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 .
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