pa hogens
A icle
Accu a e Diagnosis o Small Ruminan Len i i us In ec ion Is
Needed o Selec ion o Resis an Sheep h ough TMEM154
E35K Geno yping
Hugo Ramí ez 1,† , I ache Eche e ía2,†, Al edo A. Beni o 3, Idoia Gla ia 2, Julio Bena ides 4, Valen ín Pé ez 5,
Damián de And és2and Ramsés Reina 2,*


Ci a ion: Ramí ez, H.; Eche e ía, I.;
Beni o, A.A.; Gla ia, I.; Bena ides, J.;
Pé ez, V.; de And és, D.; Reina, R.
Accu a e Diagnosis o Small
Ruminan Len i i us In ec ion Is
Needed o Selec ion o Resis an
Sheep h ough TMEM154 E35K
Geno yping. Pa hogens 2021,10, 83.
h ps://doi.o g/10.3390/
pa hogens10010083
Recei ed: 30 No embe 2020
Accep ed: 13 Janua y 2021
Published: 19 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/).
1Vi ology, Gene ics and Molecula Biology Labo a o y, Facul y o Highe Educa ion, Cuau i lan,
Ve e ina y Medicine, Campus 4, Na ional Au onomous Uni e si y o Mexico, Km. 2.5 Ca e e a
Cuau i lán-Teoloyucan, San Sebas ián Xhala, Cuau i lán Izcalli Es ado de México C.P. 54714, Mexico;
[email p o ec ed]
2Animal Heal h Depa men , Ins i u e o Ag obio echnology (IdAB), CSIC-Go e nmen o Na a a,
31192 Na a a, Spain; [email p o ec ed] (I.E.); [email p o ec ed] (I.G.);
[email p o ec ed] (D.d.A.)
3Molecula and Cell Biology Depa men , EXOPOL SL, 50840 Za agoza, Spain; [email p o ec ed]
4Moun ain Li es ock Ins i u e (IGM), CSIC-Uni e si y o León, 24346 León, Spain; [email p o ec ed]
5Depa men o Animal Heal h, Uni e si y o León, 24071 León, Spain; [email p o ec ed]
*Co espondence: amses. [email p o ec ed]; Tel.: +34-948-168022
† These au ho s con ibu ed equally (p io i y o de was decided acco ding o senio i y).
Abs ac :
Small uminan len i i uses (SRLV) cause an incu able mul io ganic disease widely sp ead
in sheep and goa s ha dis u bs animal wel a e and p oduc ion. In he absence o a accine, con ol
measu es ha e been adi ionally based on ea ly diagnosis and b eeding wi h i us-inac i a ed
colos um wi h seg ega ion o se oposi i e animals. Howe e , an igenic he e ogenei y, poo an ibody
p oduc ion due o low i al load, and single s ain design o mos a ailable ELISA, pose a h ea o
SRLV diagnosis. Genome-wide associa ion s udies ha e desc ibed TMEM154 E35K polymo phism
as a good gene ic ma ke o selec ion o esis an animals in some Ame ican and Eu opean b eeds.
In his s udy, a mul i a ge ed se ological and i ological sc eening o mo e han 500 animals om
ou di e en b eeds (la xa, aza Na a a, assa , and chu a) a ending o SRLV in ec ion s a us
was pe o med. Then, animals we e geno yped o cha ac e ize TMEM154 E35K polymo phism.
ELISA p ocedu es, indi idually conside ed, only iden i ied a p opo ion o he se oposi i e animals,
and PCR de ec ed a ac ion o se onega i e animals, globally o e ing di e en animal classi ica ions
acco ding o SRLV in ec ion s a us. TMEM154 allele equency di e ed subs an ially among b eeds
and a posi i e associa ion be ween se op e alence and TMEM154 geno ype was ound only in one
b eed. Selec ion based on TMEM154 may be sui able o speci ic o ine b eeds o SRLV s ains,
howe e gene aliza ion o he whole SRLV gene ic spec um, o ine b eeds, o epidemiological
si ua ion may need u he alida ion.
