Technology offer. Safe and effective Salmonella vaccines for poultry

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1 Tchnology offr Saf and ffctiv Salmonlla vaccins for poultry Attnuatd vaccins basd on Salmonlla dltion mutant strains (with dfctiv multi drug rsistanc (MDR) fflux pump systms) Targt markt and valu Th two major zoonotic Salmonlla srotyps ar Entritidis and Typhimurium, of which th first is typically associatd with layrs and ggs, whil both srotyps ar colonizing broilrs. Bcaus of trad issus of ggs and mat whn flocks ar contaminatd, ths infctions can rsult in svr conomical losss (in addition to halth car costs for humans). Anothr srotyp that is conomically important worldwid is Salmonlla Gallinarum. This srotyp causs fowl typhoid, a svr spticamic disas in layrs that is still a major problm in th middl East, Asia (including China), South Amrica and Africa (and still occasionally in th EU and US). It can induc high mortality in both chicks and adult hns, with mortality rats abov 50%. Salmonlla vaccins hav bn dvlopd, but ths ar mostly strains carrying undfind mutations. As an xampl, th dominant commrcial Salmonlla Entritidis and Typhimurium vaccins usd in th EU ar Avipro VacE and VacT (and th combind Vac DUO) ar so-calld mtabolic drift mutants. Anothr xampl is th dominant Salmonlla Gallinarum vaccin usd worldwid, SG9R. In countris with a low prvalnc of fowl typhoid th us of this vaccin strain has causd som outbraks du to rvrsion of th vaccin strain to a virulnt phnotyp (Van Immrsl t al., 2013). This clarly highlights th importanc of using wll dfind gn dltion mutants as vaccins in th futur instad of undfind strains with unknown mutations. With our tchnology w can targt th following markt sgmnts: Food safty : o Vaccination of layrs to rduc contamination of ggs with zoonotic Salmonlla srotyps Entritidis o Colonization inhibition to rduc contamination of mat with zoonotic Salmonlla srotyps Entritidis and Typhimurium Animal halth: vaccination of layrs to protct thm against fowl typhoid Salmonlla Gallinarum gnrating hug conomic losss du to high mortality. Whil Salmonlla Gallinarum is a major problm in rgions lik th middl East, Asia (including China), South Amrica and Africa (and still occasionally in th EU and US) th control of Salmonlla as a zoonotic pathogn in poultry is a global issu. Salmonllosis is on of th most prvalnt zoonotic disass in humans. In Europ alon ovr 100,000 cass ar rportd ach yar and in th Unitd Stats thr ar approximatly 40,000 cass rportd annually. Sinc many

2 Tchnology offr mildr cass ar not diagnosd or rportd, th actual numbr of infctions may b ovr thirty tims highr. Th most important srovars found ar Salmonlla Entritidis and Salmonlla Typhimurium, both of which ar oftn linkd back to poultry products. Exampls includ th 2010 massiv gg rcall in th US du to Salmonlla Entritidis-contaminatd ggs, and mor rcntly, th Salmonlla Hidlbrg contamination of ground turky mat in th US. Ths incidnts prov that Salmonlla control in poultry rmains important to guarant safr food production for consumrs. Th poultry industry and rgulatory bodis howvr do not rspond to it in th sam mannr vrywhr. In Wstrn as wll as xporting countris control masurs hav bn implmntd, somtims voluntary by th poultry industry, somtims forcd by a govrnmntal rgulation. Th importanc of controlling Salmonlla in poultry is gaining mor intrst and is mainly drivn by: Incrasd rgulation for th control of Salmonlla such as in th EU and US Incrasd xport rgulations and barrirs, making control of Salmonlla vital for broilr intgrators xporting broilr mat Incrasd branding of poultry mat and ggs maks that Salmonlla contamination can do grat damag to th brand and th company, affcting futur sals Human salmonllosis outbraks ar th triggr for strong rgulations and control masurs. Tchnology Our ar ky targts for th dvlopmnt of saf liv attnuatd vaccin strains ar th multi drug rsistanc fflux pump systms (MDR). Saf and ffctiv liv vaccins ar obtaind by th gnration of dltion mutants (MDR gns). Indd, ths dltion mutant strains cannot surviv in a harmful nvironmnt, such as littr, in tissus of th vaccinatd birds and in ggs cannot rvrt to virulnt phnotyps Th tchnology can b applid to svral Salmonlla srotyps, yilding liv attnuatd vaccins for diffrnt applications. Salmonlla Entritidis mutants as liv vaccins for laying hns to control gg contamination Salmonlla Entritidis/Typhimurium mutant combination vaccin to rduc shdding and colonization of Salmonlla in th broilr gut. This concpt of vaccinating broilrs is not basd on adaptiv immunity induction, but on a colonization-inhibition phnomnon. Attnuatd Salmonlla Gallinarum mutant vaccins with incrasd safty to control fowl typhoid

