Salmonella infection in nursery piglets and its role in the spread of salmonellosis to further production periods

pa hogens A icle Salmonella In ec ion in Nu se y Pigle s and I s Role in he Sp ead o Salmonellosis o Fu he P oduc ion Pe iods Ma ía Be nad-Roche 1, Alejand o Casano a-Higes 2, Cla a M. Ma ín-Alcalá2, Albe o Cebollada-Solanas 3,4 and Raúl C. Maina -Jaime 1,*   Ci a ion: Be nad-Roche, M.; Casano a-Higes, A.; Ma ín-Alcalá, C.M; Cebollada-Solanas, A.; Maina -Jaime, R.C. Salmonella In ec ion in Nu se y Pigle s and I s Role in he Sp ead o Salmonellosis o Fu he P oduc ion Pe iods. Pa hogens 2021,10, 123. h ps:// doi.o g/10.3390/pa hogens10020123 Academic Edi o s: Joana Mou ão and Pa ícia An unes Recei ed: 10 Decembe 2020 Accep ed: 21 Janua y 2021 Published: 25 Janua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Depa amen o de Pa ología Animal, Facul ad de Ve e ina ia, Ins i u o Ag oalimen a io de A agón-IA2, Uni e sidad de Za agoza-CITA, 50013 Za agoza, Spain; mbe nad [email p o ec ed] 2 Unidad de P oducción y Sanidad Animal, Cen o de In es igación y Tecnología Ag oalimen a ia de A agón, Ins i u o Ag oalimen a io de A agón-IA2, Uni e sidad de Za agoza-CITA, 50059 Za agoza, Spain; [email p o ec ed] (A.C.-H.); cma in@uniza .es (C.M.M.-A.) 3 G upo de Gené ica de Micobac e ias, Depa amen o de Mic obiología, Medicina P e en i a y Salud Pública, Uni e sidad de Za agoza, 50009 Za agoza, Spain; albe o@uniza .es 4Unidad de Biocompu ación, Ins i u o A agonés de Ciencias de la Salud (IACS/IIS A agón), Cen o de In es igación Biomédica de A agón (CIBA), 50009 Za agoza, Spain *Co espondence: cmaina @uniza .es; Tel.: +34-976762088 Abs ac : Few s udies ha e ocused on assessing Salmonella in ec ion in he nu se y and i s ole in u he pig p oduc ion pe iods. Mesen e ic lymph nodes, in es inal con en , and mea juice om 389 6-week-old male pigle s in ended o human consump ion om i e b eeding a ms and 191 pooled loo ecal samples om gil de elopmen uni s (GDU) om he same a ms we e analyzed o es ima e and cha ac e ize (by pulsed- ield gel elec opho esis and an imic obial esis ance analyses) Salmonella in ec ion. The p e alence o in ec ion and shedding among pigle s was 36.5% and 37.3%, espec i ely, shedding being signi ican ly associa ed wi h in ec ion (Odds Ra io = 12.7; CI 7.3–22.0). Salmonella Rissen; S. 4,[5],12:i:-; and S. De by we e he mos common se o ypes. A low le el o Salmonella-speci ic ma e nal an ibodies a he beginning o he nu se y pe iod sugges ed i was a pe iod o high isk o in ec ion. Resis ance o 3 d- and 4 h-gene a ion cephalospo ins was de ec ed in pigle isola es al hough he pigle s ne e ecei ed an ibio ics, indica ing hey could be ec o s o an imic obial esis ance. The same Salmonella clones we e de ec ed in pigle and GDU isola es, sugges ing ha in ec ed pigle s play a signi ican ole in he in ec ion o gil s and consequen ly o inishing pigs in he case o p oduc ion a ms. The con ol o Salmonella in ec ion in nu se y pigle s may dec ease he isk o aba oi and ca cass con amina ion. Keywo ds: nu se y pigle s; p e alence; Salmonella; swine; shedding; PFGE; zoonoses 1. In oduc ion Salmonella in ec ion is qui e common in pigs in he Eu opean Union [ 1 ]. I usually cou ses asymp oma ically, bu in ec ed pigs may shed he bac e ium h ough hei eces, making hem a majo isk ac o o ca cass con amina ion a slaugh e [ 2 ]. Since he p esence o Salmonella du ing he g owing– inishing phase is di ec ly ela ed o ca cass con amina ion a aba oi s, main mi iga ion measu es ha e been usually di ec ed owa d his p oduc ion pe iod [ 3 – 6 ]. Howe e , ew s udies ha e ocused on he pe iods p e ious o his one, such as he nu se y, al hough ac i e Salmonella in ec ions ha e been iden i ied in pig nu se ies [7]. The so-called nu se y is a pe iod ha comp ises he ime om weaning a 3–4 weeks o age o app oxima ely 10 weeks o age (jus be o e en e ing he g owing uni ). This is a c i