Keywo ds: small uminan len i i uses; TMEM154; ELISA; PCR
1. In oduc ion
Small uminan len i i us (SRLV) in ec ion widely a ec s animal heal h and p o-
duc ion by causing a was ing disease cha ac e ized by ch onic in lamma ion o ca pal
join s, udde , cen al ne ous sys em, and/o lungs [
1
]. In ec ion akes place h ough
colos um/milk consump ion om in ec ed ewes, and/o by di ec con ac wi h espi a-
o y sec e ions om in ec ed animals [
2
,
3
]. Once in ec ion occu s, immune esponses esul
in p oduc ion o an ibodies ha gene ally emain de ec able, o he whole animal’s li e,
which is a he basis o he cu en con ol p og ams [
4
,
5
]. S a egies o con ol SRLV a e
based on he iden i ica ion o se oposi i e animals since no accine is a ailable, despi e
Pa hogens 2021,10, 83. h ps://doi.o g/10.3390/pa hogens10010083 h ps://www.mdpi.com/jou nal/pa hogens
Pa hogens 2021,10, 83 2 o 16
p o use ials [
6
]. Howe e , an igenic he e ogenei y o ci cula ing s ains may be wide
han he co e ed by a ailable ELISA es s [
7
–
9
] making se ological esponse no always
de ec able. Acco dingly, he desc ip ion o new in ec ion ou b eaks in ELISA-con olled
locks [
9
–
11
] ha e p ac ically ques ioned cu en con ol s a egies. Molecula diagnosis
by PCR may add diagnos ic alue o se odiagnosis since se onega i e animals may show
PCR posi i e esul s due o low an ibody p oduc ion [
12
,
13
]. New molecula me hods a e
being desc ibed ocused on he design o uni e sal p ime s, he eby inc easing sensi i i y
o enable he iden i ica ion and emo al o animals wi h low i al load in i o [14–17].
In addi ion o hei use in con ol p og ams, he de ec ion o an ibodies h ough ELISA
es s along wi h molecula ools has been used o iden i y associa ion be ween b eeds
and suscep ibili y pa e ns o SRLV in ec ion. Genome wide associa ion s udies (GWAS)
ha e opened he possibili y o apply gene ic selec ion p og ams by desc ibing a numbe
o candida e genes associa ed o SRLV se o eac ion [
18
–
21
] o p o i al load [
22
]. Among
hem, di e en s udies sugges ha TMEM154 haplo ypes 1, 2, and 3, he mos common
haplo ypes ound in sheep, ha e an e ec on SRLV suscep ibili y. Sheep wi h a copy o
ei he haplo ype 2 o 3, bo h o which encode a glu ama e amino acid esidue a posi ion
35 (E35) o he ex acellula po ion o TMEM154, ha e an inc eased isk o SRLV in ec ion.
Con e sely, sheep homozygous o haplo ype 1, which encodes a lysine esidue a posi ion
35 (K35), ha e a dec eased isk o in ec ion in sheep b eeds om No h Ame ica and
Ge many [
18
,
23
,
24
], bu also in Asian sheep b eeds [
25
,
26
]. Wi h he excep ion o he
men ioned coun ies, he e is li le in o ma ion abou he TMEM154 haplo ype composi ion
in p oduc i e b eeds and i s associa ion wi h SRLV suscep ibili y.
In his s udy, we analyzed TMEM154 E/K geno ype associa ion wi h SRLV in ec-
ious s a us in app oxima ely 500 sheep belonging o di e en locks, p oduc ion sys ems,
and b eeds acco ding o h ee di e en ELISAs and a PCR-based molecula es .
2. Resul s
2.1. Se odiagnosis
SRLV diagnosis h ough ELISA was ca ied ou using h ee di e en comme cial es s
based on di e en s ains and an igens. An animal was conside ed in ec ed in he To al
ELISA classi ica ion when es ed posi i e o a leas one o he ELISAs.
A he popula ion le el, ELISA es ing indica ed ha all he locks we e in ec ed wi h
a se op e alence anging om 3.5% o 100%. Indi idually conside ed, he di e en ELISAs
showed impo an di e ences when examining in a lock se op e alence. In e es ingly,
wo locks o he chu a b eed we e classi ied as unin ec ed aking in o accoun esul s om
ELISA#1. Howe e , To al ELISA analysis indica ed a se op e alence o 60% (Table S1).
Conside ing animals by b eed, assa locks we e he mos in ec ed showing a se o-
p e alence up o 89%, depending on he ELISA es ed. When conside ing he esul s om
he h ee ELISA es s, se oposi i e animals eached 97.3% (Table 1), being all locks abo e
90% o se op e alence. Raza and la xa Na a a b eeds showed a mode a e se op e alence
acco ding o single ELISA es s, howe e , when applying he h ee ELISAs se oposi i e
animals eached 50%. Chu a sheep showed mode a e o high se op e alence alues, each-
ing a o al ELISA a e o 66.3% (Table 1). ELISA e iciencies, calcula ed as he p opo ion
o se oposi i e animals de ec ed by a single ELISA, eached 56%, 74%, 92%, and 91% in
aza Na a a, la xa Na a a, assa , and chu a animals, espec i ely. ELISA#3 was clea ly
mo e pe o man in chu a animals, whe eas in ec ion in la xa Na a a animals was be e
de ec ed by ELISA#1 (Table 1).
