Factors associated with the introduction of mycobacterium avium spp. Paratuberculosis (map) into dairy herds in galicia (north-west Spain): The perception of experts

animals A icle Fac o s Associa ed wi h he In oduc ion o Mycobac e ium a ium spp. Pa a ube culosis (MAP) in o Dai y He ds in Galicia (No h-Wes Spain): The Pe cep ion o Expe s F ancisco Ja ie Villaamil 1, Edua do Yus 2, Bibiana Bena ides 3, Albe o Allepuz 4,5 , Sebas ián Jesús Moya 4, Jo di Casal 4,5 , Ca melo O ega 6and F ancisco Ja ie Diéguez 7,*   Ci a ion: Villaamil, F.J.; Yus, E.; Bena ides, B.; Allepuz, A.; Moya, S.J.; Casal, J.; O ega, C.; Diéguez, F.J. Fac o s Associa ed wi h he In oduc ion o Mycobac e ium a ium spp. Pa a ube culosis (MAP) in o Dai y He ds in Galicia (No h-Wes Spain): The Pe cep ion o Expe s. Animals 2021,11, 166. h ps:// doi.o g/10.3390/ani11010166 Recei ed: 2 Decembe 2020 Accep ed: 4 Janua y 2021 Published: 12 Janua y 2021 Publishe ’s No e: MDPI s ays neu- al wi h ega d o ju isdic ional clai- ms in published maps and ins i u io- nal a ilia ions. Copy igh : © 2021 by he au ho s. Li- censee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and con- di 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/). 1ADSG Cos a da Mo e, 15124 A Co uña, Spain; [email p o ec ed] 2Ins i u e o Food Analysis and Resea ch, San iago de Compos ela Uni e si y, 27002 Lugo, Spain; [email p o ec ed] 3Animal Heal h Depa men , Uni e sidad de Na iño, San Juan de Pas o 52001, Colombia; [email p o ec ed] 4Depa men o Heal h and Animal Ana omy, Au onomous Uni e si y o Ba celona, 08193 Ba celona, Spain; [email p o ec ed] (A.A.); [email p o ec ed] (S.J.M.); [email p o ec ed] (J.C.) 5Cen e de Rece ca en Sani a Animal (CReSA), UAB, IRTA, 08193 Ba celona, Spain 6Animal Pa hology Depa men , Ve e ina y Facul y, Za agoza Uni e si y, 50013 Za agoza, Spain; epidemio@uniza .es 7Ana omy, Animal P oduc ion and Clinical Ve e ina y Sciences Depa men , Ve e ina y Facul y, San iago de Compos ela Uni e si y, 27002 Lugo, Spain *Co espondence: anciscoja ie [email p o ec ed]; Tel.: +34-9828-22400 Simple Summa y: Pa a ube culosis emains one o he mos impo an diseases o ca le wo ldwide. Con ol o disease is di icul and o e s impo an challenges a bo h diagnos ic and managemen le els. This pape desc ibes a s udy aimed a quan i ica ion o expe opinion on isk ac o s o pa a ube culosis in ec ion in dai y he ds in Spain. Fo his pu pose, a panel o nine expe s wo king in he ield o pa a ube culosis was selec ed. Risk ac o s we e also included in o a ques ionnai e ha was esponded o by 93 a ms whose sani a y s a us was known. The mos impo an isk ac o s o he in oduc ion o MAP, acco ding o expe opinions, we e ela ed o pu chase and g azing p ac ices. The sco es ob ained o each a m, based on he expe opinions, allowed MAP posi i e/MAP nega i e a ms o be disc imina ed wi h 68.8% sensi i i y and 68.7% speci ici y. Despi e inc eased awa eness o he disease and he ac ha se e al coun ies a e implemen ing con ol p og ams, he e is s ill incomple e unde s anding o he epidemiology o he disease. This, oge he wi h he lack o comple ely eliable diagnos ic me hods, makes i o i al impo ance conside ing he in e -he d ansmission ac o s in o de o p e en he in oduc ion o he disease. P io i izing he mos impo an ac o s should be use ul o ocusing u u e aining ini ia i es and imp o ing isk- educ ion s a egies in his economically impo an indus y. Abs ac : This s udy aimed a quan i ying expe opinions on he isk ac o s in ol ed in Mycobac e ium a ium subsp. pa a ube culosis (MAP) in ec ion in dai y ca le he ds. Fo his pu - pose, po en ial isk ac o s associa ed wi h he in oduc ion o MAP in o dai ies we e chosen based on a li e a u e e iew and discussions wi h esea che s and e e ina ians. Fo each ac o , a decision ee was de eloped, and key ques ions we e included in each. Answe s o hese key ques ions led o di e en e en s wi hin each decision ee. An expe opinion wo kshop was o ganized ( ollowing he ecommenda ions o he OIE), and o dinal alues anging om 0 o 9 (i.e., a null o e y high likelihood o in ec ion) we e assigned o