ical p oduc ion phase in which pigle s a e e y suscep ible o a a ie y o en e ic in ec ions. A common consequence o weaning is he modi ica ion o he pigle s’ in es inal mic obio a, cha ac e ized by a signi ican educ ion in he numbe o lac obacilli [ 8 ], mos ly Pa hogens 2021,10, 123. h ps://doi.o g/10.3390/pa hogens10020123 h ps://www.mdpi.com/jou nal/pa hogens Pa hogens 2021,10, 123 2 o 14 due o sudden changes in hei die , which goes om mos ly liquid (sow milk) du ing lac a ion o a solid-based die (p es a e eed) a he beginning o he nu se y, and en i- onmen [ 9 ]. These changes, along wi h he decay o ma e nal an ibodies [ 10 ], he only immune p o ec ion a his age, and he usual animal s ess linked o he pigle ’s sepa a ion om i s dam and i s commingling wi h new pigle s, make weaned pigle s highly p one o G am-nega i e bac e ial in ec ions, such as Esche ichia coli and Salmonella spp. [11]. While E. coli in ec ion has been widely s udied and is con i med as a p e alen en e ic pa hogen a his age [ 12 ], he p e alence o Salmonella in ec ion has been ba ely s ud- ied. Howe e , i is accep ed ha weaning- o pos -weaning-age pigs would be among he mos clinically a ec ed had hey become in ec ed by Salmonella [ 2 ]. Bo h a e closely ela ed bac e ia ha a e suscep ible o he same class o an ibio ics, so he ea men s agains colibacillosis may be hinde ing Salmonella in ec ions a his age. Indeed, un il e- cen ly, he use o an imic obials as p ophylac ics, mos ly aminoglycosides and polymyxins ( colis in ), was a common p ac ice in in ensi e pig husband y sys ems, pa icula ly du ing he nu se y pe iod [ 13 , 14 ]. In Spain, colis in was commonly adminis e ed, o as long as 15 days, as an in- eed an imic obial o yea s, due o i s high e icacy agains G am-nega i e bac e ia [15]. In a ecen s udy, we obse ed ha Salmonella p e alence in suckling pigle s om se oposi i e b eeding a ms was high, con i ming ha appa en ly heal hy 4-week-old (wo) pigle s may ac as ca ie s o in ec ion a leas un il weaning [ 16 ]. Conside ing he likely high p opo ion o Salmonella-in ec ed pigle s a weaning, i is likely ha he nu se y will be hea ily in ec ed by his pa hogen as well. The e o e, i seems logical o hink ha du ing he pas ew yea s, Salmonella in ec ion has been o e looked du ing he nu se y pe iod due o p e en i e an imic obial ea men s. The ala ming inc ease in an imic obial esis ance igge ed he Eu opean heal h au- ho i ies o se up new Eu opean Union egula ions on he use o colis in in e e ina y medicine, and since 2015, colis in has been banned o use as a p ophylac ic [ 14 ]. These new egula ions may a o an inc easing incidence o salmonellosis in he nu se y. Howe e , o he bes o he au ho s’ knowledge, no ield s udies on Salmonella p e alence du ing he nu se y pe iod ha e been ca ied ou so a . Thus, in his s udy, we i s assess he p e alence o Salmonella in ec ion a he beginning o he nu se y pe iod, i.e., wo weeks a e weaning, in a pigle popula ion ha came om a g oup o Salmonella-se oposi i e b eeding a ms. Fu he , we cha ac e ize by pulsed- ield gel elec opho esis (PFGE) and an imic obial suscep ibili y analyses he Salmonella isola es ob ained and compa e hem o hose isola ed om gil de elopmen uni s (GDU) om he same he ds. Resul s om his s udy may help o shed some ligh on he ole ha Salmonella in ec ion in nu se y pigle s may play in subsequen p oduc ion pe iods, such as g owing/ inishing, and he e o e in aba oi and ca cass con amina ion. 2. Resul s 2.1. Salmonella Isola ion, Se o yping, and Se ology in Pigle s A o al o 389 weaned pigle s we e sampled om Salmonella-se oposi i e b eeding a ms (an a e age o 78 pigle s pe a m). Pigle s we e sampled in all seasons (35.7% in win e , 23.4% in sp ing, 22.4% in summe , and 18.5% in au umn). Table 1shows he dis i- bu ion o he sampling by a m and he co esponding p e alence o in ec ion (Salmonella- posi i e in mesen e ic lymph nodes) and shedding (Salmonella-posi i e in in es inal con en ). The p e alence o in ec ion a ied signi ican ly be ween a ms, anging om 17.5% ( a m E) o 59.2% ( a m D), wi h an a e age o 36.5% (95% CI 31.9–41.4). The p e alence o shedding pigle s also a ied signi ican ly be ween a ms, wi h a simila mean alue (37.3%; 95% CI 32.6–42.2). Pa hogens 2021,10, 123 3 o 14 Table 1. Resul s o Salmonella isola ion * om in es inal con en (IC) and mesen e ic lymph nodes (MLN) in 6-week-old nu se y pigle s. Fa m No. o Pigle s No. o MLN-Posi i e (%) No. o IC-Posi i e (%) No. o IC- and MLN-Posi i e (%) ** A 96 39 (40.6) 47 (48.9) 31 (56.4) B 75 34 (45.3) 26 (34.7) 20 (50.0) C 89 26 (29.2) 34 (38.2) 22 (57.9) D 49 29 (59.2) 22 (44.9) 20 (64.5) E 80 14 (17.5) 16 (20.0) 11 (57.9) To al 389 142 (36.5) 145 (37.3) 104 (56.8) * In e na ional O ganiza ion o S anda diza ion (ISO) 6579:2002/A1:2007. ** Pe cen age es ima ed om posi i e (ei he IC o MLN) pigle s. A median o 14.4 g (95% CI 13.96–15.00) o MLN was collec ed. No signi ican di - e ence was obse ed be ween he weigh o MLN-posi i e and MLN-nega i e samples (median o 15.0 and 14.1 g, espec i ely; p= 0.31). All Salmonella isola es (145 om IC samples and 142 om MLN samples) we e se o yped. The dis ibu ion o Salmonella se o ypes by a m and ype o sample is shown in Table 2.Salmonella Rissen was he mos equen se o ype (42.8%) eco e ed om IC samples, ollowed by he monophasic a ian o S. Typhimu ium (S. 4,[5],12:i:-) (40.0%), and S. De by (4.8%). A simila dis ibu ion o se o ypes was obse ed in MLN-posi i e samples, wi h S. 4,[5],12:i:- (40.8%) and S. Rissen (31.0%) being he mos p e alen , ollowed by S. B andenbu g (10.6%). Salmonella 4,[5],12:i:- was he only se o ype p esen in all a ms. Table 2. Dis ibu ion o Salmonella se o ypes in 6-week-old nu se y pigle s and in gil de elopmen uni s (GDU) among he 5 a ms. Fa m Pigle Isola es No. o Pigle s wi h he Same Se o ype in MLN–IC GDU Isola es IC MLN Se o ype No. (%) Se o ype No. (%) Se o ype No. (%) A Rissen 25 (53.2) Rissen 19 (48.7) 17 Rissen 11 (78.6) 4,[5],12:i:- 15 (31.9) 4,[5],12:i:- 17 (43.6) 9 Ana um 2 (14.3) De by 4 (8.5) B andenbu g 1 (2.6) 4,[5],12:i:- 1 (7.1) Kapemba 2 (4.3) Goldcoas 1 (2.6) Typhimu ium 1 (2.1) London 1 (2.6) B Rissen 12 (46.2) Rissen 8 (23.5) 5 Rissen 4 (66.6) 4,[5],12:i:- 5 (19.2) 4,[5],12:i:- 8 (23.5) 3 B andenbu g 1 (16.7) Goldcoas 5 (19.2) Goldcoas 3 (8.8) 3 4,[5],12:i:- 1 (16.7) B andenbu g 3 (11.5) B andenbu g 13 (38.2) 3 De by 1 (3.8) De by 2 (5.9) 1 C Rissen 23 (67.6) Rissen 13 (50) 12 Rissen 10 (83.3) 4,[5],12:i:- 7 (20.6) 4,[5],12:i:- 6 (23.1) 2 De by 2 (16.7) De by 2 (5.9) De by 5 (19.2) 1 Ana um 1 (2.9) Ana um 1 (3.8) 1 Kedougou 1 (2.9) Typhimu ium 1 (3.8) D 4,[5],12:i:- 19 (86.3) 4,[5],12:i:- 18 (62.1) 15 De by 7 (70.0) Goldcoas 1 (4.5) Goldcoas 9 (31.0) 1 Rissen 3 (30.0) B andenbu g 1 (4.5) B andenbu g 1 (3.4) 1 Ohio 1 (4.5) Ohio 1 (3.4) 1 E 4,[5],12:i:- 12 (75.0) 4,[5],12:i:- 9 (64.3) 8 Rissen 8 (88.9) Rissen 2 (12.5) Rissen 4 (28.6) 2 4,[5],12:i:- 1 (11.1) Ana um 1 (6.3) Ana um 1 (7.1) 1 Agona 1 (6.3) To al 145 142 86 51 Pa hogens 2021,10, 123 4 o 14 Salmonella was no de ec ed in 206 (52.9%) o he sampled pigle s, while posi i e esul s in bo h MLN and IC samples we e ob ained o 104 (26.7%) o hem. The same se o ype was de ec ed in 86 (82.7%) o he animals wi h posi i e MLN and IC cul u es (Table 2). A signi ican associa ion be ween he isola ion o Salmonella in MLN and IC samples was obse ed: an MLN-posi i e pigle had a ound 12 imes highe odds o shedding Salmonella han an MLN-nega i e pigle (OR = 12.7; CI 7.3–22.0; p< 0.001) once he season and a m e ec s we e accoun ed o (Table 3). Table 3. Associa ion be ween Salmonella shedding and Salmonella in ec ion in 6-week-old nu se y pigle s by mixed logis ic eg ession analysis *. No. o Pigle s No. (%) o IC-Posi i e Pigle s Logis ic Reg ession Pa ame e s OR 95% CI (OR) p MLN Nega i e 1247 41 (16.6) 1 - - Posi i e 142 104 (73.2) 12.71 7.33–22.05 <0.001 Season Win e 1139 33 (23.7) 1 - - Sp ing 91 26 (28.6) 1.17 0.55–2.51 0.672 Summe 87 56 (64.4) 2.69 1.31–5.55 0.007 Au umn 72 30 (41.7) 2.53 1.22–5.26 0.013 * Fa m used as g ouping ac o . 