Archive Build­ing im­mu­nity

Jim Trea­surer, Si­mon Wadsworth and An­drew Grant of Ma­rine Har­vest (Scot­land) re­port the find­ings of an ex­per­i­ment with hy­dro­gen per­ox­ide

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POS­SI­BLE devel­op­ment of re­sis­tance by sea lice to medicines in ther­a­peu­tant treat­ments and how it may be man­aged is an im­por­tant is­sue in sea lice con­trol. This is par­tic­u­larly crit­i­cal where the range of medicines avail­able to treat sal­mon is lim­ited. Un­til re­cently, dichlor­vos (Aqua­gard, No­var­tis) and hy­dro­gen per­ox­ide (Salartec, Bren­ntag; Paramove, Solvay In­terox) were the only medicines licensed in the UK for the treat­ment of At­lantic sal­mon with a sea lice bur­den. While dichlor­vos is now used rarely, aza­me­thiphos (Sal­mosan, No­var­tis) and cyper­me­thrin (Ex­cis, No­var­tis) have been licensed and are al­ter­na­tive bath treat­ments.

Re­sis­tance of in­sects to pes­ti­cides, par­tic­u­larly organophos­phates and pyrethroid­s, is well es­tab­lished, and is thought to de­velop by ge­netic se­lec­tion for in­di­vid­u­als pro­duc­ing detox­i­fy­ing en­zymes and al­ter­ations in tar­get sites and in the bal­ance of acetyl­cholineste­rasae. Re­sis­tance of sea lice to dichlor­vos has been demon­strated and its devel­op­ment de­scribed (Roth, 1999). Roth also quoted the pos­si­bil­ity of re­sis­tance to pyrethroid­s in Nor­way.

Re­cent anec­do­tal ev­i­dence from fish farms in Scot­land sug­gested that treat­ments with hy­dro­gen per­ox­ide on some farms have been less ef­fec­tive than when first used. Hy­dro­gen per­ox­ide has been used in Scot­land since 1992 and has been the main medicine used on many farms due to lice re­sis­tance to dichlor­vos.

The man­u­fac­tur­ers’ rec­om­mended treat­ment is 1500 ppm for 20 min­utes, but ef­fi­cacy de­pends on wa­ter tem­per­a­ture and this rec­om­men­da­tion would give in­ef­fec­tive treat­ments at tem­per­a­tures be­low 10 deg C. Treat­ments are rarely fully ef­fec­tive and 85 to 100 per cent of the mo­bile stages may be re­moved. Re­sis­tance has been dif­fi­cult to demon­strate con­clu­sively in farm treat­ments as the con­cen­tra­tion of per­ox­ide varies, with highly vari­able vol­umes of wa­ter en­closed within the tar­pau­lin. Tem­per­a­ture and du­ra­tion of treat­ment also af­fect the suc­cess of treat­ments.

Re­sis­tance of the sal­mon louse Lepeoph­theirus salmo­nis to hy­dro­gen per­ox­ide was in­ves­ti­gated on a farm in Scot­land fol­low­ing reg­u­lar use of hy­dro­gen per­ox­ide. Re­sis­tance of lice on fish from this farm was also com­pared ex­per­i­men­tally in bins con­tain­ing a known con­cen­tra­tion of hy­dro­gen per­ox­ide with lice from a farm where per­ox­ide had not been used.

Ef­fi­cacy of rou­tine treat­ments

The ef­fec­tive­ness of hy­dro­gen per­ox­ide was as­sessed in Jan­uary 1999 (wa­ter tem­per­a­ture 6 deg C, salin­ity 28 ppt) on farm A (to­tal stock 78,000 fish in cages 16m square and 8m deep) where the fish had been treated ex­clu­sively with hy­dro­gen per­ox­ide over six years.

Ten sal­mon were re­moved from each of four cages by hand­net 24 hours prior to treat­ment, anaes­thetised in 15 ppm ben­zo­caine and to­tal mo­bile lice (all were L. salmo­nis) counted. All cages were treated at a per­ox­ide con­cen­tra­tion of 2,500 ppm for 23 min­utes by the bath method as there was poor ef­fi­cacy in pre­vi­ous treat­ments with lower con­cen­tra­tions.