each e en . The po en ial isk ac o s we e also inco po a ed in o a s uc u ed ques ionnai e ha was esponded o by 93 a ms whe e he sani a y s a us agains MAP was known. The eby, based on he alues gi en by he expe s and he in o ma ion collec ed in he ques ionnai es, each a m was assigned a sco e based on hei MAP en y isk. F om hese sco es (con as a iable) and using a ROC cu e, he cu -o ha bes disc imina ed MAP-posi i e and -nega i e a ms was es ima ed. The mos impo an isk ac o s o he in oduc ion o MAP, acco ding o expe opinions, in ol ed pu chase and g azing p ac ices ela ed o animals unde six Animals 2021,11, 166. h ps://doi.o g/10.3390/ani11010166 h ps://www.mdpi.com/jou nal/animals Animals 2021,11, 166 2 o 19 mon hs o age. The sco es ob ained o each a m, also based on he expe opinions, allowed MAP posi i e/MAP nega i e a ms o be disc imina ed wi h 68.8% sensi i i y and 68.7% speci ici y. These da a should be use ul o ocusing u u e aining ini ia i es and imp o ing isk- educ ion s a egies in he dai y indus y. Keywo ds: dai y ca le; expe opinion; Johne’s disease; isk analysis; Spain 1. In oduc ion Mycobac e ium a ium subspecies pa a ube culosis (MAP) is he causa i e agen o pa a u- be culosis o Johne’s disease (JD), a ch onic g anuloma ous en e ic disease ha a ec s domes ic and wild uminan s; he disease seems o espond o an ibio ics as symp oms weaken bu ecu a e an ibio ics a e no longe adminis e ed. The o ganism is an acid- as , G am-posi i e and acul a i e in acellula pa hogen ha equi es i on o g ow h, depen- den on mycobac in, and he e o e is incapable o en i onmen al eplica ion [ 1 ]. Howe e , MAP is ex emely esis an and, in ideal condi ions, can su i e o up o a yea in he en i onmen [2]. This disease causes se ious economic losses in dai y a ming, mainly as a esul o educed milk yield [ 3 , 4 ], inc eased suscep ibili y o o he diseases, pa icula ly mamma y in ec ions [ 5 ], loss o bodyweigh [ 6 ] and consequen ial p ema u e culling [ 7 ]. Pa a ube - culosis has also been ela ed o educed e ili y a es [ 4 , 8 ]. In any case, and especially on a ms wi h a low p e alence, he economic impac may be di icul o he a me o app ecia e. MAP is also a long-suspec ed cause o C ohn’s disease in humans and a ecen ly p oposed cause o ulce a i e coli is [9]. The p ima y means o MAP ansmission is he ecal–o al ou e. The bac e ia a e usually in oduced in o he ds h ough he pu chase o in ec ed bu clinically inconspicuous ca le. In oduced animals may be subclinically in ec ed, shed he bac e ia and con amina e he en i onmen o se e al mon hs o e en yea s be o e clinical signs appea . O he ou es, such as he in oduc ion o con amina ed eces by ehicles, equipmen and isi o s; con amina ed pas u e o wa e sou ces; o he uminan s; and so on, a e less common bu may also be in ol ed in he sp ead o he disease [ 10 – 12 ]. Once in he he d, younge animals a e he mos suscep ible o MAP in ec ion, especially sho ly a e bi h, al hough clinical signs ypically do no appea o 3–5 yea s, e lec ing he long incuba ion pe iod and slow cou se o he disease. A p e ious s udy s a ed conside able di e ences in age suscep ibili y o in ec ion be ween adul s and cal es less han six mon hs o age and be ween adul s and cal es aged 6–12 mon hs [13]. Bo ine pa a ube culosis p og esses h ough ou main s ages, depending on he se e i y o clinical signs, he po en ial o shedding o ganisms in o he en i onmen and he ease wi h which he disease may be de ec ed using cu en labo a o y me hods. S age I is asymp oma ic (i.e., subclinical) wi h he pa hogen no de ec able in eces. This is ollowed by a s age (S age II) in which he disease emains asymp oma ic and he pa hogen is o en shed in insu icien quan i y o be de ec able by con en ional es s. The in ec ed animal may ha e an al e ed immune esponse. In he clinical s age (S age III), nea ly all animals a e de ec able by PCR o cul u e o ecal samples and usually ha e inc eased an ibody de ec able by a comme cial ELISA es . The animal has g adual weigh loss and dia hea. The appe i e emains no mal and in e mi en dia hea is o en p esen o weeks. Finally, animals