymouth Laboratory experimental facility on separate flow-through systems under conditions of strict biosecurity.

Sera were separated within 4h of collection, pooled by tank and species origin, filter sterilised, divided into 0.4ml samples, and stored at -80 o C.

Isolates were tested for sensitivity to both pooled non-immune (naive) Rainbow Trout serum (PNRTS) and pooled non-immune (naive) Atlantic Salmon serum (PNASS), as described by Davies (1991b) with minor modifications. The test organisms were inoculated onto TSA and grown overnight at 22 o C. Resultant fresh colonies were then suspended in 10ml PBSa to an optical density of 0.4  0.01 at 610nm (equivalent to approximately 5x10 8 c.f.u ml -1 ). A total of 0.1ml of this suspension was then added to 0.4ml of test serum and incubated at 22ºC. Duplicate 0.1 ml samples were serially tenfold diluted and dilutions inoculated onto TSA at 0, 1.5 and 3h.

Plates were incubated for 48h at 22 ºC and resultant colonies counted. All isolates were also tested with PNRTS and PNASS that had been heated at 46ºC for 20 min to abolish complement activity (Sakai 1981). All isolates were tested in triplicate with both PNRTS and PNASS.

Serum-resistant isolates were defined as those that increased in number by 3h. Serum-sensitive isolates were defined as those that decreased in number by 3h. 3) were tested for resistance to antibody-mediated (classical complement) killing. The method used was that of [START_REF] Boesen | Vibrio anguillarum Resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide[END_REF] with minor modifications. Antisera used for the assays were samples of convalescent serum obtained from 5-10g salmon in experiment 2 that survived exposure to serotype O1 isolates 06041, 06059 and 07039, serotype O2 isolate 06060 and serotype O5 isolate 05094. Also included was serum collected from 250g trout that survived exposure to isolate 06041 in experiment 3. All sera were collected 5-6 weeks after exposure to the test organisms. For testing, an approximately 1 x 10 8 cfu ml -1 bacterial suspension of each isolate were first prepared, as described for the non-immune serum killing assay. A sterile swab was then introduced into a 1:10 dilution of the suspension in PBS. Surplus moisture was removed from the swab by rotating several times while pressing firmly against the inside wall of the tube. The swab containing the inoculum was then streaked over the surface of a TSA plate. After streaking over the entire surface the plate was rotated 60º and the plate streaked again. Following a second rotation of 60º, the agar surface was streaked for a third time. This was then left to dry for 1h, producing a semi-confluent growth of the test organism. Samples of 3μl heat-decomplemented antisera were placed as drops on the agar and allowed to bind with the bacteria at 10ºC as a source of specific antibody. After 1h, 3 μl of the species-specific pooled non-immune serum (as a source of complement) was added to the previously applied drops of antiserum. The plates were incubated for 48h and examined for zones of clearing. Samples to which 3μl of heat inactivated and normal PNRTS and PNASS were initially added, as opposed to heat-decomplemented antisera, were also included as controls.

A c c e p t e d M a n u s c r i p t 9

Statistical Analysis

Data for serum-susceptibility were logarithmically transformed and the log10-change of each isolate between 0h and 3h calculated. Two-way ANOVAs were carried out to confirm that the average log-change did not differ between species but did significantly differ between the naïve and heated (negative control) serum, significant for P-values <0.05. All statistical analyses was performed using Stata 10 StataCorp 2007).

Results

intraperitoneal (i.p.) injection

All ten isolates tested, including the ERM-causing trout isolate 06041, caused high levels of mortality in juvenile salmon by i.p. injection, with 60% or more mortality by the time the experiment was terminated after 5 days (data not shown).

Bath challenge of salmon

Bath exposure of salmon fry and juveniles to the ERM-causing trout isolate 06041 at 16ºC caused high levels of mortality in both age groups tested (60% and 59% and respectively; Table 2). When tested at 16ºC, the salmon O1 serotype isolate 06059 was also highly virulent to salmon by this challenge route, causing 74% in fry and 63% mortality in parr (Table 2). The other salmon isolates also caused mortality in both size classes at this challenge temperature (16 o C). Although there was evidence of both tank-to-tank variation in mortality and apparent differences in susceptibility between fry and older fish. For instance, the serotype O2 isolate 06060 caused 42% mortality in fry, but only 4% older salmon parr that were challenged died.

Similarly, 33% fry challenged with the serotype O5 isolate tested 05094 died, while only 13% the 5-10g juveniles exposed to this organism died, and these were all in only one of the two test tanks. Typical clinical signs included inappetance, darkening, exophthalmia, and haemorrhaging around the vent and fin bases. Internally, fish showed severe haemorrhaging of the internal A c c e p t e d M a n u s c r i p t 10 organs and ascites. Mortalities by bath exposure to salmon fry typically showed the pattern seen in Figure 1 for isolates 06059 and 05094. The response for the larger fish challenged was delayed, with mortalities peaking at d 9-10 post-challenge.

Bath challenge of rainbow trout

Both trout fry and larger 150-250g fish were confirmed as highly susceptible to the serotype O1 ERM-causing isolate 06041 by immersion exposure, with an average of 78% of larger fish and 34% fry killed (Table 2). The salmon isolates were avirulent or of low virulence towards either size class of trout challenged), with fry seemingly moderately more susceptible than 150-250g fish (Table 2). Although the larger fish were not killed by the other test isolates, a number of individuals, particularly those exposed to isolate 06059, exhibited signs of disease two to three weeks post-exposure. Signs included bilateral exophthalmia, ascites and darkening. When the experiment was terminated five weeks post exposure, all fish appeared healthy.

Effect of temperature

Both serotype O1 isolate 06059 and serotype O5 isolate 05094 caused much greater mortality in salmon when tested at 16 o C (other factors kept constant) compared to 12 o C (Figure 1). Isolate 06059 caused the highest mortality at both temperatures.

Carriage of Y. ruckeri by survivors

There was some evidence of persistent infection in fish of both species challenged with the different isolates (Table 2). In experiment 4, Y. ruckeri was detected five weeks post-challenge in the head kidneys of a higher proportion of larger trout bath-challenged with serum-resistant serotype O1 isolates, compared to those challenged with non-serotype O1 isolates.

Serum Susceptibility

Five out of the 15 tested isolates grew, or at least did not decrease in viable cell concentration, after 3h incubation in PNRTS (isolates 06018, 06041, 06076,06059, 06051; Table 1). The other A c c e p t e d M a n u s c r i p t 11 ten isolates were all killed by PNRTS, showing average log 10 cell concentration reductions of between -0.12 and -3.4 after 3 h (Table 1). Biotype 1 and biotype 2 examples of rainbow trout serotype O1 isolates tested (isolates 06018, 06041 and 06076) were all resistant to PNRTS and PNASS serum killing. The Norwegian serotype O5 salmon isolate 06051, was resistant to PNRTS killing, but killed by PNASS (Table 1). The other organisms tested were all sensitive to both sera (Table 1). In heated (decomplemented) control serum from both species, all isolates showed a +0.1 to +0.82 log 10 c.f.u ml -1 increase in cell number after 3h, suggesting that killing was likely complement-mediated (data not shown). Preliminary experiments were also undertaken with salmon serum at 15 ºC. These confirmed there was no difference in PNASS killing effectiveness at this lower temperature (data not shown). 3).

