Tenacibaculum maritimum: Current Knowledge & Future Directions

Transcription

1 Tenacibaculum maritimum: Current Knwledge & Future Directins Wrkshp Reprt and Synpsis Jim Pwell and Tina Pdlasly, BC CAHS

2 Tenacibaculum maritimum: Current Knwledge and Future Directins Wrkshp Reprt and Synpsis Wrkshp date and lcatin: Feb 15 and 16, 2015 Campbell River Maritime Heritage Centre, 621 Island Hwy, Campbell River, BC V9W 5C1 Page 1 f 40

3 Cntents Overview... 3 Wrkshp ratinale:... 3 Target audience:... 4 Purpse... 4 Objective... 4 Outcme f the wrkshp... 4 Wrkshp agenda... 5 TM Wrkshp Schedule... 5 Shrt Bigraphical Infrmatin f the Speakers... 5 Summary f Presentatins... 8 Alicia Estévez Tranz (U. f Santiag Cmpstela USC; Spain)... 8 Tenacibaculsis f farmed fish in Suthern Eurpe... 8 Øvind Brevik, Cermaq (AS Nrway)... 9 The impact f tenacibaculsis in Nrway... 9 Sverre Bang Småge (Cermaq AS Nrway)... 9 Nvel TM species in Nrway detectin and identificatin... 9 Tim Hewisn & Michael Ness (Grieg Seafd BC Ltd.) Tenacibaculum maritimum A Fish Farm Perspective Ahmed Siah (BC CAHS) Clinical methds f detectin fr T. maritimum Jaime Tbar (Centrvet, Chile) Current situatin f Tenacibaculum in Chile and ptential preventin strategies Participants List Summary f Discussins Synpsis f Grup Discussins Recmmendatins Develpment Pririties Appendix One: Agenda Appendix Tw: Summary f Presentatins Questin panel Appendix Three: Bigraphy f Presenters Appendix Fur: Day One Presentatin Attendees Day Tw Breakut Grup Attendees Page 2 f 40

4 Tenacibaculum maritimum: Current Knwledge and Future Directins Overview The BC Centre fr Aquatic Health Sciences (BC CAHS) was apprached by industry representatives seeking additinal infrmatin and new develpments regarding Tenacibaculum maritimum, the causative agent f tenacibaculsis r yellw muth. After preliminary discussin and research int the tpic, it was sn apparent that althugh sme wrk had been dne in the early years f salmn farming in BC, there was little recent infrmatin regarding preventin and treatment. Indeed, there was n new infrmatin regarding tenacibaculsis fr Atlantic Salmn farmed in BC waters in general. Meanwhile, prducers are witnessing an increased incidence f lsses that may be due t Tenacibaculum spp. infectin. The majr issues f raised were: Accurate identificatin f the pathgen (it is difficult t islate and culture), Understanding the surce and cnditins f infectin, Mitigatin t prevent the nset f the infectin, Access t reliable and cst-effective detectin f the causative agent(s), Effective treatment, and Vaccine develpment. Tenacibaculsis has plagued Mediterranean fish culture fr sme time and the signs f infectin are similar in Atlantic Salmn. This germane wrk in the field identified T. maritimum as the etilgical agent in turbt, snapper, bream, sea bass and ther species including Ch Salmn in nrthwestern Spain. Until recently, Atlantic Salmn researchers in nrthern Eurpe were reluctant t ascribe pstsmlt lsses t tenacibaculsis because f the identificatin f an agent and the cncmitant bservatin f Tenacibaculum-like presence in winter ulcer lsses. With the advent f RT-qPCR identificatin f Tenacibaculum spp. in bth lsses after smlt and c-infectin with Mritella viscsa in winter ulcer cases, mre attentin has been paid t detectin and preventin f lsses t Tenacibaculum spp. In respnse t this grwing need fr infrmatin and t identify research gaps, BC CAHS gratefully received Aquaculture Cllabrative Research and Develpment Prgram (ACRDP) apprval t hld a wrkshp t discuss tenacibaculsis the first in BC since BC CAHS brught tgether experts frm Spain, Nrway, Chile and BC t discuss the issue and parse ut research directins and needs. The result was a tw-day wrkshp that was well attended by gvernment persnnel, industry leaders, fish health prfessinals, prducers and researchers. The fllwing reprt details the wrkshp and utcmes. Wrkshp ratinale: Lsses t T. maritimum can be significant and are largely unpredictable in pst-sw entry smlts. Salmn prducers in BC have expressed a need t learn mre abut the current status f T. maritimum in rder t eliminate r mitigate lsses t tenacibaculsis. It has been sme time since the issue has been addressed n an internatinal scale and the timing is right since the recgnitin f tenacibaculsis as an internatinal cncern in salmnid culture. It is necessary t discuss the state f the art with key researchers, prducers, fish health and gvernment persnnel in rder t determine the future directin f the research required, surveillance methds, treatment methds and future preventin techniques. This wrkshp will act as the springbard fr activating and strengthening research and develpment effrts in the near and medium term. The bjective is t identify the knwledge gaps Page 3 f 40

5 surrunding T. maritimum cncerning detectin, mnitring, surveillance and mitigatin and define future research needs t address these issues. Target audience: Prducers Vancuver Island (Campbell River, Prt Hardy and Tfin) is the hme t the majrity f BC s seawater finfish prductin; prducers have a vested interest in the current state f T. maritimum, the research directins, detectin and surveillance. Salmn prducers have expressed interest in creating this wrkshp. Fish health prfessinals With significant lsses in SW entry fish each year, fish health prfessinals have keen interest in the state f the art. Diagnsticians The latest detectin techniques will be a tpic f discussin. Fish veterinarians Fr prfessinal develpment and infrmatin n the state f the art, fish veterinarians will gain up-t-date knwledge by attending the wrkshp. Prviders f fish health prducts Prducts are required t treat current utbreaks as well there is a need t develp new prducts such as vaccines r preventative medicines. Fish health prducts will play a pivtal rle in T. maritimum cntrl. Researchers The wrkshp will be a key pprtunity fr all participants t interact with researchers n the cutting edge f T. maritimum knwledge. Gvernment and academic researchers will want t knw the needs f the industry t assist with research and discvery needs. Regulatrs and gvernment representatives Plicy and regulatin f pathgenic micrrganisms is always f cncern t the public regarding prtectin f the wild resurces and preservatin f the industry. Purpse The purpse f the wrkshp is t bring tgether user grups, scientists, researchers, fish health persnnel and gvernment in a setting where the free-flwing exchange f infrmatin leads t establishing the current knwledge base, identifies the gaps and charts a curse fr future research. Objective T review the current knwledge f T. maritimum bilgy, detectin and preventin in rder t mitigate lsses t the pathgen. At the end f the wrkshp there will be a clear understanding f the future needs fr research, identificatin f the key participants and a pathway frward t investigate and eliminate the knwledge gaps identified. Research pprtunities will be identified and reprted. Outcme f the wrkshp T establish the state f the knwledge regarding T. maritimum, identify knwledge gaps and set a curse fr detectin, surveillance and treatment. Identificatin f current, future and ptential research pririties defined. Page 4 f 40