in S age IV o he disease a e weak, emacia ed and usually ha e ch onic, p o use dia hea. In e mandibula edema o bo le jaw is cha ac e is ic o his phase o he disease [14]. Con ol o pa a ube culosis has p o ed o be highly p oblema ic due o he ela i ely poo pe o mance o diagnos ic es s in he ini ial s ages, he p olonged incuba ion pe iod and ex ensi e en i onmen al su i al [ 10 ]. The unce ain y su ounding he es ima ions o pa a ube culosis p e alence and i s impac also condi ion con ol s a egies [15]. Animals 2021,11, 166 3 o 19 Fo his eason, biosecu i y measu es ha educe ansmission oppo uni ies be ween he ds a e essen ial o con olling he disease. Howe e , due o he ecommended p ac ices o en being labo ious, and he di icul ies in unde s anding he nega i e implica ions o JD on he pa o he a me s, he la e o en ail o comply wi h he ecommended p ac ices and end up s opping he con ol measu es. Fo his eason, eliable in o ma ion on he main measu es o p e en ing he en y o MAP in o he ds, as well as an assessmen o hei ela i e impo ance, would be use ul o ocusing aining and awa eness p og ams and imp o ing isk educ ion s a egies. The objec i e o his s udy was o pe o m a semi-quan i a i e isk analysis o ac o s ela ing o he en y o MAP in o dai y he ds, based on expe opinions. 2. Ma e ial and Me hods 2.1. A ea Desc ip ion The s udy was ca ied ou in Galicia. This egion is he main dai y ca le a ea in Spain, con ibu ing 55% o he a ms and 38% o he milk p oduc ion. The mean Galician he d size pe a m is 43 cows, lowe han he na ional a e age o 59.3, and he a ms a e s ill p edominan ly amily owned and managed [ 16 ]. In addi ion, he dai y a ming popula ion is cha ac e ized by a high densi y o a ms and a low a ailabili y o g assland. Galicia was he i s egion in Spain o es ablish a olun a y pa a ube culosis con ol p og am, in 2004. Blood samples om all a ms included in he p og am we e aken annually om cows olde han wo yea s o labo a o y analysis. The se um was analyzed o an i-MAP an ibodies wi h comme cial ELISA (Pa a ube culosis Sc eening Ab; IDEXX, Wes b ook, ME, USA) and ecal samples o ELISA-posi i e samples we e analyzed by PCR (ID Gene®Pa a ube culosis Duplex, ID.Ve , G abels, F ance). 2.2. He ds Su eyed and Da a Collec ion Po en ial ac o s associa ed wi h he in oduc ion o MAP in o dai y he ds we e selec ed acco ding o a li e a u e e iew [ 15 , 17 , 18 ], as well as discussions wi h esea che s and e e ina ians a San iago de Compos ela Uni e si y and he Au onomous Uni e si y o Ba celona, egional go e nmen e e ina ians and ca le e e ina ians om di e se subspecial ies. The conside ed ac o s we e included in a s uc u ed ques ionnai e ha was gi en o 93 dai y a ms om Galicia [ 19 ]. The ques ionnai es we e comple ed, be ween July 2017 and Ap il 2018, h ough pe sonal in e iews wi h he a me and he e e ina ian esponsible o he con ol p og am a each a m ( he 93 a ms o med pa o he olun a y MAP con ol p og am). The ques ionnai e comp ised closed ques ions and included h ee main sec ions: (I) an- imal mo emen s (e.g., equency o in oduc ions and sani a y s a us in he a m o o igin, ca le ai s o compe i ions, pas u es and small uminan s on ha same a m); (II) mo e- men s and ypes o ehicles and equipmen ( o li e and dead animal anspo , manu e, slu y and eeding ehicles, machine y o ma e ials) and biosecu i y- ela ed measu es (e.g., ehicles may en e he a m pe ime e and ehicles may en e wi h o he animals); and (III) isi o s and s a (e.g., ex e nal wo ke s; equency o isi s by p o essionals, such as e e ina ians and echnicians, o non-p o essionals, such as neighbo ing a me s, who come in o con ac wi h he animals; and use o p o ec i e clo hing). The ques ionnai e ga h- e ed in o ma ion on he a ming p ac ices o e he las wo yea s, including he numbe s o bo h animal pu chases and ex e nal employees. 