For salmon serum, in all cases the homologous antiserum mediated effective killing of isolates that were resistant to PNASS (table 3). Four of the other five convalescent antisera used mediated killing of all the PNASS-resistant isolates (Table 3). The exception being the serotype O2 isolate 06060 antiserum, which failed to kill two typical O1 ERM-causing trout strains (06018 and 06041) (Table 3). Isolates that were killed when incubated in PNASS and PNRTS were also shown to be normal serum sensitive in this assay (data not shown). Although not directly quantified, it was observed that the zones of clearing in the lawns of normal serum sensitive isolates were larger and more intense when both normal serum and antiserum were added together then when only normal serum was added.

Discussion

Major differences between the virulence of different Y. ruckeri isolates to salmon and trout were shown, particularly when administered by a more natural exposure route (bath challenge), than by injection. Davies (1991b) also demonstrated host tropism in Y. ruckeri, with isolates reportedly virulent to chinook salmon (Onchorhynchus tshawytscha L.) and brook trout Salvelinus fontinalis (Mitchill, 1814) [START_REF] Cipriano | An epizootic in chinnok salmon (Onchorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri[END_REF][START_REF] Cipriano | Immunization of salmonids against Yersinia ruckeri; significance of humoral immunity and cross protection between serotypes[END_REF], avirulent to (rainbow) trout, when tested in that species by experimental bath challenge. The organisms that typically cause ERM in rainbow trout represent a closely-related subgroup of O1 serotype Y. ruckeri , the Hagerman Type strain ATCC 29473 T -like clonal complex (Davies 1991b;Wheeler et al. 2009). This work confirms that an example of this group (isolate 06041) also caused severe disease in salmon by experimental bath challenge.

Other isolates that affect salmon do not necessarily also cause disease in trout. Although the most virulent isolates were O1 serotype, significant mortalities were also seen in fry challenged with O2 and O5 serotype isolates, though these isolates were not highly virulent to older salmon.

There is evidence that serotype O2 and serotype O5 isolates genetically very similar to those tested have been circulating in UK salmon hatcheries for more than 15 years (Wheeler et al. 2009). This is consistent with reports from hatcheries that Y. ruckeri outbreaks, particularly those associated with serotype O2 and O5 isolates, normally affect fry rather than older fish. The finding that two isolates were both more virulent when tested at 16ºC than 12ºC is also consistent with reports that farmers typically see Y. ruckeri problems in salmon hatcheries when temperatures are greater than 15ºC.

Although serotype O1 isolates are typically noted as more virulent, serotype O2 isolates have been reported as causing disease in chinook salmon (Onchorhynchus tshawytscha L) and brook trout [START_REF] Cipriano | An epizootic in chinnok salmon (Onchorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri[END_REF][START_REF] Cipriano | Immunization of salmonids against Yersinia ruckeri; significance of humoral immunity and cross protection between serotypes[END_REF].

The experiments were not explicitly designed to study carriage of the different isolates by both species (carriage was only determined in one organ (the head kidney) for 17-35 days postinfection ( Table ;)).However it was interesting to note that, even for groups of fish Differences of O antigen serotype in Y. ruckeri relate to variations in LPS structure [START_REF] Davies | Biochemical and cell-surface characteristics of Yersinia ruckeri in relation to the epizootiology and pathogenesis of infections in fish[END_REF] as is typical for other Enterobacteriacae. Variations in O2 and O5 serotype isolate resistance to PNASS did not correlate with virulence. In particular, salmon serotype O5 isolate A c c e p t e d M a n u s c r i p t 14 05094 was sensitive to PNASS, while a Norwegian serotype O5 isolate tested, 06051, was very resistant. Salmon serotype O2 isolate 06060, that was moderately pathogenic to both trout and salmon fry, was sensitive to PNRTS and did not grow in PNASS. These isolates may possess other factors that allow them to survive, replicate and cause disease in salmon fry. Serotype O1 isolate 06059 was highly virulent to salmon but not to trout, even though the isolate was resistant to killing by naive serum collected from both species. Davies (1991b) studied the effect of repeated in vivo passage of eight diverse Y. ruckeri isolates through rainbow trout and observed no differences in their virulence before the first and after the fourth passage. Attempts to serial passage isolate 06059 through rainbow trout also did not increase the virulence towards that species (data not shown). Trout ERM-causing strains may well possess trout colonisation factor(s) that are absent in isolates such as 06059. Further work is required to determine the causes of observed variations in Y. ruckeri PNRTS and PNASS isolate sensitivity. This is the first demonstration that prior exposure to the pathogen likely mediates effective classical complement killing of Y. ruckeri in either species. There was evidence of in vitro crossprotectiveness, with both salmon and trout sera prepared from fish that were exposed to isolate 06041 acting as a source of antibody that mediated effective killing of PNRTS and PNASSresistant Y . ruckeri isolates. This is similar to results obtained by [START_REF] Boesen | Vibrio anguillarum Resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide[END_REF] for PNRTS resistant V. anguillarum O1 isolates, though that study also showed that homologous antibody did not mediate killing of PNRTS resistant V. anguillarum O2 isolates.

In conclusion, this study confirms hatchery reports that Y. ruckeri poses a significant risk to salmon, as well as to rainbow trout. Strains that affect salmon show diversity in their phenotypic properties (relative virulence, serotype and possession of serum-resistance factors), compared to ERM-causing serotype O1 rainbow trout isolates

Tables

Table 1 Properties of Y. ruckeri isolates Table 2. Summary of experiments 1-4 bath challenge results. For each experiment, groups a of different size rainbow trout and Atlantic salmon were exposed to ≥1.0 x 10 7 c.f.u. ml -1 of the different Y. ruckeri isolates indicated for 4 h at 16ºC. Table 3 Results of testing two rainbow trout (isolates 06018 and 06041) and one Atlantic salmon (isolate 06059) serotype O1 normal serum resistant isolates for sensitivity to antibodymediated (classical complement) salmon and rainbow trout serum killing. Isolates were incubated with different heat inactivated convalescent sera (antisera) as a source of antibody, followed by exposure to normal serum from the same species. S = normal serum resistant and antiserum sensitive, R = normal and antiserum resistant . a Cefas culture collection identification number; information on the isolates (pulsotype, previous designation, biotype, year of isolation) is as indicated in Table 1. b Antisera recovered from Atlantic salmon and rainbow trout previously bath challenged with different Y. ruckeri isolates as indicated (the three serotype O1 isolates tested and a serotype O2 isolate (06060) and a serotype O5 isolate (05094). With the exception of RTα-06041, recovered from surviving rainbow trout, all antisera were from surviving Atlantic salmon.

Figure captions

  All the challenge experiments were conducted in compliance with the requirements of the UK's Animals (Scientific Procedures) Act 1989, under a UK Home Office Project Licence.This included prior approval by the Cefas Weymouth Local Review of Ethical ProceduresCommittee.