6 Wrkshp agenda Organism: Identificatin Different sertypes per specie and gegraphical area Virulence factrs Epidemilgy Causes Risk factrs Signs f disease in different fish species Pathphysilgy f the disease What happens inside the fish Treatment Antibitic treatment Develped vaccines fr S. salar and ther species Vaccine results TM Wrkshp Schedule Day 1 travel day fr speakers t arrive in Campbell River, BC Day 2 Wrkshp 8:30 am t 4:30 pm at Maritime Heritage Centre 6 pm dinner fr speakers and veterinarians c/ MHC/Cermaq/Grieg Day 3 Wrkshp 8:30 am t 12 nn at Maritime Heritage Centre 1 pm t 5pm SW Farm tur f lcal Grieg Seafds BC site. Day 4 travel day fr speakers leaving Campbell River Shrt Bigraphical Infrmatin f the Speakers Full bigraphical infrmatin fr the speakers appears in Appendix Three. Alicia Tranz, U. f Santiag Cmpstela (Spain) Tenacibaculsis f farmed fish in Suthern Eurpe Tenacibaculsus detectin, mitigatin and treatment in Eurpean farmed fish Øvind Brevik, Cermaq (AS Nrway) The impact f tenacibaculsis in Nrway Lsses and mitigatin strategies emplyed by the Nrwegian industry Sverre Bang Småge, Cermaq (AS Nrway) Nvel TM species in Nrway detectin and identificatin Nvel TM species in Nrway detectin and identificatin Page 5 f 40

7 Tim Hewisn and Michael Ness, Grieg Seafds Canada, (Campbell River BC) Tenacibaculum maritimum A Fish Farm Perspective Overview f recent suspected fish health challenges due t T. maritimum Ahmed Siah, BC Centre fr Aquatic Health Sciences, (Campbell River BC) Clinical methds f detectin fr T. maritimum Screening methds f detectin fr T. maritimum used at BC CAHS Jaime Tbar, Chile Current situatin f Tenacibaculum in Chile and ptential preventin strategies Vaccine develpment and immunlgical bsters fr treatment in Chile Page 6 f 40

8 Page 7 f 40

9 Summary f Presentatins Ntatin f the presentatin can be fund in Appendix Tw. The full presentatins are n the BC CAHS website Alicia Estévez Tranz (U. f Santiag Cmpstela USC; Spain) Tenacibaculsis f farmed fish in Suthern Eurpe Dr. Tranz pened her talk by giving an accunt f the aquaculture peratins in Galacia, nrthwestern Spain. The main species f fish raised in n-shre tanks and sme few netpen peratins are Gilthead Seabream, Turbt, Sle, Seabass and sme Salmn. There are five species f Tenacibaculum spp. that ccur as infectin agents in Galician cultured fish: T. maritimum, T. sleae, T. disclr, T. dicentrarchi and T. gallaicum. In sme cases, A. salmnicida is a cinfectius agent and if V. anguillarum is present, it will vercme the grwth f T. maritimum in culture. Fr a presumptive diagnsis, T. maritimum is abundant, lng thin rd shaped in wet munt and Gram stain negative. It is difficult t culture and must be grwn n media cntaining high levels f salt. If grwn n Marine Agar, the grwth is rapid and can be cnfused with ther rapidly-grwing clnies. If FFM is used, the grwth is slwer and at lwer density, which is useful in recgnizing T. maritimum clnies. Phentypic variance amng the species is nt cmpletely defined as there are several sertypes and gentypes that must be defined by PCR, prtemically (MALDI-TOF) and antigenically r Dt Blt. RAPD-PCR analysis was used t separate strains int distinct gentypes that are strngly assciated with the sertypes described. MLSA studies f 11 lci defined frm 73 strains revealed that there was a statistically-significant assciatin between gentypes and infrmatin related t the hst fish and gegraphic lcatin. This infers that there is endemic clnizatin f farms by lcal strains. During infectin studies, Turbt required 18h f expsure t induce the disease that caused eruptins f external lesins. Bacteria were als lcated in the kidney. In fish that are symptmatic and surviving expsure, the bacteria can nly be recvered frm the mucus. Virulence factrs are assciated with adherence via the capsular material f the bacterium. There is enzyme-assciated up-regulatin related t cell t cell cmmunicatin that influences virulence. In vaccine develpment, a cmmercial plyvalent vaccine was develped fr Turbt fr immersin applicatins. By accunts it is effective when used as a bath at 1-2g fllwed by a bster injectin at 20-30g. N vaccines have been develped fr ther species. Therapeutic cntrl is dne by using Enrflxacin, but resistance builds quickly. Flrfenicl is the drug f chice. Prphylactic treatments using hydrgen perxide are effective, but stress the fish. Freshwater baths are effective when the salinity is drpped t 5.0 fr 24h. Her final remarks nted research gaps invlving the rute f transmissin, survival strategies and reservirs f the bacterium, discvering the genetic basis f the virulence mechanism, establishing adequate vaccinatin prgrammes and the imprtance f identifying the same species f Tenacibaculum in different cuntries and the predminant sertypes at play. Page 8 f 40