2.3. Expe G oup Wo kshop Mo eo e , o each ac o conside ed, a decision ee was de eloped, and key ques ions we e included in each. Answe s o hese key ques ions led o di e en e en s wi hin each decision ee. Figu e 1shows he decision ee o he pu chase o animals. The o he decision ees a e included in Figu e A1 (Appendix A). Fo example, e en E3 in Figu e 1 co esponds o a he d o which ca le had been pu chased o e he p eceding wo yea s, Animals 2021,11, 166 4 o 19 he sani a y s a us o he o igin a m/s was nega i e and he mean numbe o pu chased ca le was ≥3/yea . Figu e 1. Pu chase o ca le decision ee. Subsequen ly, an expe opinion wo kshop was o ganized o ga he opinions on he isk ha each e en om he decision ees would pose o he possible en y o MAP in o dai y he ds. This was implemen ed ollowing he ecommenda ions o he Handbook on Impo Risk Analysis o Animals and Animal P oduc s [20]: - Ini ially, nine e s om di e en subspecial ies (labo a o y pe sonnel in cha ge o p ocessing MAP con ol p og am samples, p ac i ione s, e e ina ians in cha ge o MAP con ol p og ams, esea che s specializing in epidemiology and in ec ious dis- eases in ca le, ca le associa ion e e ina ians and e e ina ians om pha maceu ical labo a o ies) we e selec ed, based on hei expe ience, p oximi y and knowledge o he disease. In o ma ion on he backg ound o he pa icipan s is p o ided as Table A1 (Appendix B). - Nex , he decision ees we e sen o each e e ina ian, oge he wi h an app op ia e explana ion o he me hod, whe e each e en was o be assigned a sco e (on an o dinal scale om 0 o 9) based on he isk ha , in he opinion o each expe , his e en could pose o he en y o he disease in o a dai y he d [21]. - Finally, a wo kshop o expe s was held on Decembe 2019 in he Ve e ina y Facul y o Lugo (Spain). Du ing he wo kshop, doub s and possible misunde s andings we e discussed and esol ed. In addi ion, his og ams showing he sco es assigned by he pa icipan s we e p esen ed. These his og ams we e discussed wi h he en i e g oup, and he pa icipan s had he chance o change hei assigned sco es i hey conside ed ha hey had o e - o unde es ima ed any o he e en s. Animals 2021,11, 166 5 o 19 2.4. Da a Analysis Ini ially, he mean sco e (along wi h median, maximum and minimum sco es) assigned by he pa icipa ing expe s o each e en om he decision ees was calcula ed. F om hese means, and using he da a collec ed om he 93 ques ionnai es, he sco e ha each a m would ob ain o each decision ee was eco ded. Fo example, in he case o a a m ha had pu chased ≥ 3 animals/yea om nega i e he ds (E en 3 in he decision ee o he pu chase o animals, Figu e 1), he mean sco e ha expe s assigned o ha e en would be eco ded o his a m. Finally, by adding up all he sco es ob ained by each a m o all o he decision ees, a o al sco e was assigned o each a m. The 93 he ds included in he s udy we e classi ied as ei he MAP posi i e o MAP nega i e based on he labo a o y esul s ob ained in he wo annual sampling campaigns pe o med p io o he comple ion o he ques ionnai e. He ds wi h no se oposi i e cows in ei he annual sampling we e classi ied as nega i e (n= 67). He ds wi h he p esence o he bac e ium con i med by ei he PCR o se oposi i i y ≥ 15% wi hou his being con i med in ei he o he annual sampling campaigns we e classi ied as posi i e (n= 16). To minimize misclassi ica ion, he emaining he ds, which we e se oposi i e ≤ 15% wi hou con i ma ion o he bac e ium o se onega i e he ds wi h only one annual sampling campaign (as hey had ecen ly joined he p og am) (n= 10), we e no included in his pa o he analysis. To al sco es in posi i e and nega i e a ms we e compa ed using ANOVA. Addi ion- ally, om he o al sco es (con as a iable) and using a ROC cu e, he cu -o ha bes disc imina ed posi i e and nega i e a ms was es ima ed. Addi ionally, o each posi i e a m, he wo highes sco ing e en s eco ded on ha a m, acco ding o expe opinion, we e also p o ided. 3. Resul s The mean, median, maximum and minimum sco es gi en by e e ina ians o each e en in each decision ee a e p esen ed in Table 1. Acco ding o he opinions o expe s, he e en s wi h he highes assigned sco es we e: pu chase ≥ 3 animals/yea om posi i e a ms (mean sco e 8.89 ou o 9); pu chase < 3 animals/yea om posi i e a ms (8.11/9); MAP-posi i e small uminan s on he a m (8/9); animals < 6 mon hs pas u ed wi h possible con ac wi h ca le