  A c c e p t e d M a n u s c r i p t 7 2.6Confirmation that challenge mortalities were the result of Y. ruckeri infection Kidney swabs from challenge mortalities and survivors were made onto TSA to detect Y. ruckeri.

  assay for antibody-mediated (classical complement) killing Three Y. ruckeri isolates (Table

  -mediated serum killing assay Convalescent serum recovered from trout that had survived exposure to a virulent biotype 2 serotype O1 isolate (06041) mediated effective antibody (likely classical complement) serum killing of 06041 and other PNRTS serotype O1 isolates tested (06018 and the virulent salmon isolate 06059) (Table

  .L. 1990. O-serotyping of Yersinia ruckeri with special emphasis on European isolates. Veterinary Microbiol. 22, 299-307. Davies, R.L. 1991a. Clonal analysis of Yersinia ruckeri based on biotypes, serotypes and outer membrane protein-types. J. Fish Dis.14, 221-228. Davies, R.L. 1991b. Virulence and serum-resistance in different clonal groups and serotypes of Yersinia ruckeri. Veterinary Microbiol.. 29, 289-297 Horne,M.T., Barnes,A.C. 1999. Enteric Redmouth disease. In: Woo,P.T.K, Bruno,D.W. (Eds.) Fish Diseases and Disorders, CABI Publishing, pp 456-477. Sakai, D.K. 1981. Heat inactivation of complement and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, goldfish and tilapia. Bull. Jpn. Soc. Fish. 47, 565-571. Stevenson, R.M.W., Airdrie, D.W. 1984. Serological variation among Yersinia ruckeri strains. J. Fish Dis.7, 247-254. Wheeler,R., Davies,R.L., Dalsgaard, I. Garcia,J. Welch,T.W., Wagley, S., Bateman,K.S., Verner-Jeffreys,D.W. (2009) Yersinia ruckeri biotype 2 isolates from mainland Europe and the UK likely represent different clonal groups. Dis. Aquat Org. 84, 25-33.

Figure 1 .

 1 Figure 1. Cumulative mortality of 5-10g Atlantic salmon exposed to two Y. ruckeri isolates at

  Results of testing two rainbow trout (isolates 06018 and 06041) and one Atlantic salmon (isolate 06059) serotype O1 normal serum resistant isolates for sensitivity to antibody-mediated (classical complement) salmon and rainbow trout serum killing. Isolates were incubated with different heat inactivated convalescent sera (antisera) as a source of antibody, followed by exposure to normal serum from the same species. S = normal serum resistant and antiserum sensitive, R = normal and antiserum resistant .

Acknowledgements

Funding was provided by the UK government's Department for Environment Food and Rural Affairs through projects FC1178, FB001 and a placement studentship for S.J.H. (FC1172).

 2009). b All isolates were tested, in triplicate, for resistance to PNRTS and PNASS. Values reported are the average log 10 change in bacterial concentration (c.f.u. ml -1 ) after 3h incubation in the respective sera. R = resistant to normal serum killing, S = sensitive to normal serum killing. c nk = not known; all isolated prior to 1986. a Cefas culture collection identification number; other information on the isolates (pulsotype, previous designation, biotype, year of isolation) is as indicated in Table 1


Peter Simon P L Travis
email: simon.travis@ndm.ox.ac.uk

Dr Tim Higgins

C Orchard

Remo Janneke Van Der Woude

Alain Panaccione

Colm A Bitton

Julian O'morain

Andreas Panes

Walter Sturm

Reinisch

C J Van Der Woude

Erasmus Janneke

Mc

Spl Travis

Pdr Higgins

T Orchard

R Panaccione

A Bitton

C O'morain

J Panés

A Sturm

W Reinisch

M A Kamm

G D'haens

Dr Simon Travis

Dphil Frcp

Defining remission in ulcerative colitis

Keywords: Ulcerative colitis < Disease-based, Inflammatory bowel disease < Disease-based, Large intestine < Organ-based, Outcomes research < Topics, Symptom score or index < Topics, Endoscopy < Topics, Histopathology ulcerative colitis (UC), remission, definition, long-term outcomes, clinical trial, guidelines, endoscopy, mucosal healing, 5-ASA

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

There is no international agreement on clinical, endoscopic, or histopathological scoring systems in ulcerative colitis (UC). Nor is there a fully validated definition for disease remission. Definitions vary depending on whether remission is applied in a trial, regulatory, guideline, clinical, or patient context.

For example, the clinician may choose a particular therapeutic strategy to achieve remission, which is rarely objective and usually based on the best balance that they can achieve between symptom control and adverse events from treatment. In contrast, remission in the context of quality of life is important to the patient. Furthermore, the treatment goal or expectation of the patient may differ from that of the physician. Conversely, clinical trials generally use a definition of remission commensurate with drug registration, which is usually the objective of the study [START_REF] Travis | Remission in trials of ulcerative colitis: What does it mean? In: Katz S Ed. Inflammatory Bowel Disease: A Practical Approach[END_REF][START_REF] Stange | European evidence-based Consensus on the diagnosis and management of ulcerative colitis: definitions and diagnosis[END_REF]. Consequently there are several definitions of remission:

•
Clinical remission: used in practice, meaning cessation of rectal bleeding and normal stool frequency

•
Complete remission: used in clinical trials to demonstrate safety/efficacy; normal stool frequency and no rectal bleeding as well as a normal or quiescent appearance of the mucosa at sigmoidoscopy.

•

Registration remission: used in trials to gain drug licence, currently used by regulatory authorities; requires cessation of rectal bleeding and a sigmoidoscopy score of 0 or 1 of the Mayo Clinic score or Ulcerative Colitis Disease Activity Index (normal appearance of the rectal mucosa or erythema only)

These definitions do not include the patient perspective, which may simply mean the ability to enjoy normal social, recreational and occupational activity without undue interference from their symptoms, or mean an even higher level of expectation, such as cure of their colitis. There is, therefore, no standard way of evaluating clinical outcomes or providing explicit recommendations for guidelines [START_REF] Cooney | Outcome measurement in clinical trials for ulcerative colitis: towards standardisation[END_REF].

It is quite possible that the absence of a standardised definition of remission has contributed to a selfperpetuating cycle of suboptimal therapy in UC (Figure 1). Long-term prognostic studies show low rates of remission (<50% of patients), and therefore new and better (or better use of old) therapies are needed [START_REF] Travis | Remission in trials of ulcerative colitis: What does it mean? In: Katz S Ed. Inflammatory Bowel Disease: A Practical Approach[END_REF][START_REF] Katz | Mind the Gap": an unmet need for new therapy in IBD[END_REF]. Measurement of disease activity is critical in determining a therapy's efficacy (usually measured as the ability to induce or maintain remission). Remission rates can vary by more than twofold depending on the definition of remission used for data analysis [START_REF] Katz | Different definitions of remission for ulcerative colitis result in large variations of clinical outcome scores[END_REF] making it difficult to interpret trial data and compare results [START_REF] Travis | Remission in trials of ulcerative colitis: What does it mean? In: Katz S Ed. Inflammatory Bowel Disease: A Practical Approach[END_REF]. This may lead to the introduction of medication which does not adequately maintain remission. Remission must therefore be clearly defined to ensure that treatment is aligned to a recognised outcome target and to standardise clinical practice. This review discusses

REMISSION AS DEFINED IN CLINICAL TRIALS

No single disease activity index is used for UC, not least because there are at least seven different symptom-based activity scores, two composite scores and four patient evaluation scoring systems [START_REF] Cooney | Outcome measurement in clinical trials for ulcerative colitis: towards standardisation[END_REF][START_REF] Haens | A review of activity indices and efficacy end points for clinical trials of medical therapy in adults with ulcerative colitis[END_REF]. Furthermore, the inconsistent use of names for different indices leads to considerable difficulty and confusion when comparing publications. In addition, disease measures such as the Physician's Global Assessment, patient general well being and sigmoidoscopy are subjective evaluations that vary depending on interpretation [START_REF] Travis | Remission in trials of ulcerative colitis: What does it mean? In: Katz S Ed. Inflammatory Bowel Disease: A Practical Approach[END_REF][START_REF] Cooney | Outcome measurement in clinical trials for ulcerative colitis: towards standardisation[END_REF]. Some indices have never been used in a clinical trial of substantial size (e.g. Lichtiger or Modified Truelove Witts Severity Index, [START_REF] Lichtiger | Cyclosporine in severe ulcerative colitis refractory to steroid therapy[END_REF]). The lack of agreement about the best activity index for clinical trials in UC means that agreement on the assessment of remission is needed at the very least, so that the efficacy of different treatments can be compared.