10 Øvind Brevik, Cermaq (AS Nrway) The impact f tenacibaculsis in Nrway Dr. Brevik began with a brief verview f Cermaq Atlantic Salmn farming peratins in Nrway. He cntinued by utlining the presentatin f the disease, particularly winter ulcers, and nted that these ulcers increase in prevalence as farms are lcated nrthward. He als nted ther factrs that are seen in assciatin: when the fish were entering seawater, whether they were an S0 r S1 smlt and the seasn the fish entered seawater with the early r middle mnths seeing the highest lsses. The first cases f identified lsses in smlts were in 2011 in land-based SW tanks where the inlet was at 20 r 80m depth and in fish f g. The lsses are seen in February and mrtalities are arund 5%. In seawater netpens with spring and autumn transfers, lsses are seen within weeks f entering seawater. The infectin is first seen in the muth area and prgresses t the fins and then skin t frm ulcers. An utbreak can ccur quickly riginating in ne pen and spreading t the entire site. Antibitic treatment can help abate the impact. In larger fish, the utbreak can start in autumn and cntinue int the winter. The presentatin f the infectin fllws that f smlts, starting in the muth and spreading t lesins. An utbreak in ne pen can lead t fish lsses in ther pens. The ecnmic impact f lsses and treatment in fish f this size is cnsiderable. Identificatin f the utbreak is by culture n marine r bld agar. RT-qPCR assay specific fr the husekeeping genes f T. maritimum is used. This des nt detect the ther bacterial such as M. viscsa and it can be used n slime r skin swabs as a nn-lethal methd f detectin. Treatment is using Flrfenicl and has immediate effect. There is n current vaccine available fr Atlantic Salmn. Sverre Bang Småge (Cermaq AS Nrway) Nvel TM species in Nrway detectin and identificatin Mr. Småge discussed the cnundrum f perhaps false identificatin f winter ulcers attributed t M. viscsa which may have caused by T. maritimum. A vaccine develped fr M. viscsa did nt end the ulcers and T. maritimum was islated frm the margins f the ulcer whereas M. viscsa is fund in the pen wund. Cmmn factrs assciated with the nset f ulcers is handling and stress related t seawater transfers at lw temperatures. Mr. Småge then went int detailed discussin n the islatin f Tenacibaculum spp. and the culture f pure clnies that are best seen frm the presentatin slides. Genetic sequencing detailed a phylgenetic tree amng strains based n husekeeping genes n 16s rrna. The bacterium is Flrfenicl sensitive. There may be a causal relatinship t medusa prevalence, sea lice infestatin, herring presence and the use f cleanerfish (wrasse). In summary he ntes that tw islates were able t repeat the disease in a challenge mdel t cause ulcers. He pstulates that ne f the islates may be a different species f bacterium than T. maritimum. Page 9 f 40

11 Tim Hewisn & Michael Ness (Grieg Seafd BC Ltd.) Tenacibaculum maritimum A Fish Farm Perspective Mr. Hewisn began by reiterating that T. maritimum is ne f 20 members in the genus Tenacibaculum and that psitive cnfirmatin f the causative agent here in BC will have t be cnfirmed bichemically and by PCR and genetic sequencing. Mr. Ness presented graphic infrmatin f mrtality vs. day vs. fish weight that shwed there was an initial spike in mrtality abut 7-14 days pst entry int seawater and anther when fish mass was abut 400g. Data were shwn regarding bld chlride levels f smlts between 149, 150 and 160mml/l that supprts the ntin that level f smregulatin in higher salinity receiving waters may influence the severity f the utbreak; lwer salinities can trigger a plateau in lsses. They als felt that higher temperatures may play a rle in levels f mrtality. In the last eight years, mrtality has been increasing n a percentage basis by year. Respnse t an utbreak is treatment with ptentiated sulphnamides r Flrfenicl in feed fr six days and results are nticed after three. The number f treatments required varies, but the use is cncentrated within the first tw mnths after smlt entry. They nte that the respnse t treatment (sparked by a spike in mrtality) drives the need t medicate and the mrtalities drp ff after three days. Un-medicated feed is presented thrugh the day t satiate aggressive-feeding fish and keep them satiated t ensure all fish receive treated feed at sme pint. Mr. Hewisn stated that they use antibitics t nly treat tenacibaculsis and n ther infectin. The level f treatments is between tw and three times per site which is within ASC guidelines. The cst f treatment is abut $70,000 annually and causes a prfit lss f 2% fr mrtality and little less than that fr lss bimass thrugh grwth. The annual cst f the utbreaks is ver $1.6 millin. Ahmed Siah (BC CAHS) Clinical methds f detectin fr T. maritimum Dr. Siah pened with a brief histry f the detectin and identificatin f putative T. maritimum bacteria stemming frm wrk in 1977 cntinuing t the taxnmic descriptin based n the GyrB and 16s rrna sequences. Kent (1992) bserved that Pacific yellw muth infectins were bserved as yellw pigment arund the muth with ndules that culd end in degradatin f the upper and lwer jaws. Islatin requires cllectin f samples frm % salinity seawater and C. FMM appears t be the preferred culture media at ph Detectin f the sample can be by serlgical methds, flurescent antibdy techniques r nested PCR using mucus as the target tissue. RT-qPCR is als a gd methd. Hwever, fr Pacific islates, the Bader and Schtts (1998) methd is preferred ver ther Eurpean primers which d nt detect Pacific strains. T this effect, it will be necessary t cnduct whle genme sequencing. In this way it will be easier t design accurate detectin methds and identify virulence factrs. It will als assist in defining hst-pathgen relatins. Page 10 f 40

12 Jaime Tbar (Centrvet, Chile) Current situatin f Tenacibaculum in Chile and ptential preventin strategies Tenacibaculsis in Chile ccurs in bth Rainbw Trut and Atlantic Salmn. It appears that infectin ccurs at tw life stages: ne mnth after smlt entry int seawater and in adults f 1.5kg mass. The causative bacterium is fund in seawater and brackish water and at a range f temperatures indicating it has a high adaptive capacity. Trigger factrs are stress, lw water quality, salinity variatins and immunsuppressin. Treatment f utbreaks is by antibitic, usually OTC. Fish can recver frm infectin, but there is an impact measured by lss f prductin in FCR, early mrtality, external lesins that have healed (affecting specific markets skin-n fillets) withut lss t fillet quality. The infectins appears t mdel the SRS immune respnse mechanism and the treatment regimen is adpted here. Unvaccinated fish witness tw peaks in mrtality in bth small (80-120g) and larger (1.5kg) fish. The strategy fr vaccinatin is t revaccinate; in vaccinated fish the peak mrtality is reduced, but nt eliminated, prbably due t a lack f specific antibdy prductin. If a bster vaccinatin is given the secnd peak is decreased. There are prblems with a secnd vaccinatin due t handling and increased stress that may pse a risk f secndary infectins and cst because the fish is larger. Centrvet Chile is marketing an ral bster t vercme the injectin-related side effects and reduce vaccine csts. By using the ABH ral delivery system f a Micrmatrix technlgy, the gut is permeated by the antigen and an immunstimulant. This methd cmpares well in efficacy with an injectin methd. By using flw cytmetry n lymphcytes, it is demnstrated that fish with higher lymphcyte cunts had higher survival rates whether r nt they were revaccinated. This culd be a future tl t determine immune respnses t vaccinatin. Participants List As nted abve, the wrkshp ccurred ver tw days. There were 57 registered attendees and six drp-ins fr a ttal f 63 participants n day ne. On the secnd day there were 50 peple participating in the Breakut Grups and discussins. The list f participants and their affiliatins appears in Appendix Fur. Summary f Discussins The questins asked at the end f each presentatin (if time allwed) are listed, with answers, in Appendix Tw: Presentatins. The mrning f Day Tw f the wrkshp was dedicated t Breakut Grup Discussin and fllwed the frmat f questins psed t participants. Each grup had a variety f participants frm varius sectrs f prductin, administratin, service industry, regulatry and fish health. Facilitatrs frm the DFO, Industry and fish health were asked t facilitate and use a flip chart (r ther) t capture discussin pints. The names f grup leaders and participants appear in Appendix Fur. Given the subject matter and after a recap f the previus day, participants were asked t address the tpics cited belw: Page 11 f 40