1Re e ence ca ego y. IC: in es inal con en ; MLN: mesen e ic lymph nodes; OR: odds a io. Rega ding se ological esul s, he median op ical densi y pe cen age (OD%) alue o he 389 sampled animals was 4.7 (95% CI 3.0–6.0). No signi ican di e ences we e obse ed be ween MLN-nega i e and MLN-posi i e pigle s (median o 4.8 and 4.7, espec i ely; p= 0.5 ). No signi ican di e ences we e obse ed ei he in OD% alues be ween IC- nega i e and IC-posi i e pigle s (median o 5.2 and 4.0, espec i ely; p= 0.1). 2.2. Salmonella Isola ion, Se o yping, and Se ology in Gil De elopmen Uni s (GDU) A o al o 191 pooled loo ecal samples om 5 GDU we e collec ed. Salmonella was isola ed om 51 o hem (26.7%; 95% CI 20.9–33.4), bu he p opo ion o posi i e samples in he g owing uni s a ied among a ms, anging om 17.5% o 43.8% (Table 4). The mos equen se o ype was S. Rissen (70.6%), which was p esen in all a ms (A, B, C, D, and E), ollowed by S. De by (17.6%; a ms C and D) and S. 4,[5],12:i:- (5.9%; a ms A, B, and E). In all he a ms, excep a m D, he se o ypes ound in he GDU we e also de ec ed in he nu se y pigle s (Table 2). Thus, he same se o ypes, i.e., S. 4,[5],12:i:- and S. Rissen in a m A; S. 4,[5],12:i:-, S. Rissen, and S. B andenbu g in a m B; S. Rissen and S. De by in a m C; and S. 4,[5],12:i:- and S. Rissen in a m E, we e obse ed in pigle s and loo samples om gil uni s (Table 2). Fou hund ed and wen y-one gil se um samples we e a ailable om ou a ms (A, B, C, and E; Table 4). The mean se op e alence was 24.2% (95% CI 20.4–28.5). Among gil uni s, se op e alence a ied sligh ly ( om a minimum o 19.3% in a m E o a maximum o 31.9% in a m A). Table 4. Resul s o Salmonella isola ion * in loo ecal samples om gil de elopmen uni s and Salmonella se op e alence ** in gil s om he 5 a ms. Fa m No. o Floo Fecal Samples No. o Posi i e Floo Fecal Samples (%) * No. o Se um Samples No. o Se oposi i e Samples (%) ** A 32 14 (43.8) 91 29 (31.9) B 24 6 (25.0) 90 25 (27.8) C 32 12 (37.5) 90 19 (21.1) D 57 10 (17.5) NA - E 46 9 (19.6) 150 29 (19.3) To al 191 51 (26.7) 421 102 (24.2) * ISO 6579:2002/A1:2007. ** Conside ing a cu o alue o OD% ≥ 40% (He dcheck Swine Salmonella ELISA es , IDEXX Labo a o ies, USA); NA: no a ailable. Pa hogens 2021,10, 123 5 o 14 2.3. Pulsed-Field Gel Elec opho esis (PFGE) PFGE analysis was pe o med when he same Salmonella se o ype was de ec ed in pigle s and loo ecal samples om gil uni s om he same a m. Thus, a o al o 37 Salmonella isola es om IC-posi i e pigle s (15 om a m A, 9 om a ms B and C, and 4 om a m E) and 17 om he GDU (5 om a m A, 2 om a m B, 6 om a m C, and 4 om a m E) we e submi ed o PFGE analysis. Isola es o S. B andenbu g om he gil uni o a m B we e no a ailable. PFGE analysis o hese 54 Salmonella isola es showed 12 di e en XbaI pa e ns (based on a simila i y cu o o ≥ 90%) (Figu e 1). Obse ed PFGE clus e s ma ched well wi h se o ypes and an imic obial esis ance (AMR) p o iles. When analyzed by se o ype, six main clus e s we e obse ed o S. 4,[5],12:i:-, ou o S. Rissen, and wo o S. De by. Figu e 1. Dend og am showing he main XbaI pulsed- ield gel elec opho esis (PFGE) pa e ns ( ≥ 90% homology) o 54 Salmonella s ains isola ed om pigle s’ in es inal con en and GDU om 4 a ms (A, B, C, and E). A, aminopenicillins; C, phenicols; S, aminoglycosides; Su, sul onamides; T, e acyclines; Na, quinolones; C , 3 d- o 4 h-gene a ion cephalospo ins. Pa hogens 2021,10, 123 6 o 14 Salmonella isola es om he GDU we e g ouped in o six di e en PFGE pa e ns, and wi hin i e o hem isola es om pigle s om he co esponding a m we e included (pa e ns 1, 7, 8, 9, and 11; Figu e 1). O e all, 75.7% o he pigle isola es analyzed we e included wi hin hese i e clus