The first instrument to measure disease activity in UC was created in 1955 to quantify the clinical symptoms of UC [START_REF] Truelove | Cortisone in ulcerative colitis. Final report on a therapeutic trial[END_REF]. This comprised five objective variables: the number of bloody stools per day (although it may be debated whether any symptom is 'objective'); temperature; pulse; haemoglobin and erythrocyte sedimentation rate (ESR). The index was followed by a plethora of clinical, endoscopic, histological and quality of life indices and their composites (Table 1; for details see [START_REF] Haens | A review of activity indices and efficacy end points for clinical trials of medical therapy in adults with ulcerative colitis[END_REF]).

Endoscopic indices evolved from the Baron Score, initially developed for rigid proctoscopy in ambulatory patients with mild to moderate disease [START_REF] Baron | Variation between observers in describing mucosal appearances in proctocolitis[END_REF]. Subsequent endoscopic indices became more complex and incorporated the presence of ulcers, mucopus, granularity and light scattering in addition to bleeding and friability [START_REF] Powell-Tuck | Correlations between defined sigmoidoscopic appearances and other measures of disease activity in ulcerative colitis[END_REF][START_REF] Schroeder | Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study[END_REF][START_REF] Rutegard | A simple index for assessment of disease activity in patients with ulcerative colitis[END_REF]. Modifications were intended to improve the capture of disease activity, but they invariably increased the subjectivity of the scoring system and may have undermined reproducibility. Whilst endoscopy is likely to add stringency to the definition of remission, there is wide inter-observer variation in the assessment of mucosal appearance even by experienced investigators. • Gebboes Index (Gebboes 2000)

• Chicago Index [START_REF] Rubin | Increased degree of histological inflammation predicts colectomy and hospitalisation in patients with ulcerative colitis[END_REF] For details, see [START_REF] Haens | A review of activity indices and efficacy end points for clinical trials of medical therapy in adults with ulcerative colitis[END_REF] Some indices deserve more detailed consideration, because they are commonly used in clinical trials.

The Rachmilewitz index (clinical activity index, CAI) [START_REF] Rachmilewitz | Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial[END_REF] has been used in trials of 5-aminosalicylic acid (5-ASA) [START_REF] Kruis | Once daily versus three times daily mesalazine granules in active ulcerative colitis: a double-blind, double-dummy, randomised, non-inferiority trial[END_REF]. The Sutherland Index (also known as the Disease Activity Index or the UC Disease Activity Index; UC-DAI) is a simplified composite score incorporating four variables: stool frequency, rectal bleeding, mucosal appearance, and physician's rating of disease activity [START_REF] Sutherland | 5-Aminosalicylic acid enema in the treatment of distal ulcerative colitis, proctosigmoiditis, and proctitis[END_REF]. By employing a four point scoring scale for each variable, the relative simplicity of the index reduces the impact of physician and patient subjectivity in disease scoring. The index has been adopted in large clinical studies [START_REF] Marteau | Combined oral and enema treatment with Pentasa (mesalazine) is superior to oral therapy alone in patients with extensive mild/moderate active ulcerative colitis: a randomised, double blind, placebo controlled study[END_REF]. Of particular note, a score <2.5 points correlates with patient-defined remission [START_REF] Higgins | Patient defined dichotomous end points for remission and clinical improvement in ulcerative colitis[END_REF],

although the index has not been formally validated.

The Mayo Clinic score [START_REF] Schroeder | Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study[END_REF] is another composite index that includes endoscopy and has been most

widely used in large clinical trials of UC [START_REF] Rutgeerts | Infliximab for induction and maintenance therapy for ulcerative colitis[END_REF]. As with the UC-DAI, the stool frequency score is not an absolute number, but relative to 'normal' for that subject, which may itself introduce variation between observers that has yet to be quantified. The FDA has recognised a relatively liberal definition of remission as a Mayo Clinic score ≤2. This is less stringent than others (see below), but until the long-term predictive value for different endpoints is validated, this is unlikely to change.

The weighting of individual measures varies between scoring systems and since 'remission' is set at a particular score of the combined parameters, variation arises between the indices. Conversely, remission may be considered as the absence of disease activity, which again may be set at an arbitrary threshold below that which symptoms exist. The patient might not equate such a threshold with remission and the predictive value of different thresholds has not been examined.

Impact of different definitions on the assessment of clinical trial outcomes

In clinical trials the definition of remission is often specific to the scoring system applied. As a result, the threshold for setting remission has a substantial impact on the remission rates in both the active and the placebo arms of clinical trials [START_REF] Su | A meta-analysis of the placebo rates of remission and response in clinical trials of active ulcerative colitis[END_REF]. Placebo remission rates vary from <5 to >40% and are influenced by factors including trial duration, number of study visits, design features used to enrol patients with more active disease and intensity of endoscopic follow-up, but a stricter remission definition drives down placebo rates [START_REF] Su | A meta-analysis of the placebo rates of remission and response in clinical trials of active ulcerative colitis[END_REF]. The variety of endpoints used to define remission in clinical trials of patients with active disease is given in Table 2. The range is large and includes complete remission (DAI=0), a modified UC-DAI ≤ 1, UC-DAI ≤ 2, CAI ≤ 4, and a Mayo Score ≤2 with no individual subscore >1 [START_REF] Kruis | Once daily versus three times daily mesalazine granules in active ulcerative colitis: a double-blind, double-dummy, randomised, non-inferiority trial[END_REF][START_REF] Marteau | Combined oral and enema treatment with Pentasa (mesalazine) is superior to oral therapy alone in patients with extensive mild/moderate active ulcerative colitis: a randomised, double blind, placebo controlled study[END_REF][START_REF] Rutgeerts | Infliximab for induction and maintenance therapy for ulcerative colitis[END_REF][START_REF] Hanauer | Delayed-release oral mesalamine at 4.8 g/day (800 mg tablet) for the treatment of moderately active ulcerative colitis: the ASCEND II trial[END_REF][START_REF] Kamm | Once-daily, high-concentration MMX mesalamine in active ulcerative colitis[END_REF].

Differences in defining remission between clinical trials complicate comparisons of drug efficacy.