13 Synpsis f Grup Discussins There was cnsensus amng the grups that we d nt knw if Tenacibaculum spp. are the causative agents f the disease presentatin in BC. It is unclear if we are dealing with the same species r anther species and whether r nt there are different sertypes. The questin f whether r nt tenacibaculsis is caused directly r indirectly by Tenacibaculum spp. r if it is an pprtunistic infectin. T date, the ability t culture BC Tenacibaculum spp. has been difficult and is hampered by a methd f abslute identificatin. Until the bacterium can be cultured successfully and repeatedly we have nly bichemical methds t identify the bacterium preliminarily. Cnventinal PCR has been successful here, but the primers used in Eurpe are nt the same cmpared t primers used in Japan that have been successful in BC. Hwever it is unclear if we have a variant f Tenacibaculum r a new species. Only sequencing f the genme frm culture preparatins with assistance frm Genme BC will allw us t determine if we have the same pathgen as identified elsewhere. The next step in this prcedure is t replicate the cnditins f the disease in fish held under captive cnditins. Currently the nly wet lab with this capacity is the DFO Pacific Bilgical Statin (PBS). It is pssible that if we are able t successfully grw the bacterium we culd demnstrate a methd f infectin and transmissin. These studies wuld g a lng way t develping mitigatin and preventin strategies. Once an infectin mdel is established, the gateway t understanding the pathgen is pen and interactin with the hst. Anther cmmn theme in the discussin grups was the understanding f the envirnment in which the fish are becming infected. Examinatin f fish health data is difficult because recrds are nt cmplete when lking fr specific utbreaks; tenacibaculsis is nt a reprtable disease and is difficult t diagnse clinically. A thrugh data dive f envirnmental recrds may r may nt prvide insight, but future infrmatin cllectin frm a brad spectrum f sites frm diverse gegraphic lcatins culd be intrduced t help better understand why sme sites are mre prne t utbreaks than thers. This is a cmplex multivariate analysis t be undertaken by an expert. In the shrt term, the structure f envirnmental mnitring prgrammes can be develped t help define if envirnmental factrs can be ruled ut. After pathgen and envirnment cmes hst. Smlt quality culd be a factr in susceptibility t infectin and relate t the transmissibility f the infectin. Bth lab and field studies wuld be invlved t examine smlt fitness and mnitring thrugh the critical stages f infectin and expressin. Page 12 f 40

14 Recmmendatins Recmmendatin 1: Identificatin f the pathgen It was clear frm the presentatins that there are different species in the Pacific waters than in Eurpean. Identificatin f the causative agent will be necessary and fundamental t any future wrk. A suite f identificatin tls stemming frm islatin, culture, serlgy, bichemical and mlecular technique develpment will be essential. By sequencing the genme, cmparisns f knwn sequences can be undertaken t identify virulence factrs, strain differences and susceptibility t treatment. The first step is t islate and culture pure lines. Recmmendatin 2: Develp methds f culture and diagnsis f the pathgen and determine the rle f agents in the disease presentatin Once the pathgen has been identified, culture f the pathgen will lead t streamlined methds f detectin. There is cncern that there is an interactin between Tenacibaculum spp. and M. viscsa that is at play with winter ulcers; T. maritimum is present n the margins f the ulcer and M. viscsa is present within the ulcer. Understanding the rles f the pathgens in winter ulcer utbreaks is imprtant and this can nly be determined by identifying the rles each play in the presentatin f the infectin. A cst-effective methd f culturing and specifically detecting bth Tenacibaculum spp. and M. viscsa is imprtant. Recmmendatin 3: Determine the rle f envirnmental factrs in the expressin f the disease The cnsensus f the grup discussins was that variability in the smlting envirnment can affect the nset and severity f the disease. Accurate envirnmental data exists fr sites affected that culd be cupled with incident reprts. This may lead t insight as t preferred smlting sites r ther mitigatin methds. It was recmmended that envirnmental and fish health data be examined with the view t determining if the infrmatin is sufficient t use as a predictive index fr mitigatin plans. Recmmendatin 4: Investigate the epidemilgy f the disease There was discussin within the wrkshp where researchers indicated that seawater tank r net pen bifilms culd be the reservir fr Tenacibaculum spp. There was als sme evidence presented where the spread f the disease culd be seen in a pattern as the utbreak prgressed. It wuld be extremely valuable t prducers t understand the patterns, causes, and effects f health and disease cnditins in cultured fish ppulatins. Recmmendatin 5: Develp additinal wet lab capacity The wet lab capacity fr study is limited t the federal facility at the Pacific Bilgical Statin. There was verwhelming supprt t increase capacity fr a lcal r reginal facility that can address the needs expressed in the wrkshp t augment the research and develpment at the Statin. It is recmmended that effrts be made t develp additinal wet lab capacity lcally. Recmmendatin 6: Investigate mitigatin and treatment strategies This recmmendatin will fllw the identificatin f the pathgen, culture and detectin methds. In the interim, small-scale, farm-level investigatins that lk int the hst, pathgen and envirnment can be undertaken by peratrs while the larger scale prjects are underway. Drug efficacy wrk is needed t determine the use f prphylactic treatments, dsage levels and alternative therapies. Vaccine develpment is unlikely due t the nerus regulatry csts while the use f feed supplements t bst immune respnse r ptentiate prir vaccinatin effrts shuld be investigated. Page 13 f 40