e s. A leas one gene ic ela ionship be ween Salmonella isola es om pigle ecal samples and loo ecal samples om he co esponding GDU was de ec ed in all ou a ms. In he case o isola es om pigle s, gene ically simila se o ypes we e de ec ed on se e al occasions wi hin he same a m and some imes mo e han 150 days apa ( i.e., S. 4,[5],12:i:- and S. Rissen in a m A, S. Rissen in a m B, and S. Rissen and S. De by in a m C). When looking a pai s o homologous Salmonella isola es coming om pigle s and GDU, isola es o i e o hese pai s we e collec ed wi hin a sho (<1 mon h) pe iod o ime, namely one S. Rissen pai om a m A, one S. 4,[5],12:i:- om a m B, one S. Rissen and one S. De by om a m C, and one S. Rissen om a m E. In ano he i e pai s, he pigle isola es we e collec ed a be o e (>150 days be o e) he gil samples (one S. Rissen om a m A, one S. 4,[5],12:i:- om a m B, one S. Rissen and one S. De by om a m C, and one S. Rissen om a m E). In wo mo e (one S. Rissen om a m B and one om a m C), he pigle isola es we e collec ed a a e gil samples had been ob ained (Figu e 1). 2.4. An imic obial Resis ance All 54 isola es submi ed o PFGE we e also es ed o AMR agains 17 an imic obial agen s, as desc ibed below. All bu wo S. De by displayed mul id ug esis ance (MDR), bo h isola ed om a m C (Table 5). A o al o 11 mul i-AMR p o iles we e de ec ed, he mos common being ACST (n= 17 31.5%) and ACSSuT (n= 15, 27.8%), bo h o hem mos ly associa ed wi h S. Rissen and de ec ed in all he a ms (Figu e 1). The mos com- mon pheno ypic esis ance was agains lo enicol (92.6%), e acycline (90.7%), ampicillin (83.3%), and ime hop im-sul ame hoxazole (42.6%). Table 5. An imic obial esis ance (AMR) pa e ns ound in he 54 Salmonella isola es om 6-week-old nu se y pigle s and gil de elopmen uni s. AMR Family Pa e n * No. o S ains Se o ypes In ol ed (No. o S ains) Fa m ACS 1 4,[5],12:i:- E ACSSu 1 Rissen A ACSSuT 15 Rissen (13), De by (2) A, B, C, E ACSSuTC 1 Rissen C ACSSuTC Na 5 Rissen A, B, C ACST 17 4,[5],12:i:- (10), Rissen (7) A, B, C, E ACSTC 1 Rissen A AST 4 4,[5],12:i:- A, E CS 2 De by C CSC 1 Rissen E CSSuT 1 Rissen B CST 5 Rissen (4), 4,[5],12:i:- (1) A, B, E * A, aminopenicillins; C, phenicols; S, aminoglycosides; Su, sul onamides; T, e acyclines; Na, quinolones; C , 3 d- o 4 h-gene a ion cephalospo ins. Rega ding esis ance o an imic obials o c i ical impo ance o humans, no AMR agains ca bapenems (imipenem) o polymyxins (colis in) was de ec ed in any o he isola es analyzed. Howe e , AMR agains cephalospo ins o 3 d (ce io u ) and 4 h (ce quinome) gene a ions was de ec ed in 13.0% (7/54) and 5.6% (3/54) o he isola es, espec i ely. All o hem we e S. Rissen. 3. Discussion To p ope ly assess he ue p e alence o Salmonella in ec ion in nu se y uni s a a pig a m is qui e challenging and expensi e, as i equi es he killing o a la ge numbe o young animals (be ween 4 and 10 weeks o age) ha a e usually in ended o o he pu poses, Pa hogens 2021,10, 123 7 o 14 i.e., ei he o slaugh e a ma ke age (5–6 mon hs old) o as eplacemen animals. Fo his s udy, pigle s came om i e b eeding a ms whe e emale weaned pigle s we e aised as e- s ocking gil s o o he pig p oduc ion a ms, while male pigle s we e a ened up o 7–9 kg li e weigh and hen slaugh e ed o human consump ion. These male pigle s we e weaned when hey we e 4 wo and ed o wo mo e weeks, un il slaugh e ing. This managemen allowed us o analyze MLN, IC, and mea juice samples in o de o ob ain a good assessmen o he ue Salmonella incidence s a us o hese pigle s. In addi ion, hese pigle s came om Salmonella-se oposi i e b eeding he ds [ 16 ] and had no been ea ed wi h an ibio ics, as hey we e o be slaugh e ed. Thus, his app oach could somewha e lec wha may be happening in nu se y pigle s om Salmonella-se oposi i e p oduc ion he ds. The o e all pe cen age o Salmonella-in ec ed (MLN-posi i e) pigle s was 36.5% (95% CI 31.9–41.4) bu was a iable ac oss pig