3 Tchnology offr Proof of concpt and dvlopmnt status PoC1: Prvntion of gg contamination by Salmonlla Entritidis aftr oral vaccination of laying hns with Salmonlla Entritidis mutants Two diffrnt groups (n=30) wr orally immunizd at day of birth, at 6 wks of ag and at 16 wks of ag through crop instillation of 0.5 ml containing 10 8 cfu of Salmonlla Entritidis 147 mutant 1 (singl dltion ; group 1) or Salmonlla Entritidis 147 mutant 2 (multipl dltion ; group 2). A third group of birds (n=30) was kpt as non-immunizd but Salmonlla challngd positiv controls (group 4). At 24 wks of ag, all th animals wr intravnously inoculatd in th wing vin with 0.5 ml containing 5 x 10 7 cfu of th Salmonlla Entritidis strain S1400/94. Tabl 1: Th prcntag of gg contnt batchs positiv for th challng strain during th two wks following infction. Rsults btwn brackts show th prcntag of batchs positiv aftr nrichmnt in ttrathionat brilliant grn broth (37 C, ovrnight). Diffrnt suprscripts indicat significant diffrncs btwn th groups (p<0.05). Group Wk 1 Wk 2 Non-vaccinatd 70 a (74 a ) 0(17) a singl dltion 0 c (0) c 0(0) c multipl dltion 0 c (0) c 0(0) c singl dltion multipl dltion Figur 1: Th prcntag of Salmonlla positiv sampls in spln (A), caca (B), oviduct (C) and ovary (D) in non-vaccinatd and vaccinatd animals, aftr nrichmnt. Statistical

4 Tchnology offr significant diffrncs (p<0.05) in prcntag of positiv organ sampls btwn vaccinatd groups and th non-vaccinatd control group ar markd with an astrix. PoC2: Salmonlla Entritidis and Salmonlla Typhimurium dltion mutant strains ar saf for us in practic and protct broilrs against Salmonlla infction Forty on-day-old chicks wr dividd into 4 groups of 10. Two groups wr givn 0.5 ml of a mixtur containing 2 x 10 8 CFU/ml of th Salmonlla Entritidis mutant strain and 2 x 10 8 CFU/ml of th Salmonlla Typhimurium mutant strain by oral gavag on day 1 of th xprimnt. Th two othr groups wr givn 0.5 ml of Hank s Balancd Salt Solution by oral gavag as a control on day on of th xprimnt. On day two of th xprimnt, on control group and on group tratd with th mixtur wr givn 0.5 ml of a solution containing 2 x 10 5 CFU/ml of th Salmonlla Entritidis 76Sa88 nal R challng strain by oral gavag, whil th othr two groups wr challngd by administring 0.5 ml of a solution containing 2 x 10 5 CFU/ml of th Salmonlla Typhimurium MB2136 strpt R challng strain by oral gavag. To valuat colonization by th challng strains, thir numbrs in cacum and spln wr dtrmind at day 7 of th xprimnt. Shdding of th challng strains was valuatd by bactriological analysis of cloacal swabs takn on days 3 and 7. Figur 2: Cacal (A & C) and spln (B & D) colonization by Salmonlla Entritidis (A & B) or Salmonlla Typhimurium (C & D) wild-typ strains on day 7 of ag aftr xprimntal infction of broilr chickns tratd with a CI cultur. Th valus shown rprsnt log10 of th CFU/g sampl. Th horizontal lins rprsnt th man, th rror bars rprsnt th standard rror of man (SEM). Th numbr of sampls quals 10 in all groups.

5 Tchnology offr PoC3: Safty of a Salmonlla Gallinarum mutant strains as a potntial liv vaccin strain for us in laying hns Forty on-day-old chicks wr dividd into 2 groups of 20. On day 35 of ag, on group was givn 1 ml of a mixtur containing 10 6 CFU/ml of th Salmonlla Gallinarum SG9R strain by oral gavag as a control, whil th othr group was givn 1 ml of a solution containing 10 6 CFU/ml of th Salmonlla Gallinarum mutant strain. Aftr inoculation, th wight of chickns was dtrmind daily. Thr was no diffrnc in wight btwn th group tratd with th SG9R strain and th group tratd with th Salmonlla Gallinarum mutant strain, indicating that th Salmonlla Gallinarum mutant strain dos not caus a growth dprssion whn compard to th SG9R strain that is currntly frquntly usd in practic. mutant Figur 3: Avrag wight of 20 laying hns orally inoculatd with ithr 10 6 CFU of a SG9R strain or 10 6 CFU of a Salmonlla Gallinarum mutant strain.

6 Tchnology offr IP-position A patnt application has bn fild and is currntly pnding: application numbr EP Partnring W ar looking for partnrs that ar intrstd to us th tchnology for th dvlopmnt and markting of commrcial vaccins and w ar opn to assist during th transfr of th tchnology and during th dvlopmnt phas. W can offr a challng modl for Salmonlla (broilrs and layrs ; Salmonlla Entritidis, Typhimurium and Gallinarum) diagnostics for monitoring Salmonlla on farm and in infctd animals (isolation, quantification) diagnostics to valuat immun rsponss to vaccination/infction Rfrncs Van Immrsl F, Mthnr U, Rychlik I, Nagy B, Vlg P, Martin G, t al. Vaccination and arly protction against non-host-spcific Salmonlla srotyps in poultry: xploitation of innat immunity and microbial activity. Epidmiol Infct 2005;133: Dsin TS, Kostr W, Pottr AA. Salmonlla vaccins in poultry: past, prsnt and futur. Exprt Rv Vaccins 2013;12: Van Immrsl F, Studholm DJ, Eckhaut V, Hyndrickx M, Dwulf J, Dwal I, t al. Salmonlla Gallinarum fild isolats from laying hns ar rlatd to th vaccin strain SG9R. Vaccin 2013;31: Contact Svn ARNOUTS Svn.Arnouts@UGnt.b