om o he a ms (7.67/9); pu chase ≥ 3 animals/yea om a m(s) wi h unknown sani a y s a us (7.33/9); pa icipa ion in ai s/con es s wi h e u n o he animals (7/9); and animals < 6 mon hs pas u ed wi h possible con ac wi h small uminan s om o he a ms (7/9) (Table 1). The maximum o al sco e ha a a m could ob ain, i all he decision ees had he highes sco ing e en , was 77.90. The mean o al sco e (by adding up he sco es ob ained o all o he decision ees) was 23.26 (S.D. = 5.92) o nega i e a ms and 30.61 ( S.D. = 10.72 ) o posi i e a ms (p< 0.001). Acco ding o he ROC cu e, he cu -o sco e ha bes disc imina ed posi i e and nega i e a ms was 25.94. This cu -o p o ides 68.8% (46/67) sensi i i y and 68.7% (11/16) speci ici y (Table 2). Fi e ou o 51 a ms (9.8%) wi h sco es below 25.94 we e posi i e, while his pe cen age inc eased o 34.4% (11/32) among hose a ms wi h sco es equal o o highe han 25.94 (Table 2). Addi ionally, all a ms wi h a o al sco e highe han 42.99 (n= 3) we e posi i e. Fo each posi i e he d, he wo highes sco ing e en s acco ding o he expe g oup a e p esen ed in Table 3. In 5 ou o 16 posi i e a ms (31.2%), he highes sco ing e en was ela ed o he pu chase o ca le. In e es ingly, in 6 ou o 16 (37.5%), i was ela ed o he en y o a ious ehicles wi h no implemen a ion o cleaning and disin ec ion p o ocols. Animals 2021,11, 166 6 o 19 Table 1. Mean o dinal alues o each e en oge he wi h he, minimum, median and maximum alues acco ding o expe opinion. Mean Median Maximum Minimum Pu chase o ca le E1 0 0 0 0 E2 2 2 3 1 E3 3.11 3 4 2 E4 6.22 6 8 5 E5 7.33 7 9 6 E6 8.11 8 9 7 E7 8.89 9 9 8 Pa icipa ion in ca le ai s o compe i ions E1 0 0 0 0 E2 3.44 3 5 1 E3 5.22 5 7 4 E4 7 7 9 5 Sheep/goa s on he a m E1 0 0 0 0 E2 2.22 2 3 0 E3 5.44 5 7 4 E4 8 9 9 6 Pas u ing E1 0 0 0 0 E2 1.44 1 3 0 E3 4.44 5 5 3 E4 3.78 4 5 2 E5 1.33 1 3 0 E6 6.22 6 7 5 E7 5.55 6 7 3 E8 2.22 2 5 0 E9 7.67 7 9 6 E10 7 7 9 4 Sha ed manu e ehicle E1 0 0 0 0 E2 3 3 5 1 E3 6.89 7 9 4 Sha ed slu y ehicle E1 0 0 0 0 E2 2.89 2 5 1 E3 6.67 7 9 4 Vehicle o li e animal anspo (animals o slaugh e house o eed lo ) E1 0 0 0 0 E2 1.11 1 2 0 E3 2.67 2 5 1 E4 2.11 2 4 1 E5 3.44 3 5 2 E6 3.22 3 5 2 E7 4.78 5 6 3 E8 4.44 4 7 2 E9 6.22 7 8 3 Vehicle o dead animal anspo E1 0 0 0 0 E2 2.11 2 4 1 E3 4.55 5 6 3 Sha ed eed ehicle E1 0 0 0 0 E2 2.55 2 4 2 E3 4.78 5 6 3 Sha ed machine y/ma e ials E1 0 0 0 0 E2 2.67 3 4 2 E3 5.33 5 7 4 (Ex e nal) employees E1 0 0 0 0 E2 0.55 1 1 0 E3 1.78 2 3 0 E4 5.22 5 7 3 E5 2.22 3 4 0 E6 5.78 6 7 3 Visi o s pe mon h ha could con ac animals E1 2.11 2 3 0 E2 4.55 5 5 4 E3 2.78 3 5 0 E4 6.11 7 7 4 Animals 2021,11, 166 7 o 19 Table 2. C oss-classi ica ion o he MAP s a us o he a ms (ob ained om he labo a o y es s pe o med as pa o he con ol p og am) and he o al sco e o he a ms (ob ained om he a m managemen p ac ices acco ding o he opinions o a g oup o expe s). Fa m MAP S a us To al Nega i e Posi i e To al sco e <25.94 46 5 51 ≥25.94 21 11 32 To al 67 16 83 Table 3. Highes wo sco ing e en s acco ding o he mean alues assigned by he expe g oup in he 16 in ec ed a ms in Galicia (mean sco e assigned o his e en ). Fa m Highes Sco ing E en Second-Highes Sco ing E en 1 Sha ed manu e ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.89) Sha ed slu y ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.67) 2Animals < 6 mon hs go o pas u e wi h possible con ac wi h ca le om o he a ms (7.67) Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) 3Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) Sha ed manu e ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.89) 4 Sha ed manu e ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.89) Visi o /mon h > 4 wi hou p o ec i e clo hing (6.11) 5Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) Sha ed manu e ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.89) 6Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e helps wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (6.22) 7 Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e helps wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (6.22) Sha