a ms ( om 17.5% o 59.2%). A simila p opo ion o pigle s (37.3%; 95% CI 32.6–42.2) could be conside ed Salmonella shedde s as he bac e ium was ound in hei IC (Table 1). Few ield s udies ha e been ca ied ou on Salmonella p e alence a his s age, and when pe o med h ough he sampling o li e pigle s, esul s sugges ed ac i e in ec ion bu he eco e y le els o ecal Salmonella we e usually much lowe [ 7 , 17 ]. Two majo issues would help o unde s and he di e ences be ween his and p e ious s udies. Fi s , we analyzed 25 g o ecal con en , he amoun equi ed acco ding o he ISO 6579:2002/A1:2007 s anda d and signi ican ly la ge han ha collec ed h ough swabbing. Second, his s udy only included pigle s om b eeding a ms wi h high le els o Salmonella se oposi i i y. A good ma ch was also ound be ween animal in ec ion and shedding a his age, wi h he odds o shedding Salmonella being mo e han 12 imes highe o an MLN-posi i e pigle compa ed o an MLN-nega i e one (Table 3). This ela ionship was u he suppo ed by he de ec ion o he same Salmonella se o ype on MLN and IC in mos (82.7%) o he pigle s ha had been posi i e in bo h ypes o samples. Shedding he bac e ium h ough eces is he main mechanism o ansmission o Salmonella and is common when animals a e in ec ed o he i s ime o when in ec ed animals su e episodes o s ess, such as commingling o anspo [ 11 ]. Gi en he age o hese pigle s, hey had p obably been in ec ed e y ecen ly, besides su e ing such s ess ac o s as hey had been sen o slaugh e . In addi ion, an unknown numbe o new in ec ions may ha e also occu ed du ing anspo and/o lai age [ 18 , 19 ]. Al oge he , his would help o explain he high le el o shedding among hese pigle s. F om hese esul s, i can be de i ed ha mos o he pos -weaning pigs ha become in ec ed wi h Salmonella a e also asymp oma ic, and in con as o wha was expec ed [ 2 ], i appea s ha when compa ed o olde pigle s, hey would no be mo e clinically a ec ed. Thus, salmonellosis may be easily o e looked du ing he nu se y pe iod as well, ega dless o he use o an imic obials. In any case, his high p e alence o in ec ion and shedding was no a su p ising esul , since simila indings we e obse ed in a p e ious s udy on weaned (4-wo) pigs om he same a ms [16]. No di e ences we e ound ega ding se ological OD% alues be ween in ec ed and non-in ec ed pigle s (median OD% o 4.7 and 4.8, espec i ely), hese alues being simila o hose ound in pigle s o he same age in o he s udies [ 20 ]. Howe e , on a e age, OD% alues we e much lowe han hose ound in he p e ious s udy on 4-wo pigle s om he same a ms (15.9%; [ 16 ]), e idencing he likely signi ican decay o ma e nally de i ed IgGs agains Salmonella wi hin he wo weeks a e weaning [ 7 ]. I seems ha he beginning o he nu se y may be a pe iod o high suscep ibili y o he in ec ion and ha measu es o p e en exposu e o Salmonella should be encou aged a his ime. One o he aims o his s udy was o de e mine whe he Salmonella in ec ion om nu se y uni s will be able o each u he p oduc ion phases, ha is, he g owing uni s. Fo ha pu pose, loo ecal samples om he GDU, om each o co esponding b eeding a ms unde s udy, we e collec ed in o de o de ec Salmonella. All he gil uni s we e Salmonella posi i e, wi h an o e all p opo ion o posi i e samples o 26.7% (Table 4). This igu e would be wi hin he expec ed ange o Salmonella-posi i e ecal samples o g owing/ inishing pig uni s in Spain [ 1 , 21 ] bu p obably unde es ima es he ue p opo ion Pa hogens 2021,10, 123 8 o 14 o posi i e samples, gi en he ela i e low sensi i i y o he bac e iological cul u e when pe o med on ecal samples om asymp oma ic animals [5]. The main se o ypes de ec ed in hese gil uni s we e S. Rissen, ollowed by S. De by and S. 4,[5],12:i:-. The h ee o hem we e also he mos common se o ypes de ec ed in he pigle s, sugges ing ha hese se o ypes we e ci cula ing be ween bo h p oduc ion pe i- ods e en when he GDU we e loca ed kilome e s away om