This is because a patient in remission in trial A may not be in remission according to criteria applied in trial B [START_REF] Travis | Remission in trials of ulcerative colitis: What does it mean? In: Katz S Ed. Inflammatory Bowel Disease: A Practical Approach[END_REF]. The lack of standardisation makes interpretation of trials difficult, because important symptoms such as bleeding or increased stool frequency can be hidden in low scores. The differences in defining remission within a clinical trial can therefore have a major impact on the apparent efficacy of a drug (Table 3) [START_REF] Katz | Different definitions of remission for ulcerative colitis result in large variations of clinical outcome scores[END_REF][START_REF] Sinha | Epidermal growth factor enemas with oral mesalamine for mild-to-moderate left-sided ulcerative colitis or proctitis[END_REF]. When the effect of epidermal growth factor (EGF) on UC was assessed by three different indices of disease activity (the Powell-Tuck, UC-DAI and a unique 'Simplified Symptom Score'), remission thresholds were set at score ≤4, 0-1 and 0 respectively. Remission rates varied between 33% and 83%, depending on the index [START_REF] Sinha | Epidermal growth factor enemas with oral mesalamine for mild-to-moderate left-sided ulcerative colitis or proctitis[END_REF].

A trial endpoint is more easily reached with a lower threshold (ie a higher score) for remission. This is apparent from the ACT trials (Active ulcerative Colitis Trials [ACT] I and II) of infliximab for patients with UC refractory to standard therapy. The definition of remission was a DAI ≤2, with no individual subscore >1 [START_REF] Rutgeerts | Infliximab for induction and maintenance therapy for ulcerative colitis[END_REF]. When this definition was applied (albeit to a population of patients without treatment-refractory disease) in a retrospective analysis of two large trials of mesalazine [START_REF] Hanauer | Delayed-release oral mesalamine at 4.8 g/day (800 mg tablet) for the treatment of moderately active ulcerative colitis: the ASCEND II trial[END_REF][START_REF] Hanauer | Delayed-release oral mesalamine 4.8 g/day (800 mg tablets) compared to 2.4 g/day (400 mg tablets) for the treatment of mildly to moderately active ulcerative colitis: The ASCEND I trial[END_REF], the remission rate for 2.4g mesalazine increased from 22% (according to the original trial definition) to 50% [START_REF] Katz | Different definitions of remission for ulcerative colitis result in large variations of clinical outcome scores[END_REF] (Table 3).

Impact of different definitions on patients

The lack of a standardised definition of remission has considerable implications for patients. Specific symptoms that are important to patients, such as urgency, tenesmus, or incontinence are often not addressed by indices. Although most indices contain some measure of patient well being, only the Simple Clinical Colitis Activity Index (SCCAI) incorporates urgency and incontinence [START_REF] Walmsley | A simple clinical colitis activity index[END_REF]. In registration trials, where the main aim is to obtain a drug licence, urgency and incontinence are not assessed. This means that the drug registration process overlooks the control of major symptoms that are extremely important to patients. It is only now becoming apparent that a stringent endpoint for remission such as defined in guidelines (clinical plus endoscopic remission [START_REF] Stange | European evidence-based Consensus on the diagnosis and management of ulcerative colitis: definitions and diagnosis[END_REF]) is related to longer duration of remission. For example, an endoscopic score of 0 (defined as complete mucosal healing) applied to a post-hoc analysis of the ACT I and II trials revealed that patients with healing at week 8 had a four-fold increased likelihood of remission at week 30 of infliximab treatment [START_REF] Colombel | Mucosal healing in patients with ulcerative colitis associates with a reduced colectomy risk, high incidence of symptomatic remission and corticosteroid-free state[END_REF]. This is as might be expected, but needs to be confirmed if clinical practice is to change. By way of example, patients assessed by an index that did not incorporate endoscopy were less likely to be in remission at one year follow-up than those whose remission was defined more stringently by endoscopic, as well as clinical criteria (Figure 2) [START_REF] Dignass | Mesalamine once daily is more effective than twice daily in patients with quiescent ulcerative colitis[END_REF][START_REF] Paoluzi | Comparison of two different daily dosages (2.4 vs. 1.2 g) of oral mesalazine in maintenance of remission in ulcerative colitis patients: 1-year follow-up study[END_REF],

Impact of definitions of remission on long term disease outcomes

although caution is necessary, since it compares different studies. Finessing the endoscopic mucosal friability component of the Sutherland Index to develop the more stringent 'Modified UC-DAI' has affected long term remission rates in prospective clinical studies. Using the Modified UC-DAI, patients receiving Multi-Matrix System (MMX) mesalazine achieved and maintained remission rates >60% at four months and one year follow-up [START_REF] Kamm | Randomised trial of once-or twice-daily MMX mesalazine for maintenance of remission in ulcerative colitis[END_REF][START_REF] Kamm | Effect of extended MMX mesalamine therapy for acute, mild-to-moderate ulcerative colitis[END_REF].

The concept of 'deep remission' is developing in Crohn's disease, but has not yet transferred to ulcerative colitis. Nevertheless there are data suggesting that the more criteria by which remission is defined, the better the long term outcome. The combination of clinical, endoscopic and histological remission has been reported to be associated with a 70% likelihood of remaining in steroid-free remission over the next two and a half years (Odds Ratio for steroid-free remission 0.20, 95% CI 0.08-0.47,p=0.002 compared to patents without remission at the start of a cohort study of 87 patients).

Furthermore, histological remission was associated with a lower rate of hospitalisation (OR 0.27, 95% CI 0.07-0.95, p=0.048) during the median 29 month follow up in this small cohort [START_REF] Burger | Depth of remission may not predict outcome of ulcerative colitis over 2 years[END_REF]. These preliminary data need confirmation, but if histopathology can act as a surrogate marker for a lower rate of hospital admission in future years, then this may be a relevant outcome to include in clinical trials.

Role of non-invasive surrogate markers of mucosal healing in clinical trials

Non-invasive surrogates of mucosal healing would help lower study costs and might increase patient participation in clinical trials [START_REF] Higgins | Is endoscopy necessary for the measurement of disease activity in ulcerative colitis[END_REF]. Neutrophil-derived proteins in faeces can act as biomarkers of endoscopic inflammation [START_REF] Langhorst | Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices[END_REF]. Specifically, patients with active UC have higher levels of faecal lactoferrin, calprotectin, or neutrophil-elastase. These biomarkers appear to be able to differentiate active from inactive disease [START_REF] Langhorst | Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices[END_REF]. Indices such as the Seo Index [START_REF] Seo | An index of disease activity in patients with ulcerative colitis[END_REF] use multiple biomarkers and can identify patients meeting the 'regulatory definition' of remission (i.e. a score <120 identifies patients with no more than grade 1 on a modified Baron endoscopic score and the absence of visible blood) [START_REF] Higgins | Is endoscopy necessary for the measurement of disease activity in ulcerative colitis[END_REF]. Although the Seo index correlates with other disease activity indices [START_REF] Higgins | Is endoscopy necessary for the measurement of disease activity in ulcerative colitis[END_REF][START_REF] Seo | Evaluation of disease activity in patients with moderately active ulcerative colitis: comparisons between a new activity index and Truelove and Witts classification[END_REF] and endoscopic findings [START_REF] Seo | Correlation between endoscopic severity and the clinical activity index in ulcerative colitis[END_REF], as well as having some predictive value for patient outcomes [START_REF] Seo | Evaluation of disease activity in patients with moderately active ulcerative colitis: comparisons between a new activity index and Truelove and Witts classification[END_REF][START_REF] Seo | Evaluation of the clinical course of acute attacks in patients with ulcerative colitis through the use of an activity index[END_REF], it is too complex to use in practice. Clinical trials are now using faecal biomarkers of mucosal inflammation, but Histopathology may yet turn out to be a more useful and accessible measure.