15 Recmmendatin 7: Develp user awareness and educatin This wrkshp was a watershed event in raising the awareness f the scpe and magnitude f an emerging cncern. If ceanic cnditins cntinue, there will be a cntinued need fr cmmunicatin and awareness amng stakehlders, regulatrs and the public. It is recmmended t establish a regular reprting mechanism, utreach capacity and cntinued wrkshps. Develpment Pririties Develpmental pririties are derived frm the questin perids f the first day f the wrkshp and frm grup discussins n the secnd day. 1. Characterizatin and identificatin f the pathgen (species, sertype) This develpment is universally seen as the first pririty. The issue is allcating respnsibility and funding pprtunity. Capacity exists t ptimize culture techniques if the supprt is identified and the identificatin can then mve frward. 2. Develp a validated assay fr lcal strains Subsequent t the characterizatin f the sequences is the develpment f PCR techniques fr specific detectin f the putative Pacific species r strain f Tenacibaculum spp. In rder t develp a specific and validated assay, the crrect primers amplifying the specific target genes f interest shuld be identified. Once accmplished, further develpment will flw. 3. Challenge studies (culture and ID and wet lab) It is clear that additinal wet lab space needs t be develped lcally. With the capacity t run challenge studies cmes the ability t validate diagnstic assays, study hst/pathgen interactin and develp mitigatin strategy as well as vaccine develpment. The facilities at the Pacific Bilgical Statin are running at capacity and new lcal facilities accessible by additinal researchers, diagnsticians, the prducers and their veterinarians are needed presently. 4. Case presentatin (yellw rt, winter ulcer) Further wrk is required by fish health staff t bserve and recrd the incidence f utbreaks and the cllectin and crdinatin f data by all prducers. It was suggested that the BCSFA fish health database be adapted t include infrmatin that culd be retrieved fr analysis. A risk and envirnmental factr analysis wuld stem frm the database infrmatin. 5. Epidemilgical studies The glbal BC picture f the patterns, causes and effects f tenacibaculsis needs t be determined. This wrk can begin right away. Epidemilgical studies will give insight and directin t additinal wrk as well as direct mitigatin strategy. Subsequent infrmatin frm cncmitant studies can be fed int the mdel fr further insight. 6. Mitigatin and vaccine develpment Vaccine develpment is an nerus and lengthy task and has a defined rad map f prgressin t apprval. In the interim, ther mitigatin strategies need t be develped. By incrprating physilgical infrmatin abut the hst (bld chlride, ATPase, crtisl, immuncmpetency), the envirnment (physical characteristics f salinity, temperature, turbidity), and husbandry cnditins (transprt, smelting site, timing, prphylactic treatment), mitigatin strategies can be develped t limit lsses until a vaccine can be develped. Page 14 f 40

16 7. Virulence factrs With the exceptin f a few studies, virulence factrs(s) f tenacibaculsis are relatively unknwn. It is apparent that there may be different species f bacteria causing tenacibaculsis in different parts f the wrld. It is als clear that different sertypes f Tenacibaculum spp. are present in different areas. It will be imprtant t identify these differences t help determine the best apprach t preventin and treatment f tenacibaculsis. 8. Chemtherapy There are few antibitics available t prducers t treat diseases. It is prpitius t use chemical treatments in a judicius manner t prevent resistance and limit envirnmental and fd safety impacts. Frm the ecnmic perspective it is imprtant that the use f antibitics be used in prprtin t effective treatment t limit lsses t mrtality and grwth in additin t the cst f the drug. Rbust testing f the available prducts t determine drug f chice, dse and duratin will be required. Page 15 f 40

17 Appendix One: Agenda Page 16 f 40

18 Page 17 f 40

19 Appendix Tw: Summary f Presentatins TM Wrkshp Summary Please nte: the fllwing is ntatin frm transcripts f the presentatins; spelling and grammatical errrs may be present. 9:00-9:10 Jim Pwell (BC CAHS) Intrductin General prgramme verview and husekeeping 9:10-10:00 Alicia Estévez Tranz (U. f Santiag Cmpstela USC; Spain) - Tenacibaculsis f farmed fish in Suthern Eurpe Attempts t culture A salmn in NW Spain Galicia (1975+) Inland Culture (indr and utdr tanks) Gilthead seabream (lwer TM) than the ther fish (turbt, sle, sea bass, salmn) Main Ten species recvered frm marine fish cultures in suthern Eurpe T. maritimum, T Sleae, T Disclr, T dicentrarchi, T gallacium In sme cases a mixed f TM and A Sal. was diagnsed The systemic infectin f V anguillarum causes an inhibitin in the grwth f TM which difficult in its recvery frm internal rgans Presumptive diagnsis: micrscpic examinatin f abundant lng, thin, rd-shaped bacteria win wet munts r Gram preps btained frm skin lesins and gills. Cultured in ligthrphic media elabrated with SW (30-100% strength SW) Marine Agar vs FMM MA rapid grwth and mre density, can be cnfused with ther bacterium FMM slw grwth and lw density, useful t recg the TM Clnies All strains prduce cytchrme xidase and catalase The phentypic char des nt allw the diff f TM frm the ther T species assciated t fish diseases. PCR MUCUS ideal nn destructive sample fr a sensitive detectin by PCR in carrier fish (up t 100 cels/ml) Rapid ID by prtemic (MALDI-TOF) frm clnies (can be separates frm ther species) Antigenically hetergeneus species (3 sertypes described in marine fish: there are nt a strict hst specificity Seabream and sea bass sertype O1 is predminant Page 18 f 40

20 Turbt O2 Sle, O1 y O3 (O2 is increasing) Salmn O3 is recent althugh O2 was predminant Dt Blt analysis (simple and accurate methd fr sertyping) All strains have the same surface prtein pattern (regardless f the O sertype) Marine Agar (nt useful t distinguish TM sertypes) FMM des thugh. Gentyping studies RAPD-PCR analysis strains can be sep int distinct gentypes that are strngly assc with the sergrups described. MLSA (cncatenated analysis f 11 lci frm 73 strains revealed that Statistically significant assciatin between gentypes and inf related t the hst fish Same bacterial lineages can be fund in distinct hsts in the same gegraphic area An endemic clnizatin f fish farms by lcal strains Virulence and challenge Mdel in turbt 18 hurs t induce the disease Bacteria can be recvered frm external lesins and kidney Symptmatic fish surviving expsure, the bacteria can be nly recvered frm mucus Survival experiments Sterile SW TM viable and culturable fr 160 days+ Natural cnditins 5 days+ SW wuld nt be an imprtant rute fr the TM Risk factrs f disease High temp, lw water quality, Virulence assciated factrs Adherence: (capsular material) Qurum-sensing system: cell-cell cmmunicatin system regulates the expressin f genes invlved in virulence factrs such as the exenzyme prductin Page 19 f 40