hei co esponding nu s- e ies (i.e., a m E) . To u he con i m his hypo hesis, PFGE analysis was pe o med on Salmonella isola es when he same se o ype was ound in pigle s and gil uni s om a gi en a m. PFGE analysis showed ha mos o he Salmonella isola es om pigle s we e g ouped wi h gil isola es, indica ing a high le el o gene ic simila i y ( ≥ 90%) be ween hem. Despi e he small numbe o isola es analyzed, his ma ch was ound a leas once in all ou a ms whe e hese isola es we e eco e ed and i was de ec ed o he h ee se o ypes, suppo ing he main enance o Salmonella in ec ion be ween he nu se y and he g owing uni and he ole ha pigle s may play in i . Wi hin a gi en nu se y uni , he same clone o Salmonella could be de ec ed o a long pe iod o ime (>150 days apa ) in ol ing di e en ba ches o pigle s. Besides, no pa icula empo al pa e n o in ec ion be ween pigle s and gil uni s was de ec ed ei he (a gi en Salmonella isola e could be ound a he same ime o i s in nu se y pigle s and la e in he gil uni s, o ice e sa). These esul s sugges ed ha c oss-con amina ion be ween uni s wi hin a ms and/o a lack o p ope pen hygiene a e majo issues in hese a ms, highligh ing he di icul ies o elimina ing Salmonella om he a m en i onmen despi e high in e nal hygiene and biosecu i y s anda ds. These esul s would also help o explain, in pa , he o igin o he Salmonella in ec ion in he GDU and he u he Salmonella in ec ion in sow he ds [22]. I is wo h no ing ha S. De by appea s as one o he mos equen se o ypes ound in b eeding a ms (~30%) [ 23 ] and is commonly ound h oughou he pig p oduc ion py amid, which would suppo he hypo hesis ha his se o ype is easily ansmi ed by he ans e o animals be ween uni s o he ds [ 24 ]. Wi h ega d o S. Rissen and S. 4,[5],12:i:-, bo h we e also epo ed in he Eu opean su ey men ioned abo e and bo h ha e expe ienced a wo ldwide expansion in he pas ew decades [ 25 , 26 ]. In a ecen epo om he Spanish Minis y o Ag icul u e, Fish and Food, he monophasic a ian s o S. Typhimu ium and S. Rissen we e he mos p e alen se o ypes ound in a na ional su ey ca ied ou on slaugh e ed pigs in 2019 (35.4% and 23.8%, espec i ely), ollowed by S. De by (13.1%) [ 27 ]. Bo h S. De by and S. 4,[5],12:i:- a e among he main pig- ela ed se o ypes associa ed wi h human salmonellosis in he EU in he pas ew yea s [ 28 – 32 ], while S. Rissen is conside ed a signi ican cause o oodbo ne salmonellosis in Asia and sou he n Eu opean coun ies [ 26 , 33 ]. To he bes o he au ho s’ knowledge, he e a e no epo s so a on he main Salmonella se o ypes associa ed wi h con amina ion o pigle mea o showing possible links be ween consump ion o pigle mea and human salmonellosis. Howe e , conside ing hese esul s, heal h au ho i ies should be awa e o he po en ial isk o con amina ion o pigle ca casses due o he a i al o in ec ed pigle s a he aba oi . Nine y-eigh pe cen o he Salmonella isola es displayed mul id ug- esis an pheno- ypes, wi h mos o hem showing esis ance p o iles commonly epo ed in Salmonella (ACSSu, ACSSuT, ACST, e c.). No AMR was ound agains an ibio ic classes conside ed o c i ical impo ance o humans, such as ca bapenems and polymyxins [ 34 ], bu a signi i- can p opo ion o isola es (12.9%) p esen ed esis ance o 3 d-gene a ion cephalospo ins and e en o ce quinome (5.5%), a 4 h-gene a ion cephalospo in. This ype o esis ance, de ec ed only in S. Rissen isola es, was much mo e p e alen han ha epo ed in pigs o he EU (1.1%) [ 35 ]. The wo ldwide eme gence o esis ance o ex ended-spec um cephalospo ins in non- yphoidal Salmonella is al eady a ma e o conce n [ 36 ], pa icula ly when ha bo ed by Salmonella se o ypes o po en ial zoono ic cha ac e [ 37 ]. In his case, mos o he esis an isola es (75%) we e ound in pigle s in ended o human consump ion. Thus, hey could be conside ed po en ial ec o s o he ansmission o his esis ance o humans, al hough hey ne e ecei ed an ibio ics. Pa hogens 2021,10, 123 