Long-term implications of a stringent definition of remission for clinical trials

The lack of microscopic inflammation on a mucosal biopsy effectively excludes active UC and this is an important measure for validating active disease when recruiting patients to clinical trials, since it can be assessed independently from endoscopy. Given the problems associated with inappropriate recruitment as a result of subjective over-scoring of clinical and endoscopic appearances, including histopathology in the definition of remission might ensure its presence amongst inclusion criteria. This is, however, complex. There are logistic constraints on the time to histological analysis, especially by a central reader, that effectively prevent it being used as an inclusion criterion, other than to exclude in retrospect those patients with inactive disease. Nevertheless, histological healing (from active inflammation to quiescent disease) is potentially a useful measure of treatment success, especially if histopathology predicts long-term remission. Were trials of maintenance therapy only to include patients in histological (as well as clinical and endoscopic) remission, however, the risk of relapse may then become too low for an effect of treatment to be detected. The role of histopathology appears best to be confined to confirming disease activity at trial entry and predicting the risk of relapse or long term remission at trial completion.

Clinical trials are typically six to eight weeks' duration and many have relied on clinical rather than endoscopic (mucosal healing) endpoints, which may have lead to artificially high remission rates [START_REF] Kamm | Effect of extended MMX mesalamine therapy for acute, mild-to-moderate ulcerative colitis[END_REF].

Given the impact of a stringent definition on the longer term remission rate, future trials should adopt stringent endpoints in order to identify new treatments that are effective in preventing relapse. The particular questions that need to be addressed by future clinical trials are whether endoscopy adds sufficient value to clinically-defined remission to justify routine endoscopic confirmation of remission in practice, whether histological remission is really a surrogate marker for steroid-and hospitalisationfree remission and whether less invasive biomarkers are as reliable. This can then reasonably be expected to impact on the defined incidence of future flares, hospitalisation, colectomy and colon cancer in routine practice [START_REF] Bewtra | Trends in hospitalization rates for inflammatory bowel disease in the United States[END_REF][START_REF] Lichtenstein | The importance of mucosal healing in ulcerative colitis[END_REF].

REMISSION AS DEFINED IN GUIDELINES

Clinical guidelines should provide independent and practical recommendations for best clinical practice. Current guidelines offer some direction in setting a definition of remission, despite the lack of validated definitions of either remission or active disease. On the other hand, different activity indices and definitions may lead to substantial differences between guidelines for using the various classes of drugs.

Definitions of remission used in current guidelines

All guidelines discuss disease remission, but not all are explicit in their definition, which compromises their utility. The World Gastroenterology Organisation (WGO) Global Guidelines [START_REF] Bernstein | World Gastroenterology Organization Practice Guidelines for the diagnosis and management of IBD in 2010[END_REF] define remission as a UC-DAI ≤ 2, which implies modest control of symptoms. In contrast, both the European Crohn's and Colitis Organisation (ECCO) [START_REF] Stange | European evidence-based Consensus on the diagnosis and management of ulcerative colitis: definitions and diagnosis[END_REF], and the International Organisation for the study of IBD (IOIBD) [START_REF] Haens | A review of activity indices and efficacy end points for clinical trials of medical therapy in adults with ulcerative colitis[END_REF] define it as 'complete resolution of symptoms and endoscopic mucosal healing'. Nevertheless, neither the Practice Parameters Committee of the American College of Gastroenterology (ACG) [START_REF] Kornbluth | Ulcerative Colitis Practice Guidelines in Adults: American College of Gastroenterology, Practice Parameters Committee[END_REF] nor British Society of Gastroenterology (BSG) [START_REF] Carter | Guidelines for the management of inflammatory bowel disease in adults[END_REF] define remission.

Long-term implications of guideline recommendations

The implication for clinical practice is wide variation in treatment, wide variation in outcomes and, more importantly, acceptance of a low level of efficacy by physicians on behalf of their patients. A major aim of guidelines should be to raise the expectations and standard of care for both patients and physicians. Not all recommended therapies have been assessed by precise definitions of remission, because clinical trials typically fail to adopt a definition of remission supported by guidelines.

Furthermore, guidelines quickly become outdated. Current guidelines were generally compiled before clinical studies on active UC used stringent definitions of remission that were associated with long term maintenance of remission [START_REF] Dignass | Mesalamine once daily is more effective than twice daily in patients with quiescent ulcerative colitis[END_REF][START_REF] Kamm | Randomised trial of once-or twice-daily MMX mesalazine for maintenance of remission in ulcerative colitis[END_REF]. Guidelines may therefore recommend sub-optimal treatment, unless reviewed frequently. On the other hand, guidelines should be based on robust evidence to avoid frequent change, since this is otherwise unlikely to be adopted in clinical practice. There are no figures on how rapidly guidelines change practice, but it seems likely that guidelines currently change more rapidly than the community is able to follow.

REMISSION IN CLINICAL PRACTICE

Assumptions about standardised clinical care may be based on guidelines, but in practice, the standard question to the patient is: 'are you feeling better?' When the answer is 'yes', this alone can be considered 'success' by the physician and the treatment will continue. Too often no routine measurements are undertaken to translate 'feeling better' into a quantifiable disease parameter or objective definition of 'remission'. Consequently, clinical treatment 'success' may be a long way from disease 'remission'. Remission as an endpoint in UC requires prospective validation and should display a strong negative predictive value for the future occurrence of clinical flares, hospitalisation, use of steroids, complications such as dysplasia, or colectomy. Remission of symptoms defined entirely on clinical criteria may not be sufficient to prevent relapse, because treating symptoms alone is not enough to achieve optimal long-term outcomes [START_REF] Rutgeerts | Mucosal healing in inflammatory bowel disease: impossible ideal or therapeutic target?[END_REF][START_REF] Frøslie | Mucosal healing in inflammatory bowel disease: results from a Norwegian population-based cohort[END_REF]. Clinical practice is not least influenced by the (un)acceptability of sigmoidoscopy to the patient, but this is partly due to the lack of systematic study. Patients in clinical remission may have persistent mucosal inflammation, which are associated with a higher risk of relapse. Mucosal healing assessed by the macroscopic appearance of the mucosa at endoscopy appears to predict long-term remission [START_REF] Lichtenstein | The importance of mucosal healing in ulcerative colitis[END_REF][START_REF] Rutgeerts | Mucosal healing in inflammatory bowel disease: impossible ideal or therapeutic target?[END_REF][START_REF] Egan | Re-defining outcomes and re-evaluating remission in inflammatory bowel disease: Assessing key evidence[END_REF][START_REF] Kane | Endoscopic healing should be a goal for everyone with ulcerative colitis[END_REF], although this has yet to be quantified in a way that is meaningful to patients or clinicians [START_REF] Rubin | Impact of ulcerative colitis from patients' and physicians' perspectives: results from the UC:NORMAL survey[END_REF]. Mucosal healing may also reduce the risk of dysplasia or cancer, predict lower rates of hospitalisation and surgery, or improve quality of life [START_REF] Feagan | The effects of infliximab therapy on health-related quality of life in ulcerative colitis patients[END_REF], which are all outcomes that matter to patients. In a population-based study in Norway, patients with endoscopy-assessed mucosal healing of UC after one year of treatment still had a lower risk of colectomy 7 years later [START_REF] Frøslie | Mucosal healing in inflammatory bowel disease: results from a Norwegian population-based cohort[END_REF]. Considerable variation exists in European endoscopy clinical practice [START_REF] Lakatos | Management of inflammatory bowel diseases in Eastern Europe[END_REF]. Nevertheless, endoscopy may not reveal mucosal damage even when microscopic inflammation exists and it has been reported that endoscopy contributes little additional information to scoring clinical activity [START_REF] Burger | Depth of remission may not predict outcome of ulcerative colitis over 2 years[END_REF], which further questions the value of endoscopy in clinical practice. Long-term outcomes on the contribution that endoscopy makes to defining remission still need to be investigated.