21 Secretin f enzymes: extracelluar enzymes (prteases and chnditinase) High affinity irn-uptake mechanisms (2 systems) Synthesis f siderphres Utilizatin f heme grups as irn surces by direct binding Vaccinatin studies Cmmercial vaccine fr turbt (als plivalent vaccine available) Autvaccines (sle) Strategy recmmended: bath immunizatin at 1-2g fllwed by an IP bster at 20-30g N vaccines develped fr seabream and sea bass Cntrl (Chemtherapy) Enrflxacin (rapid resistance detected) and flrfenicl (best) Prphaylatic treatments H2O2 increases stress in the fish 240ppm can be a general preventative methd fr treating surface f tanks befre the intr f the fish. FW Baths Decrease f salinity t 5ppt fr 24 hurs can be a useful strategy Final remarks Several gaps Rte f transmissin, survival strategies and reservirs Genetic basis f the virulence mechanism Establishment f adequate vaccinatin prgrams fr ecnmically imprtant marine fish (seabass and salmn) Knwledge f the predminant sertypes affecting A salmn in the different cuntries Need t talk abut the same species as they vary amng cuntries Questins t be at the end f the day (write them dwn) 10:00-10:50 Øyvind Brevik (Cermaq AS Nrway) - The impact f tenacibaculsis in Nrway. Cermaq wned by Mitsubishi crp (4400 emplyees) Page 20 f 40

22 Cermaq Nrway: 27 licenses (Finnmark) & 22 licenses (Nrland) Fish Health R&D (Bergen) : cases f skin ulcers during prductin in SW annually Nt mandatry t reprt ulcers, hwever is included in the annual FH reprt. Mrtalities (includes all mrtalities frm any cause) reprted annually Lsses (due t ulcers) increase as yu g further nrth Reprted lsses vs lcatin reprted lsses vs date fr sea transfer generatin, cunties and # f mnths in SW (middle r beginning mre likely fr lsses) first time reprted in 2010 in Nrway (apprx. 20 cases) paper published in 2011 (page 15 f PPT) recmmended. Pst smlt case First cnfirmed case in 2011 Full SW in tanks n land Water inlet 20 and 80m g Outbreak always in Feb 5% mrtality Measures: Antibitics, New UV system, imprved aeratin f inlet water, reduced stress during handling Sea transfer case Spring and autumn sea transfers Within weeks after sea transfer Starts with ne netpen and spreads t whle site Starts in the muth area and prgresses (finrt and ulcers n the side) Measures: Stamp ut whle netpens, antibitic treatment Page 21 f 40

23 Causes: smlt quality, hydrza, algae Larger fish case Autumn and winter Starts in ne r tw netpens spreads t several netpens High ecnmic impact (value f fish and expensive treatment) Ulcers n muth and sides f fish Measures: Reduce infective pressure, antibitics Diagnsis islatin MA and BA is used Q PCR assay HK genes Des nt detect ther ulcer assciated bacteria Detects 0,35 cells (Tb-rpO) Mucus r Skin is a gd sample t check M viscsa q PCR Main cause is TM rather than MV (cmparisn graph) Use f antibitics Flrfenicl (1 treatment), mrtality is reduced with immediate effect Shuld all netpens be treated? Nt using mre antibitics than needed. Outbreak and treatment Mrtality at 3% = treatment (at apprx. week 38) Future aspects Trend shws increased use f antibitics Vaccines as an ptin?? Pse a challenge fr semiclsed systems Summary Nrthern Nrway and lwer temperatures 4-6 Page 22 f 40

24 Cause infectin pressure and external trigger Occurs in all stages f sea prductin T Sp predminant in Nrway Questins t be at the end f the day (write them dwn) 10:50-11:10 Break 11:10-12:00 Sverre Bang Småge (Cermaq AS Nrway) - Nvel TM species in Nrway detectin and identificatin Winter ulcer disease SW temp under 8 Celsius Main causative agent is Mritella viscsa Vaccine is develped targeting M viscsa T spp. assciated with winter ulcers in Nrway Cmmn factrs: handling and panic reactins fllwing sea transfer at lw temp 2013: prject clarify rle f t spp. In causing winter ulcers (challenge study) Mrtalities fr the shedders (graph) Bacteria re-islated frm ulcers, n M. Viscsa detected Pathlgical bservatins n Shedder Brwn circle small ulcer severe scale lss larger ulcers erded/split jaw (yellw pigment n head regin) severe fin rt ulcers n flanks and abdmen (yellw ring f pigment) Chabitant infectin (44 days) Islate Tsp 1 Mst distantly related t T dicentrarchi f the Nrwegian islates Gentypic study Inferred phylgenic tree based n 16s rrna and a cncatenated sequence f HK genes Antimicrbial drug sensitivity test Shws that mst sensitive t flrfenicl Page 23 f 40

25 Hydrzan medusa (jellyfish) Swarms present befre utbreak f Tenacibaculsis Sea Lice & Herring & Cleanerfish TM islated Summary 2 islates are primary pathgens that are able t cuase ulcers in a salmn Tsp 1 islate is a pssible new species in the genus Tm Questin perid: 1. Armin Alicia What media are yu using? FFM 2. Ahmed Alicia have yu sequenced (gentype) between the different sertypes? a. Saw different pathgens, gentypes and the sertypes b. Any sequence differences between the sertypes? 3. Simn Jnes Oyvind presented data n Q PCR cmparisn T and shwed that it was cnsistently higher did yu see a range in the develpment f lesins? Early vs late stage f infectin /lesin develpment a. Signs n the fish were mre r less the same (have nt started mnetary, have gne in the transmissin trial) water was sterilized by UV. The presence f MV is very very lw. b. Whether there was evidence that ne predispses frm the ther r d yu have an existing cnditin? i. They shw that M V present, then yu have the whle disease ccurring. Can have a start up with M V. Is that as well, but als TM within the utbreaks as well. (ne screening) 1.5 years s far. (large and small fish) Same picture all ver the line. Yes if yu d it early, a lt f wrk as well. 4. Alicia Oyvind Des the vaccine nt wrk? a. Vaccine is nt perfect is a very cmplex picture. If yu get rid f the TM, yu will reduce the winter ulcers t a lw percentage. Almst always used. b. I think that the numbers t that yu see n the annual reprts, yu get a very selective diagnsis n the fish. In very lw mnths cmpared t thse Tenac. Depends n the lcatin. Ulcers, are a prminent kind f thing. Sme ulcers, handling, nets, starts and spreads nt due t mechanical. 5. Pst smlt sites water sterilizatin cntrl Page 24 f 40