In e es ingly, he combina ion o all ELISAs e ealed a global se op e alence highe
han 65%, p ac ically doubling he pe o mance o e ed by ki s indi idually conside ed. In-
deed, e iciency o indi idual ELISAs a ied om 0% o 100% acco ding o locks (
Table S1
)
and om 38.46% o 91.67% depending on he b eed (Table 1).
Pa hogens 2021,10, 83 3 o 16
Table 1.
Small Ruminan Len i i us (SRLV) se op e alence and ELISA e iciency in aza Na a a, la xa Na a a, assa ,
and chu a o ine b eeds. To al ELISA e lec s eac i i y o any o he ELISAs used.
TEST
Raza Na a a La xa Na a a Assa Chu a
nPosi i e E iciency % nPosi i e E iciency % nPosi i e E iciency % nPosi i e E iciency %
n%n%n%n%
ELISA#1 114 29 25.4 55.8 194 76 39.2 73.8 74 66 89.2 91.7 101 32 31.7 47.8
ELISA#2 114 28 24.6 53.9 194 60 30.9 58.3 74 61 82.4 84.7 101 42 41.6 62.7
ELISA#3 114 20 17.5 38.5 194 60 30.9 58.3 74 46 62.2 63.9 101 61 60.4 91.0
To al ELISA 114 52 45.6 100.0 194 103 53.1 100.0 74 72 97.3 100.0 101 67 66.3 100.0
2.2. Molecula Diagnosis
Diagnosis h ough comme cial PCR esul ed as sensi i e as ELISA, since he o e all
PCR eac i i y was a ound 44% compa ed o 46.4%, 44.9%, and 42.7% o ELISAs #1,
#2, and #3, espec i ely (Table 2). As shown o se ological analysis, PCR eac i i y also
depended on he lock conside ed, since 77% o he animals we e de ec ed in assa locks
and only 22% in he chu a animals (Table S2).
Table 2.
Small Ruminan Len i i us (SRLV) p o i us de ec ion using eal ime quan i a i e PCR (qPCR). To al in ec ed
e e s o samples posi i e o any o he diagnos ic me hods used (ELISA and/o PCR).
TEST
Raza Na a a La xa Assa Chu a
nPosi i e E iciency % nPosi i e E iciency % nPosi i e E iciency % nPosi i e E iciency %
n%n%n%n%
qPCR 111 33 29.7 45.2 191 90 47.1 65.7 74 57 77.0 79.2 82 18 22.0 32.1
To al in ec ed 114 75 65.8 100.0 194 139 71.6 100.0 74 72 97.3 100.0 101 67 66.3 100.0
Among se onega i e samples, 54 ou o 179 (30%) we e iden i ied as qPCR posi i e,
whe eas 135 se oposi i e samples esul ed nega i e in qPCR. When conside ing each ELISA
indi idually, PCR de ec ed a 22.37%, 34.53%, and 37.94% o se onega i e animals o ELISAs
#1, #2, and #3, espec i ely (Figu e 1and Tables S4–S6).
To al in ec ed animal classi ica ion, e ealed by ELISA o PCR, allowed he e alua ion
o PCR e iciency compa ed o ELISA. qPCR e iciency eached 79% in assa animals and
dec eased o 32% in chu a locks. In a lock e iciency in assa locks peaked a 95.8%,
whe eas highly se oposi i e chu a locks we e no de ec ed by qPCR (Table S2).
Animal classi ica ion in o in ec ed and unin ec ed a e ELISA (Table S3) and qPCR
p o i al quan i ica ion is ep esen ed in Figu e 1.
2.3. TMEM154 Geno yping
O ine DNA samples (n= 10) om he s udied popula ion we e employed o ampli y
a 335bp egion o he TMEM154 gene (Table 3), including esidue a posi ion 35, ha was
cloned and sequenced (Figu e 2).
Table 3. P ime and p obe sequences, ampli ica ion p oduc size, and pu pose o he co esponding PCR me hod.