Role of endoscopy

Role of histopathology

Microscopic (histological) healing may be a better predictor than the macroscopic appearance (or clinical criteria) of time to relapse. Histological assessment revealed that indicators of acute mucosal inflammation, including crypt abscesses, mucin depletion, or an acute inflammatory cell infiltrate were associated with a two-to three-fold increase in the risk of UC relapse during 12 months' followup [START_REF] Riley | Microscopic activity in ulcerative colitis: what does it mean?[END_REF]. The presence of basal plasmacytosis (ie dense infiltration of plasma cells in the lower third of the mucosa ) in patients with quiescent UC has also been associated with a 4.5 fold increased risk of relapse [START_REF] Bitton | Clinical, biological, and histologic parameters as predictors of relapse in ulcerative colitis[END_REF] Using a novel six-point histological scoring system, Rubin et al. [START_REF] Rubin | Increased degree of histological inflammation predicts colectomy and hospitalisation in patients with ulcerative colitis[END_REF] reported that an increased level of histological inflammation can predict both colectomy and hospitalisation in patients with UC, where a one-point increment in histological inflammation grade raised the risk of hospitalisation and surgery by 52% and 90%, respectively [START_REF] Rubin | Increased degree of histological inflammation predicts colectomy and hospitalisation in patients with ulcerative colitis[END_REF]. A prospective study has shown only modest agreement between clinical, endoscopic and histological measures of remission with complete agreement in just 58% of 91 patients (kappa 0.44) and 89% agreement between endoscopy and histopathology, although a third (36%) of those with histological remission had clinical symptoms of activity [START_REF] Thomas | How much agreement is there between histological, endoscopic and clinical assessments of remission in ulcerative colitis[END_REF]. Long-term (median 29 months, range 5-35) follow up of this cohort appears to confirm the predictive value of histopathology as a measure of remission [START_REF] Burger | Depth of remission may not predict outcome of ulcerative colitis over 2 years[END_REF].

Role of surrogate markers of mucosal healing in clinical practice

A high faecal calprotectin in UC has a strong negative predictive value for remission at six weeks and three months [START_REF] Gisbert | Fecal calprotectin and lactoferrin for the prediction of inflammatory bowel disease relapse[END_REF], but it has yet to be shown that augmenting treatment based on the results alters outcome. In a prospective study of patients with UC who had been in clinical remission for six months, the probability of relapse was <10% over the next 12 months in those with a basal faecal 14 calprotectin <150 µg/g [START_REF] Gisbert | Fecal calprotectin and lactoferrin for the prediction of inflammatory bowel disease relapse[END_REF]. Similarly, follow-up of patients with UC in clinical remission revealed significantly higher basal faecal calprotectin levels in those who relapsed compared to those who remained in remission, while ESR and C-reactive protein failed to predict disease activity [START_REF] Tibble | Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease[END_REF]. Even if thresholds of faecal calprotectin have limited predictive value for groups of patients, faecal calprotectin is increasingly popular because it is non-invasive and individual patients can serve as their own control. The use of surrogate markers for periodic monitoring of inflammation (like a haemoglobin A1c for diabetes applied to IBD) could be quite valuable as a sensitive, non-invasive screening tool to identify asymptomatic patients who could benefit from endoscopy, if proven prospectively to have a high sensitivity for subclinical disease. Escalating treatment of patients with a rising calprotectin to prevent clinical relapse is appealing and might improve quality of life, but this is unproven and a long way from current practice.

REMISSION FROM A PATIENT PERSPECTIVE: MEETING PATIENT EXPECTATIONS AND SUPPORTING ADHERENCE

A patient's expectations of treatment and remission may differ considerably from those of the physician. Patients often have difficulty in communicating symptoms, leading physicians incorrectly to assume that no complaints equates to no symptoms. Part of the problem is that patients focus on different symptoms from their physicians. While the physician may implement a therapeutic strategy with endoscopic improvement as the goal, factors important to patients with UC concern the present or immediate future, especially the speed of symptom relief with few side effects [START_REF] Gray | Treatment of ulcerative colitis from the patient's perspective: a survey of preferences and satisfaction with therapy[END_REF]. Once these acute issues are addressed, the patient focus switches to the long-term (long lasting remission of symptoms), so that they can conduct their lives without the fear of a sudden relapse [START_REF] Gray | Treatment of ulcerative colitis from the patient's perspective: a survey of preferences and satisfaction with therapy[END_REF].

Many indices contain items which patients do not recognise as being troublesome [START_REF] Joyce | Identification of symptom domains in ulcerative colitis that occur frequently during flares and are responsive to changes in disease activity[END_REF][START_REF] Waljee | Patient reported symptoms during an ulcerative colitis flare: a Qualitative Focus Group Study[END_REF]. Stool mucus, weight loss and anxiety were among 14 novel items mentioned by patients, but overlooked in indices, while tachycardia, eye symptoms and skin manifestations were incorporated, but of little concern to most patients [START_REF] Waljee | Patient reported symptoms during an ulcerative colitis flare: a Qualitative Focus Group Study[END_REF]. The UC: NORMAL internet survey in the USA was designed to understand how patients perceive the impact of UC, relative to the perception of gastroenterologists [START_REF] Rubin | Impact of ulcerative colitis from patients' and physicians' perspectives: results from the UC:NORMAL survey[END_REF]. Patients reported, on average, eight (self-defined) flares per year, almost three times the number recognised by physicians; furthermore, only 42% of patients believed that being in remission could mean living without symptoms. This is remarkable, because most gastroenterologists regard 'remission' as being synonymous with 'no symptoms'. Gastroenterologists underestimated the impact of the disease on patients leading a normal life, but both groups recognised the difficulties in taking daily medication. Thus, the physician's belief that 41% of their patients were not adherent to their therapy was substantiated by the finding that 46% of patients admitted to non-adherence over the previous week. The results indicate that standard care is suboptimal in many patients, with patients adapting their lives to accommodate UC, rather than acting to optimise therapy. This is underappreciated by physicians, so better management strategies and education are necessary [START_REF] Rubin | Impact of ulcerative colitis from patients' and physicians' perspectives: results from the UC:NORMAL survey[END_REF].