26 a. Cleaning the tanks was a big part f it. Acrylic, slippery, reducing a lt f the bifilm, did nt check fr TM, a lt less bifilm (when painted the inside) b. Flw thru system (recirc likely bigger prblem) Need very strict bisecurity fr the facility. If bacteria is in the system, yu have t treat. c. What d yu use t mnitr the bifilm? We didn t check the bifilm. ATPase? We did nt measure the amunt. d. Did yu find spts ver gd mnitring pints? i. Surface area see a lt f build up just belw the water line. e. In between crps d yu have a bifilm management prgram? i. N we dn t have that prblem anymre. ii. Remving excess feces and feed and s n. f. Next crp negative? i. Cannt pinpint what we did right. 6. Mark Sverre Cmment abut the shifting mrphlgy f the bacteria and shifting pathgenicity? Smlts get a small clny speculated that there is highly pathgenic activity ging n? Did yu see any differences in the cultures? a. Degenerative state, lk cmpletely different as they disintegrate. Very strange. We did a crss sectin and culd see thse small packets sticking up - enzymes pssibly? b. Algae (Hetersigma) seems t shift in its ability t cause death as the fish matures. c. Alicia nt related t the pathgenicity. Bacteria have very few. 7. Jim P muth lesins by day 7. Flrfenicl is very effective. Hw d yu get the feed int the fish nce yu get that diagnsis? a. Oyvind - Thse already ulcered are lst causes. Just treating the nes that dn t have symptms yet. It s nt the best way t d it. Need t test ut in wetlabs hw t treat the fish will help a lt n hw we administer tday. 8. Gegraphic lcatins (tempral) did yu ntice anything t d with salinity? Tasmanian slutin tarp. a. Oyvind we have nt lked int that part. We knw that when yu d transmissin trials. (lw 30) tend t lse the capability. Recirculatin systems usually arund and yu still get prblems. Mst f ur sites are high depending n the time f year. 9. Armin in WA, we dn t see skin lesins. Nrmally we have sites (harvested) and thers that are in the range f and is still arund. Just the gills. Page 25 f 40

27 10. Mark physilgical questin gills why des this bacteria have trpism fr the gums, scales and nt the gills? a. We see them in the gills smetimes t, mats f bacteria sitting in between. Smetimes can have massive clinical signs (hypxic) with little bacteria as well. There culd be a cuple f things happening with that. We dn t see in different areas. b. Cllagen lving bacteria (desn t grw well n bld agar) (hydrzan) Jellyfish is a pssible reservir f infectin. (fd surce) 11. Stewart J Sverre When did yu first start t see infectins? 12:00-1:00 Lunch a. Difficult t see. Lesins after 7 days. Yellw pigment bifilm (scale lss) b. Fish shedding and envirnmental cntributin. When did yu first identify an infected netpen, hw many days? (17? 40? Or faster?) i. Challenge we were unable t see befre the fish get ii. Lng dses lw dses and lw expsure time. If yu did a challenge trial, Q PCR Psitive. The wetlab nly rented fr a while. Time is limited fr challenge trial (50 days). We thinkt that it is very temperature dependent. After sea transfer is very quick. Yu can get high mrtalities within 7 days. Clinical signs and starts spreading 1 week t 2 weeks. iii. Sample the water, Q PCR the water. T get the answer. If yu try filtratin, did nt wrk. iv. (Armin) 1995 Califrnia (herring gt ulcers) bacteria lks like it spread frm herring t salmn farm. Eaten by the salmn and then ldged in the gills and spread. 1:00-1:45 Tim Hewisn & Michael Ness (Grieg Seafd BC Ltd.) - Tenacibaculum maritimum A Fish Farm Perspective Currently 1 f 20 members f the genus Tenacibaculum Cnfirmatin Bichemicals- PCR-DNA sequencing Etilgy, Prgressin & Treatment Graph Mrtality vs Weight vs Days fllwing input (previus) Current difference is that the initial spike and a secnd spike (400g) Effect f smltificatin (bld chlride) difference between 160mm/l vs 150 mm/l and 140mm/l Effect f salinity Page 26 f 40

28 High salinity, spikes in mrtality (ver a 6 mnth perid) suddent drp in salinity can plateau. Envir effect and timing Highest mrtality 11% unprecedented temperature at entry was the variable that may have played a large rle in mrtalities Yearly TM mrtality % by input is getting wrse (arund 1%) 2015 (6%) Daily retrieval in rder t id. Fish health nsite Treatment 6 day in feed treatment, effects within 3 days f treatment start. Number 1 anitbitic use ccurs in the first 2 ths after smlt entry Varies 0 3 treatments Respnse t treatment Spike drives us t treat, drp ff after a few days, Effect f treatment n feeding = reduced feeding nsite. Treatment txicity Accumulative mrtality vs daily mrtality cunt Several feedings a day with nn-med feed in between t tne dwn the aggressive feeders. Scial license TM nly reasn fish are treated presently Currently 2-3 treatments per site ASC are required t have a limited number f treatments Financial Impact 69K annually Lst prfit 2% = 879K Lst annual prfit frm lst grwth 703K Annual Cst $ Dwngrades nt included in financial lss $$ Page 27 f 40