P obes/P ime s Sequences P oduc Size (Base Pai s) Pu pose
Fw 5’-CTGCCTTTGTGGGAGATTTA-3’ 335 Ampli ica ion and sequencing o e i ica ion
o geno yping esul s
R 5’-TTCTGTGGTCACTGAAGCAA-3’
Fw 5’-TTCGTCTCCATGACAAGTCTCAAT-3’
121
De e mina ion o nucleo ide subs i u ion G/A,
esul ing in amino acid subs i u ion E35K.
R 5’-GCTTAGGGCCTCTGACTCTTCA-3’
HEX-AGGACACAGAACTGT-BHQ-1
6-FAM-AGGACACAAAACTGT-BHQ-1
Pa hogens 2021,10, 83 4 o 16
Figu e 1.
Small uminan len i i us (SRLV) diagnosis. Sca e plo dis ibu ion o ELISA abso bance
(X-axis) and p o i al load in 250 ng o DNA (Y-axis) da a. Samples conco dan ly posi i e o nega i e
be ween ELISA#1 (
A
), ELISA #2 (
B
), o ELISA#3 (
C
) and PCR (
•
) and disco dan samples (O) a e
ep esen ed. The Y-axis in e cep ed he X-axis a he a e age alue o he co esponding ELISA
posi i i y h eshold.
Pa hogens 2021,10, 83 5 o 16
Figu e 2.
Iden i ica ion o TMEM154 E35K geno ype. Alignmen o pa ial TMEM154 sequences
ob ained om selec ed sheep. Numbe s e e o he animal sample and clone analyzed. Amino acid
subs i u ion a posi ion 35 is highligh ed. Iden ical esidues a e indica ed by do s.
Conside ing Sange sequencing, se en samples we e iden i ied as homozygo es o
allele 1, one as homozygo e o allele 2, and wo as he e ozygo es. Speci ic clones encoding
allele 1 o allele 2 we e used o eal ime PCR s anda diza ion.
Fluo ogenic p obes we e designed wi hin he E35K SNP, wi h ei he FAM o HEX
(Table 3), o speci ically de ec plasmids encoding he co esponding geno ype. Equimola
mixes o plasmids encoding each o he alleles we e au oma ically classi ied as he e ozy-
go es, alida ing hei applica ion in biological samples (Figu e 3).
Allelic disc imina ion analysis showed di e en allele equencies acco ding o he
b eed conside ed (Table 4). The p o ec i e geno ype (K/K) was p edominan in all b eeds
analyzed, ollowed by he e ozygo es and homozygo es (E/E), excep o he assa b eed in
which he e ozygo es and homozygo es (E/E) we e p e alen .

Pa hogens 2021,10, 83 6 o 16
Figu e 3.
TMEM E35K geno yping using luo ogenic p obes. Sca e plo dis ibu ion o ela i e
luo escence o FAM, ep esen a i e o allele 1 (X-axis) and HEX (allele 2; Y-axis) o TMEM154 clones.
O iginal animal samples a e also shown showing a he e ozygo e pa e n.
Table 4.
Allelic equency among TMEM154 E35K geno yping in aza Na a a, la xa, assa ,
and chu a o ine b eeds.
Geno ype Raza Na a a La xa Assa Chu a
n%n%n%n%
K/K 92 80.7 134 69.1 15 20.3 75 74.3
E/K 18 15.8 56 28.9 32 43.2 24 23.8
E/E 4 3.5 4 2.1 27 36.5 2 2.0
To al 114 100 194 100 74 100 101 100
2.4. TMEM154 E35K Associa ion wi h SRLV In ec ion S a us
Geno yped sheep we e dis ibu ed acco ding o ELISA abso bance and PCR p o i al
load (Figu e 4). Conside ing b eeds in which he K/K allele was p edominan , he p opo -
ion o se oposi i e and se onega i e samples in ELISAs #1 and #3 was simila in esis an
(K/K) o suscep ible (E/K and E/E) geno ypes (Table 5). Simila ly, assa animals we e
mos ly se oposi i e i espec i e o hei TMEM154 geno ype (Tables S4–S6). Howe e ,
when conside ing all b eeds as a whole, signi ican di e ence was ound be ween esis an
and suscep ible geno yped samples, mean abso bance being highe in suscep ible samples
(p< 0.05 Mann–Whi ney). Excep ions o his gene al pic u e we e e iden when analyzing
da a ob ained a e ELISA#2 es ing o la xa and aza Na a a b eeds, since di e ences we e
ound in ELISA abso bance acco ding o TMEM154 geno ype (Figu e 4B and Table S5).