ed eeding ehicle and no cleaning and disin ec ion p o ocol be ween a ms (4.78) 8Small uminan s in he a m wi h unknown sani a y s a us (5.44) Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e does no help wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (4.78) 9 Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e helps wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (6.22) Visi o s/mon h > 4 wi hou p o ec i e clo hing (6.11) 10 Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e helps wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (6.22)) Visi o s/mon h > 4 wi hou p o ec i e clo hing (6.11) 11 Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) Sha ed eeding ehicle and no cleaning and disin ec ion p o ocol be ween a ms (4.78) 12 Pu chase ≥3 animals/yea om a m wi h unknown sani a y s a us (7.33) Sha ed slu y ehicle and no cleaning and disin ec ion p o ocol be ween a ms (6.67) 13 Visi o s/mon h > 4 wi hou p o ec i e clo hing (6.11) Sha ed eeding ehicle and no cleaning and disin ec ion p o ocol be ween a ms (4.78) 14 Animals < 6 mon hs go o pas u e wi h possible con ac wi h ca le om o he a ms (7.67) Visi o s/mon h > 4 wi hou p o ec i e clo hing (6.11) 15 Visi o s/mon h > 4 wi hou p o ec i e clo hing (6.11) Vehicle o li e animal anspo en e s a m pe ime e , wi h o he animals, d i e does no help wi h loading and he e a e no cleaning and disin ec ion p o ocols be ween a ms (4.78) 16 Sha ed eeding ehicle and no cleaning and disin ec ion p o ocol be ween a ms (4.78) Visi o s/mon h < 4 wi hou p o ec i e clo hing (4.55) Animals 2021,11, 166 8 o 19 4. Discussion In Spain, o icial MAP con ol p og ams in ca le a e only ca ied ou in he Galicia egion, which, as men ioned, is he main dai y ca le a ea o he coun y. These p og ams a e olun a y and conduc ed h ough a me associa ions, called Li es ock Heal h De ense Associa ions (known as ADSG in Spanish). They a e coo dina ed by he egional go e n- men . Nowadays, ADSGs include 55.2% o he he ds and 65.0% o Galician bo ine census. The egional go e nmen co e s he cos o labo a o y es s and pa o he sala y o he e e ina ian esponsible [ 22 ]. Con ol e o s and p og ams agains MAP in ca le a e e y di e se among Eu opean s a es (al hough pa a ube culosis is no i iable in mos o hem). As in Galicia, he majo i y o coun ies ha e olun a y con ol p og ams, also coo dina ed a egional le el in many cases. In line, go e nmen unding is equen ly in ol ed, bu ope a ions end o be suppo ed by a me s and hei o ganiza ions and no by go e nmen alone [23,24] The p esen pape ocuses on ac o s ela ed o bioexclusion o ex e nal biosecu i y, ha is, ac o s associa ed wi h he en y o MAP in o a he d. In his way, i di e s om many o he p e ious s udies whe e bioexclusion was assessed oge he wi h biocon ain- men measu es. The esul s o he expe wo kshop emphasize he ela i e impo ance o he conside ed biosecu i y measu es, wi h animal mo emen (i.e., he equency o in oduc ions and sani a y s a us in he a ms o o igin, pas u e, small uminan s on he same a m) playing a cen al ole. Howe e , o he biosecu i y measu es should no be unde es ima ed, pa icula ly he con ol o sha ed ehicles/ma e ials and isi o s ha ha e con ac wi h he animals. Mos p e ious s udies based on eg ession analysis (in o he popula ions and using o he diagnos ic app oaches) coincided in poin ing ou he impo ance o animal pu chases, indica ing odds a ios anging om 1.31 o 5.44 [ 11 , 12 , 18 , 25 ]. In con as , McAllon e al. (2017) ound ha pu chasing beha io o e he p eceding 10 yea s was no signi ican [ 10 ]. In Galicia, 31% o he a ms pu chased ca le [ 26 ]. The p ima y di e ence be ween his s udy and p e ious ones is ha , in Galicia, a ms ha a e pa o he con ol p og am a e equi ed o es all newly pu chased animals using an ibody ELISA es . Howe e , gi en he limi a ions o he se um ELISA es a he indi idual le el [ 27 ], i seems necessa y o ha e comp ehensi e in o ma ion on he o e all s a us o he a m o o igin [ 11 ]. Pu chase o ca le is he highes sco ing e en in 5 ou o he 16 posi i e a ms in he s udy. Al hough o a ms in he con ol p og am i