9 o 14 In summa y, nu se y pigs can become subclinically in ec ed and ac as ac i e ca ie s o Salmonella in a a m, as he same Salmonella clones we e obse ed in pigle s and in he GDU. This inding sugges s ha pigle s play a signi ican ole in he in ec ion o inishing pigs in p oduc ion a ms as well. The e o e, Salmonella in ec ion in nu se y pigle s should be conside ed a po en ial isk ac o o aba oi con amina ion, and he con ol o Salmonella in ec ion a his s age may help o dec ease he isk o ca cass con amina ion. Conside ing ha a signi ican sou ce o Salmonella o nu se y pens would be he sows [ 16 ], sows would hen be a signi ican sou ce o Salmonella in ec ion o gil s and inishing pigs [ 38 , 39 ]. Indeed, a ecen isk assessmen model adap ed speci ically o Spain showed ha sow p e alence is a s ong indica o o slaugh e pig p e alence [ 40 ]. Ou esul s suppo hei s. De ining p ope s a egies ha p e en Salmonella shedding om sows should be a majo goal in any p oduc ion/b eeding pig a m. Fac o s ha could be ela ed o Salmonella in ec ion/shedding in sows (i.e., d y/pelle eed, ine icien disin ec ion p o ocols, ype o loo , e c.) [ 39 , 41 , 42 ] should hen be a oided. The implemen a ion o eed s a egies (o ganic acids, essen ial oils, p ebio ics, e c.) o educe somehow he le el o in ec ion and shedding in he a ms [ 43 , 44 ], o e en he accina ion o sows [ 45 , 46 ], may also be ad isable. These s a egies could be implemen ed di ec ly in nu se y pigle s as well [ 47 , 48 ]. In addi ion, minimizing he use o an ibio ics in p oduc ion/b eeding a ms would appea as ano he goal o p e en u he sp ead o AMR o a ening pigs and gil s [26] and may educe Salmonella shedding as well [49]. 4. Ma e ials and Me hods 4.1. Fa m Selec ion The s udy was ca ied ou on i e mul iplie /supplie swine b eeding a ms (he d size be ween 700 and 940 sows) om he no heas pa o Spain (A, B, C, D, and E), whe e hal o he Spanish pig census is concen a ed. Fa ms A, B, and C belonged o one company and D and E o a di e en one. All hese a ms had shown Salmonella se op e alence ≥ 50%, and Salmonella was de ec ed, on a e age, in 22% o he sow ecal samples analyzed om hem [ 16 ]. The a ms we e chosen due o he a me s’ and co esponding e e ina ians’ willingness o pa icipa e in he s udy. The sampling was ca ied ou in wo pe iods, be ween Janua y 2012 and May 2013 ( a ms A, B, and C) and be ween Feb ua y 2015 and Feb ua y 2016 ( a ms D and E). These b eeding a ms keep emale weaned pigle s as e-s ocking gil s o o he pig p o- duc ion a ms. Howe e , male weaned pigle s a e usually sen o slaugh e o mea , ei he a weaning age (4 wo) o a e a pe iod o wo weeks in he nu se y (6 wo). The samples analyzed in his s udy belonged o 6-wo male pigle s. 4.2. Collec ion o Samples Pigle samplings we e ob ained a he aba oi . On a gi en day, whole in es inal packages om a ce ain numbe o animals om a gi en ba ch om a a m we e collec ed di ec ly a he slaugh e line. The e was no ou ine sampling schedule as he numbe o a ailable pigle s and sampling imes we e dependen upon bo h pigle a ailabili y om any o he i e selec ed a ms and aba oi eadiness o collabo a ion ha day. The in es inal packages we e hen submi ed o he labo a o y o immedia e p ocessing. The maximum- possible amoun o mesen e ic lymph nodes (MLN) and a minimum o 25 g o in es inal con en (IC) we e collec ed o bac e iological analysis. A piece o he diaph agm muscle was also collec ed o se ological analysis. Du ing he pe iod o pigle sampling, and e e y 3–4 mon hs, loo ecal samples we e also collec ed by a m e e ina ians om GDU om he co esponding a ms. In addi ion, se um samples om a ep esen a i e numbe o hese gil s we e a ailable o ha pe iod om he o icial e adica ion campaign agains Aujeszky’s disease. Fecal and se um samples we e use ul in iden i ying ci cula ing Salmonella se o ypes du ing he g owing pe iod and assessing he se ological s a us o he g owing gil s, espec i ely.