29 The cmpany that develps a cst-effective TM vaccine will be ur preferred vaccine supplier. Questins & Answers Stewart J have yu nticed that there is any relatinship between the frequency f fallw time vs the magnitude f the prblem at that site (majr surces?) TM clnize at sites. Tim H 9 gd pint) we try t d a cuple f fallws at each site. Kathleen) We have had muthrt n sites that have nt been used in 2-3 years. Tim H In 2013 and 2014, we d n average a larger smlts ging ut t sea still seeing mrtalities. Dusan (Cermaq) Flrefenical? Winter ulcers? Mike N -Trend is increasing seen mre in the last few years. Smlt mrtality at a given site (cage t cage/randm ccurences?) related t lcal flw cnditins? Gd questins- we have nt lked int. Rapid nset f mrtality first few years, then lks pretty randm and is usually after an entry. Carl underwater cameras? Yes, Carl pssible that the fish rub against it and it pens up fr surce f infectin? Time f entry if yu stagger, wuld that help? Mre assciated with time f entry. Sea transfer is nt the case. We tend t see that it starts in ne net pen and then spreads ut. Takes a cuple f weeks befre yu see it in ther netpens. 14 days and then the prblem has spread thrughut. 1:45-2:30 Ahmed Siah (BC CAHS) - Clinical methds f detectin fr T. maritimum Flexibacter maritimus Histry 1977 in Japan flunder and seabream Taxnmy GyrB and 16s rrna Page 28 f 40

30 Clinical Signs - Kent, 1992 Middle infectin (yellw pigment arund muth and yellw ndules Severe cmplete face rt Islatin Need % SW, Celsius Seawater cytphaga medium vs marine agar FMM culture = best media ph 7.2 t 7.4 Detectin Serlgical Flurescent Antibdy technique PCR (1996) Nested PCR mucus best fr detectin FFM Brth and Agar and Passage #2, FFM Agar and Passage #3 At 400 bp was cnfirmed (Bader & Shtts, 1998) Real Time PCR Whle genme sequencing NBRC (3 millin base pair) nw available Will be easier t find ut the virulence factr, target genes fr diagnstic, mlecular mechanisms, relatin-hst pathgen Next generatin f sequencing Questins: Simn Jnes sertype and hst preference (link) virulence - d yu think there is an pprtunity t better understand sertyping? Nt ur specialty at BC CAHS t d sertyping. Alicia is the expert, perhaps will pen cllabratins. Maybe gentyping if I may talk abut that. Based n what we heard tday, I think there is sme differences in the genetic material f the different strains. Frmt eh virulent aspect I have n idea. Gd avenue t investigate. Stewart what is the structure f the genme? Page 29 f 40

31 N plasmids in the ne they sequenced. Any evidence that plasmids exist? Ahmed saw a paper that culture withut plasmids. Alicia des nt detect plasmids. Simn J On f the mre recent develpments in flav bacterium in FW, phage therapy use f them t cntrl the infectin? Are yu aware f any wrk in the phages in the use f this? Sverre phages as causing the transprt and are difficult t culture. Stewart J. Aermnas has 7 plasmids. Pieces f virulence assciated with plasmid lss. Circular DNA. (Genme is circulative.) 2:30-2:40 Break 2:40-3:30 Jaime Tbar (Centrvet, Chile) - Current situatin f Tenacibaculum in Chile and ptential preventin strategies Histry f Virbac-Centrvet TM in Chile A Salmn and rainbw trut Bacteria targets fish at 2 stages 1 mnth after SW transfer Adults (>1.5 kg) Hmgeneus in Chile Fund in SW and Brackish Fund at different ranges f temperatures (high adaptatin capacity) Trigger factrs Stress, lw water quality, salinity variatins, immunsuppressin Treatment Usually OTC Fish can recver, (sme fish remain suppressed as they d nt take the meds) Impact in prductin Lss f FCR Early mrtality Page 30 f 40

32 External lesins = specific markets N significant damage n the fillet Vaccine strategy 2 vaccine perids required (as 2 peaks n unvaccinated fish). small fish (80 200g) and Big fish (1.5+) Mdel SRS immunity/immune respnse Vaccinated nce= first peak is reduced but secnd peak is nt fully reduced. Lack f specific antibdies due t vaccinatin. Vaccinated twice = If a bster is given, then secnd peak is decreased. Can regain and have better prtectin. Prblems with an injectable bster Risk f infectin, csts, Oral bster vercmes injectin-related side effects, chance t apply bster, csts Prblems with ral vaccines Needs t pass thru the stmach Shrt intestine Needs t be safe Micrmatrix technlgy Bacteria are encapsulated with a matrix which prtects them frm acidic ph. Antigen, immunstimulant Cmparisn injectable vs ral vaccinatin 300 days vs 600 days (ral is as gd as injectable) Immunlgical Fllw up 1202 cmplete prductin cycles (gd set f data) bsters (Atlantic salmn) Alternative tls fr evaluating immune respnse Lymphcytes Flw cytmetry as tl fr discriminating cell types Page 31 f 40

33 Methd published in 2004 highly reprducible methd Culd this be useful? Lk fr crrelates. Fish that survived had a higher lymphcyte cunt (vaccinated r nt) Cmments Analysis t determine the rle f different factrs (species statinality, densities Questins Cmparisn f the cst f vaccine vs antibitic treatment? Mrtality will reduce - Vaccine pays several times as mre fish are saved. Chile resistant strains. Bacteria is s resistant that the antibitic des nthing if the farmer ges with antibitic treatment. Is there lss f grwth with the bster? N Stewart what wuld be the challenges f prducing a vaccine that is ging t be pssibly harder? Challenge- T develp gd media, identify the key antigens necessary fr prtectin. In terms f the vaccinatin grups, there are many presentatins. We knw that Injectable is much bigger systemic immunity. But still the Oral bster is needed and necessary. Ahmed Muthrt the fish are lsing appetite. Can the bster still be used? We need t fllw immune respnse crrelate it t estimate. its anther challenge. Simn d yu think there is merit in the applicatin f functinal feeds, designed t prmte immunstimulatin (nn-specifically) beneficial in the case f TM? There are sme functinal feeds that wrk but needs t be validated case by casein terms f what elements they are treating. (persnal pinin) Significantly reduce the level f the utbreak by empwering the innate immune respnse. If yu apply functinal feed at the right time, there shuld be sme effect. Oyvind we treat with antibitics and vaccines. Culd yu speculate n the effects f thse antibdies (SRS), increase f cases f TM, what wuld yu think wuld happen if we didn t have SRS and we used a lt f antibitics n fish? I tend t believe that sme f thse cases are hidden by SRS and als the treatment by SRS. SRS sure it is pssible t develp a resistance as lng as yu use the antibitic. Antibdies SRS are nt secreted. That is gd. There is n surveillance prgram. When yu see TM, yu apply antibitic. If the antibitic wn t wrk Page 32 f 40