is compulso y o ca y ou an an ibody ELISA es on pu chased animals, he e a e no obliga ions o implemen o he biosecu i y measu es [ 28 ]. Al hough pu chased ca le a e conside ed o be he main isk ac o o disease en y o dai y a ms [ 11 , 12 , 18 , 25 ], he impo ance o o he measu es should no be unde es ima ed and may esul in less use o mone a y and labo esou ces in ended o con ol p og ams. O he p ac ices o which e e ina ians gi e high sco es (>7 ou o 9) a e less equen ly obse ed in he s udy popula ion [ 19 ]. Al hough i has been sugges ed ha in e species ansmission o some MAP s ains may no be a a e e en i he e is close con ac be ween di e en species a he a m le el [ 29 ], a small p opo ion o a ms kep small uminan s. Fu he mo e, animal mo emen o ca le ai s o compe i ions (wi h e u n o he animals) is a possible disease ansmission pa hway o ew a ms in he egion, and a ms ha allow cal es o g aze a e also sca ce [ 19 ]. In any case, wi hin a he d, i is he younge animals ha a e he mos suscep ible o con ac ing MAP, and his ends o mo e slowly h ough soils and emain on he g ass and in he uppe laye s o pas u e soil, posing an in ec ion haza d o g azing animals [ 30 ]. The p esence o small uminan s on he a m is he highes sco ing e en in 1 o he 16 posi i e a ms in he s udy, whe eas he g azing o animals unde six mon hs o age is he highes sco ing e en o wo posi i e a ms. Howe e , o he ac o s ha a e obse ed mo e equen ly on a ms in Galicia and which we e sco ed as >6 ou o 9, acco ding o expe opinion, should also be conside ed, pa icula ly sha ed ehicles ha en e he a m pe ime e wi h no cleaning and disin ec ion implemen ed be ween a ms (i.e., manu e, slu y o li e animal anspo ehicles) o isi o s who do no wea p o ec i e clo hing. These ac o s ha e been less well e alua ed Animals 2021,11, 166 9 o 19 in p e ious s udies bu , acco ding o he sco es gi en by he expe s, hey could be o g ea impo ance as hey a e e en s ha occu equen ly in he s udy popula ion [ 19 ]. Sha ed ehicles o isi o s wi h no p o ec i e clo hing a e he highes sco ing e en in 6 ou o 16 posi i e a ms. Ce ain en i onmen s on he a m we e mo e likely o be con amina ed wi h MAP and had highe a e age con amina ion le els. These high- isk a eas included manu e s o age a eas and sha ed alleyways, in o he wo ds pa s o he a m whe e he manu e o adul cows is mixed [ 31 ]. Manu e ehicles a e he ehicles o which he expe s assigned he highes isk. Addi ionally, isi o oo wea has p e iously been conside ed a high- isk omi e o he dispe sion o dus - ela ed MAP ou side he ba n [32]. Gi en he cha ac e is ic o JD, he main con ol poin o help a oid he in ec ion o he a ms should be he implemen a ion o obus biosecu i y measu es ha p e en he in oduc ion o he bac e ia o he a m. Based on he opinions o he pa icipa ing expe s, i was possible o assign a sco e o each a m based on i s le el o biosecu i y, which disc imina ed a ms wi h a high and low isk o in ec ion, wi h mo e han 68% sensi i i y and speci ici y. The esul s sugges ha con ol p og ams could iden i y and emphasize he p ac ices ha pose he highes isk o each a m. A possible limi a ion o he me hod is ha PCR es s we e only pe o med on se opos- i i e animals. The e is he possibili y ha he cows a e ELISA-nega i e bu ecal cul u e posi i e; his could be he esul o passi e shedding due o he h ough-pass o MAP in he in es inal ac [ 33 ]. Howe e , i has been indica ed ha ELISA-posi i e cows we e up o 8.8 imes mo e likely o shed he bac e ia [ 34 ]. In addi ion, o he s udy, he labo a o y da a we e assessed a he he d, no indi idual, le el. 5. Conclusions Gi en he impo ance o isk ac o managemen in he con ol o MAP, conduc ing isk ac o s udies in he egions whe e con ol p og ams a e o be implemen ed is highly ad isable. Despi e he inhe en limi a ions o his s udy, we belie e i p o ides a comp e- hensi e o e iew o he main ac o s ela ed o he en y o MAP in o dai y a ms. These da a should be use ul o ocusing u u e aining ini ia i es and imp o ing isk- educ ion s a egies in his economically impo an indus y. Au ho Con ibu ions: Concep ualiza ion, F.J.D. and A.A.; me hodology, F.J.D. and C.O.; so wa e, F.J.D.; alida ion, J.C., E.Y., B.B. and S.J.M.; o mal analysis, F.J.D.; in es iga ion, F.J.V.; esou ces, F.J.V. and A.A.; da a cu a ion, F.J.D.; w i ing—o iginal d a p epa a ion, F.J.D. and E.Y.; w i ing— e iew and edi ing, F.J.V., F.J.D. and A.A.; isualiza ion, B.B. and S.J.M.; supe ision, J.C. and E.Y.; and p ojec adminis a ion, A.A. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was suppo ed by a p ojec om he Minis y o Science and Inno a ion o Spain (AGL2016-77269-C2-2-R). Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : The da a p esen ed in his s udy a e a ailable on eques om he co esponding au ho . Con lic s o In e es : The au ho s decla e no con lic o in e es . Animals 2021,11, 166 16 o 19 Figu e A1. Con . Animals 2021,11, 166 17 o 19 Figu e A1. Decision ee diag ams. The sani a y s a us o he o igin he d was conside ed known i i had a es ing p o ocol compa able o supe io o ha in he he ds in he con ol p og am in Galicia (nega i e when all animals es ed nega i e in all es s pe o med in he las sampling campaign p io o he pu chase o he animals o posi i e i a leas one animal es ed posi i e in any es ). The sani a y s a us o he o igin he d was conside ed unknown in any o he case (ei he no es ing p o ocols o es ing p o ocols in e io o hose implemen ed in Galicia). The median equency o en ies in hose a ms ha pu chase ca le in he s udy popula ion is h ee. 1 T ac o s, wagons, cis e n, slu y whisk, bale o cal ing, cleaning, o ha es ing ma e ials. 2 The median numbe o isi o s in he s udy popula ion is ou . Adequa e p o ec i e clo hing was de ined as clean co e alls o o e coa s and boo s ha a e p o ided by he a me be o e en e ing he ca le a ea. Animals 2021,11, 166 18 o 19 Appendix B Table A1. Backg ound and expe ise o he di e en expe s who pa icipa ed in he wo kshop. Posi ion. Ins i u ion Expe ise 1 Head o he molecula biology se ice. Animal heal h and p oduc ion labo a o y (Xun a de Galicia). A ound 28 yea s wo king on se ology and molecula biology o in ec ious ca le diseases. 2 Chie e . Sani a y De ense G oup o Galicia Responsible o he MAP con ol p og am in 4 municipali ies in Galicia o e he pas 12 yea s. P e iously 15 yea s o expe ience as ee ca le p ac i ione . 3 Chie e . Sani a y De ense G oup o Galicia Responsible o he MAP con ol p og am in 5 municipali ies in Galicia o e he pas 15 yea s. 4P o esso and esea che . Animal Heal h. Ve e ina y Facul y. Za agoza Uni e si y A ound 30 yea s wo king on in ec ious uminan diseases, mainly wi h isk analysis and ansmission models. 5P o esso and esea che . Animal Heal h. Ve e ina y Facul y. Au onomous Uni e si y o Ba celona A ound 20 yea s wo king on in ec ious ca le diseases mainly wi h isk and spa ial analysis. 6P o esso and esea che . Animal Heal h. Ve e ina y Facul y. San iago de Compos ela Uni e si y A ound 35 yea s wo king on in ec ious ca le diseases mainly wi h diagnos ic echniques and ansmission models. 7 Head o Dai y He d Imp o emen P og am, Galicia A ound 33 yea s o expe ience wi h dai y ca le. Has wo ked as a p ac i ione , nu i ionis , head o a a ming coope a i e and du ing he las 23 yea s as head o he Dai y He d Imp o emen P og am in Galicia. 8 Ca le ield echnician. Pha maceu ical labo a o y A ound 19 yea s o expe ience wi h dai y ca le. F ee p ac i ione and has spen he las 5 yea s as a dai y ca le ield echnician a a pha maceu ical labo a o y. 9 F ee ca le p ac i ione A ound 9 yea s o expe ience as a ee dai y ca le p ac i ione . Re e ences 1. Lamb ech , R.S.; Collins, M.T. Mycobac e ium pa a ube culosis ac o s ha in luence mycobac in dependence. Diagn. Mic obiol. In ec . Dis. 1992,15, 239–246. [C ossRe ] 2. Whi ing on, R.J.; Ma shall, D.J.; Nicholls, P.J.; Ma sh, I.B.; Reddacli , L.A. Su i al and do mancy o Mycobac e ium a ium subsp. pa a ube culosis in he en i onmen . Appl. En i on. Mic obiol. 2004,70, 2989–3004. [C ossRe ] [PubMed] 3. Richa dson, E.K.B.; Mo e, S.J. Di ec and indi ec e ec s o Johne’s disease on a m and animal p oduc i i y in an I ish dai y he d. I . Ve . 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