The nets were first raised from 8 to 3m depth, oxy­gen was sup­plied, the cage en­closed with a ‘wedge’ tar­pau­lin and hy­dro­gen per­ox­ide, 50 per cent w/v di­luted 1:1 sea­wa­ter, pumped into the cage to give a con­cen­tra­tion checked by titra­tion. The treat­ment was ter­mi­nated and the tar­pau­lin re­moved af­ter 23 min­utes. Lice were counted 24 hours af­ter treat­ment and the per­cent­age re­duc­tion in num­bers cal­cu­lated.

De­spite the high con­cen­tra­tion and long ex­po­sure time, an av­er­age of only 63 per cent of motile lice was re­moved. Th­ese poor results con­firmed ver­bal re­ports of re­duced ef­fi­cacy from farm­ers and were fur­ther tested in ex­per­i­men­tal tri­als in large bins, car­ried out in Jan­uary 1999.

Devel­op­ment of re­sis­tance

From th­ese ex­per­i­ments it can be con­cluded that Lepeoph­theirus were re­sis­tant to hy­dro­gen per­ox­ide in ex­per­i­men­tal bin treat­ments on a farm treated reg­u­larly com­pared with a farm where hy­dro­gen per­ox­ide had not been used. Th­ese results sup­port the find­ings of less ef­fec­tive farm treat­ments. In the fullscale treat­ments, only 63 per cent of lice were re­moved, even when high con­cen­tra­tions of hy­dro­gen per­ox­ide were used, 2500 ppm for up to 23 min­utes.

Re­sis­tance of in­sects to pes­ti­cides de­vel­ops through ge­netic se­lec­tion of in­di­vid­u­als and, in lice, this may be se­lec­tion for in­di­vid­u­als with cu­ti­cle that pro­vides a bar­rier to pen­e­tra­tion by hy­dro­gen per­ox­ide or the pres­ence of detox­i­fy­ing en­zymes, for ex­am­ple, cata­lase and glu­tathione re­duc­tase.

Lice may have been pre-ex­posed to in­ef­fec­tive doses of hy­dro­gen per­ox­ide, and con­cen­tra­tions used to treat sal­mon do not kill most lice. A treat­ment that im­mo­bilises the par­a­site with­out a killing ac­tion at the treat­ment dose is likely to de­velop re­sis­tance. How­ever, re­sis­tance may also be en­cour­aged by poor ap­pli­ca­tion meth­ods.

Hy­dro­gen per­ox­ide can­not be used at a stan­dard con­cen­tra­tion and du­ra­tion in dif­fer­ent wa­ter tem­per­a­tures, and treat­ment is a bal­ance be­tween achiev­ing ef­fi­cacy and min­imis­ing tox­i­c­ity to the fish.

In the first farm treat­ments with hy­dro­gen per­ox­ide in Scot­land in 1992, only 83 per cent of lice on av­er­age were re­moved. A pro­por­tion of the res­i­dent lice pop­u­la­tion there­fore re­mained af­ter treat­ment, giv­ing scope for se­lec­tion of char­ac­ter­is­tics likely to pro­mote re­sis­tance.

Im­pli­ca­tions of re­sis­tance for sea lice con­trol

With in­creas­ing re­sis­tance to hy­dro­gen per­ox­ide, more treat­ments are re­quired at higher con­cen­tra­tions to achieve a use­ful re­duc­tion in sea lice num­bers and this may in­crease the risk of fish mor­tal­i­ties. An ef­fec­tive sea lice con­trol strat­egy re­quires in­ter­ven­tion be­fore the oviger­ous fe­males shed eggs, and re­sis­tance to hy­dro­gen per­ox­ide and the re­duced sus­cep­ti­bil­ity of oviger­ous lice to treat­ment com­pared with other mo­bile stages, even on a farm that had not used per­ox­ide, may have re­duced the ef­fec­tive­ness of the na­tional sea lice con­trol strat­egy in Scot­land in 1998 and 1999. More ef­fec­tive treat­ments, par­tic­u­larly in-feed com­pounds, are re­quired to im­prove the suc­cess of the strat­egy.

A range of medicines that can be used al­ter­nately could re­duce the devel­op­ment of re­sis­tance. The in­dus­try in Scot­land and all ma­jor sup­pli­ers of sea lice medicines are there­fore dis­cussing the best strat­egy for us­ing medicines in an at­tempt to achieve this.

Left: A sea lice treat­ment us­ing hy­dro­gen per­ox­ide in progress

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