Remission and adherence

Paradoxically, the patient in remission may be at particular risk of relapse, because short-term treatment success may lead to long-term failure if, in the absence of symptoms, the prospect of a patient skipping therapy increases [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF][START_REF] Kane | Systematic review: adherence issues in the treatment of ulcerative colitis[END_REF]. Unsurprisingly, the likelihood of maintaining remission is significantly lower in patients who are not compliant with therapy. Two and a half years into 5-ASA treatment, almost 90% of those who were adherent remained in remission compared with 39% of nonadherent patients [START_REF] Kane | Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis[END_REF]. There are many reasons for lack of adherence in UC, including regimen complexity, male gender, new patient status, work pressures and shorter disease duration among predictors of non-adherence in UC [START_REF] Kane | Systematic review: adherence issues in the treatment of ulcerative colitis[END_REF][START_REF] Hawthorne | Review article: medication non-adherence in ulcerative colitis -strategies to improve adherence with mesalazine and other maintenance therapies[END_REF][START_REF] Prantera | 5-ASA in ulcerative colitis: improving treatment compliance[END_REF][START_REF] Chaudhary | Impact of a patient support program on mesalamine adherence in patients with ulcerative colitis[END_REF][START_REF] Saini | Effect of medication dosing frequency on adherence in chronic diseases[END_REF][START_REF] Cross | Home telemanagement for patients with ulcerative colitis (UC HAT)[END_REF][START_REF] Kane | The challenge of compliance and persistence: focus on ulcerative colitis[END_REF][START_REF] Higgins | Systematic review: impact of nonadherence to 5-aminosalicylic acid products on the frequency and cost of ulcerative colitis flares[END_REF][START_REF] Kripalani | Interventions to enhance medication adherence in chronic medical conditions: a systematic review[END_REF]. Predicting long-term remission cannot depend just on the clinical, endoscopic and histological definition at a point on time.

KEY CONSIDERATIONS FOR A STANDARDISED DEFINITION OF REMISSION

A definition of remission should focus on predictors of improved long-term outcomes, because this goal would satisfy patients, clinicians and regulatory bodies. It is clear there is a need for standardisation, but the optimal parameters (clinical, endoscopic, histology, biomarkers) and specific terminology await resolution. Current standards of care could be improved with the use of a more stringent definition of remission. Pertinent questions include:

• Will it help predict the duration of remission?

• Will it distinguish between therapies?

• Will it increase the uniformity of clinical trials?

• Will it increase the uniformity of clinical practice? (iii) Biomarkers. The use of faecal calprotectin is currently limited, because it does not affect decision making. The negative predictive value of faecal calprotectin for the maintenance of remission requires validation in a long-term clinical trial. If calprotectin could predict the need for endoscopy, then it would become a valuable tool.

(iv) Histopathology. Histological improvement is slower to change than macroscopic appearance, but markers of acute mucosal inflammation have a reasonable negative predictive value for maintaining remission and predicting relapse. Histopathology is currently considered an impractical measure of remission in clinical practice, but if preliminary results on its value for predicting steroid-free remission and hospitalisation are confirmed, then this will be re-visited. Furthermore, the long-term association between inflammation and colorectal neoplasia means that histopathology should not lightly be disregarded.

Based on these considerations we propose a current definition that states Remission in ulcerative colitis should mean complete cessation of rectal bleeding, urgency and increased stool frequency, best confirmed by endoscopic mucosal healing

CONCLUSIONS

Wide variation in the definition of remission in clinical trials means that the effectiveness of different treatments is difficult to compare. As a consequence, guidelines vary and not all recommendations are anchored in a robust outcome from a clinical trial. The expectations of patients and their physicians need to be raised in clinical practice, to expect their treatment of ulcerative colitis to induce remission.

A standard based on clinical symptoms and endoscopy is proposed, but whether this definition of remission translates into better long-term outcomes remains to be determined. Longitudinal studies are necessary for this to be validated, especially with regard to histopathology, in conjunction with clinical and endoscopically-defined remission.

F

  in clinical trials, in guidelines and clinical practice as well as the patient perspective, before discussing the key components of a standardised definition of remission.

  Abbreviations: DAI: disease activity index. PFA: Patients Functional assessment. PGA: Physician's Global Assessment.

11 outcomes

 11 are awaited, so their value remains unclear.

Table 1 : Different indices used to measure ulcerative colitis disease activity

 1

	Disease parameter	Disease Index
	measured
	Clinical	• Truelove and Witts (Truelove	• Physician's Global Assessment
		1955)	(PGA; Hanauer 1993)
		• Powel-Tuck Index (St. Mark's	• Investigator's Global Evaluation
		Index; Powell-Tuck 1978)	(IGE; Hanauer 1998)
		• Clinical Activity Index (CAI;	• Simple Clinical Colitis Activity

Table 2 : Indices and endpoints used in recent clinical trials with patients in active disease Drug/study Index/Endpoint Endpoint Definition of Remission

 2

Table 3 : Impact of different definitions of remission threshold on clinical trial outcomes

 3

	Study	ASCEND I/II			EGF Study [22]
			[5,20,23]
	Index		Mayo/DAI		Powell-Tuck	UC-DAI	SSS
	Criteria	Stool	frequency	Bowel frequency	Stool frequency	Stool
	included	relative to normal		Stool consistency	Rectal bleeding	consistency
		Rectal bleeding		Abdominal pain	Mucosal	Blood in stool
		Sigmoidoscopy		Rectal bleeding	appearance	Nocturnal
		PFA			EIS, temperature	Physician's rating	defaecation
		PGA			Sigmoidoscopy
	Remission score	0	≤1	≤2	≤4	0-1	0
	Remission rate	22%	28% 50%	83%	33%	83%

Abbreviations: EGF: epidermal growth factor. DAI: disease activity index. PGA: Physician's Global Assessment. PFA: Patients Functional assessment. SSS: Simplified Symptom Score

  Patients would find endoscopy unacceptable at every clinic visit. However, if it could be shown that a definition of remission including endoscopy predicted outcome in the future, then this invasive procedure might become more acceptable. Studies are necessary to validate the long-term predictive value of endoscopic remission. Endoscopy can only be justified if the result will modify patient management.

	(ii) Endoscopy.
		F o r
		P
		e
		e r
		R
		e
	•	v i e Will this translate into improved quality of life, less hospitalisation and other improved outcomes for the patient? w
	Components of a definition of remission
	(i) Clinical aspects. The definition of remission should include both absence of rectal bleeding and a
	threshold for stool frequency. It is our view that 'urgency' should be included because it is a symptom
	of central importance to patients [17,57], although this might be debated since it has not been
	systematically studied. Descriptive terms such as mild, intense, or incontinence could be applied.
	Urgency might, however, be quantified in terms of the time in minutes that the call to stool can be
		16

deferred, although this would only be of value if it captured information that is not achieved by other items. Clinical symptoms alone do not relate to outcome and additional components are required in a definition of remission.

  Laboratories, Asahi, Merck. Novartis, Schering-Plough, SigmoidPharma, Vifor and Warner Chilcott.He owns no stock or relevant patents.C. Janneke van der Woude has served as a speaker, consultant, or advisory board member for Abbott Laboratories, Ferring, Merck and Shire. She has received research funding from Abbott Laboratories and Merck.

	o r F o r F o r F o r F o r F o r F o r F o r F o r
	P P P P P P P P P
	e e e e e e e e e
	e r e r e r e r e r e r e r e r e r
	R R R R R R R R R
	e e e e e e e e e
	v i e v i e v i e v i e v i e v i e v i e v i e v i e
	w w w w w w w w w
	25

Alimentary Pharmacology & Therapeutic

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