Dis ibu ion o geno yped samples acco ding o p o i al load alues was simila
among esis an (K/K) and suscep ible (E/K and E/E) samples, sugges ing poo associa ion
be ween TMEM154 geno ype and SRLV in ec ion (Figu e 4D).
Rela ionship be ween SRLV in ec ion s a us and TMEM154 geno yping was e alua ed
using associa ion and ela i e isk, and eg ession s a is ical analyses. Animals om he
assa and chu a b eeds did no show signi ican associa ion be ween TMEM154 geno ype
and SRLV an ibody occu ence, excep o ELISA#2 in chu a animals (Table 5). Simila ly,
animal classi ica ion by ELISA#2 o he aza Na a a and la xa Na a a b eeds allowed a
signi ican associa ion be ween SRLV se o eac i i y and TMEM154 geno ype. Addi ionally,
eac i i y o ELISAs #1, 2, and 3 was also associa ed o TMEM154 geno ype in la xa
Na a a animals. To al ELISA eac i i y was associa ed o geno yping in he case o he
a o emen ioned b eeds ( aza Na a a and la xa Na a a), bu no in assa o chu a sheep.
Pa hogens 2021,10, 83 7 o 16
Figu e 4. Con .
Pa hogens 2021,10, 83 8 o 16
Pa hogens 2021, 10, x FOR PEER REVIEW 8 o 17
Figu e 4.
Dis ibu ion o ELISA#1 (
A
), ELISA#2 (
B
), ELISA#3 (
C
) abso bance and qPCR p o i al load
(
D
) acco ding o TMEM154 geno yped la xa, aza Na a a, assa , and chu a sheep. Animal samples
we e classi ied acco ding o he E35K TMEM154 polymo phism in o K/K ( ) o E/K and E/E ( )
and analyzed by ELISA and qPCR. Samples we e g ouped by indi idual b eeds and combined (All
b eeds). A e age cu -o alues o indi idual ELISA a e ep esen ed as a ho izon al do ed line
(* Mann–Whi ney, p< 0.05).
Pa hogens 2021,10, 83 9 o 16
Table 5.
Small Ruminan Len i i us (SRLV) in ec ion s a us and TMEM154 geno yping associa ion. Samples classi ied in o posi i e o nega i e acco ding o di e en me hods (ELISAs and
qPCR) we e e-classi ied acco ding TMEM154 E35K polymo phism. S a is ical p obabili y associa ed o Fishe ’s exac es (p) and o ela i e isk (RR; p’) a e shown. Signi ican alues a e
in bold.
TEST SRLV Raza Na a a La xa Na a a Assa Chu a
TMEM154
Geno ype (%)
TMEM154
Geno ype (%)
TMEM154
Geno ype (%)
TMEM154
Geno ype (%)
pRR P’ pRR P’ p RR P’ pRR P’
KK EK/EE (95%CI) KK EK/EE (95%CI) KK EK/EE (95%CI) KK EK/EE (95%CI)
ELISA#1 Nega i e 69 16 0.792 1.09
(0.51–2.35) 0.824 95 23 <0.0001 2.12
(1.52–2.95) <0.0001 2 6 0.66 1.04
(0.83–1.29) 0.745 53 16 0.338 1.35
(0.74–2.48) 0.326
Posi i e 23 6 39 37 13 53 21 10
ELISA#2 Nega i e 74 12 0.024 2.32
(1.25–4.31) 0.008 111 23 <0.0001 3.59
(2.36–5.48) <0.0001 4 9 0.446 1.16
(0.84–1.60) 0.381 50 9 0.005 2.02
(1.31–3.10) 0.002
Posi i e 18 10 23 37 11 50 24 17
ELISA#3 Nega i e 78 16 0.214 1.79
(0.78–4.13) 0.171 101 33 0.007 1.83
(1.22–2.75) 0.0037 5 23 0.772 0.91
(0.60–1.38) 0.674 31 9 0.644 1.11
(0.79–1.56) 0.530
Posi i e 14 6 33 27 10 36 44 17
TOTAL
ELISA Nega i e 52 10 0.475 1.25
(0.80–1.96) 0.003 73 18 0.002 1.54
(1.20–1.98) 0.0007 0 2 10.97
(0.92–1.01) 0.157 28 6 0.232 1.23
(0.93–1.61) 0.142
Posi i e 40 12 61 42 15 57 47 20
qPCR Nega i e 62 16 10.90
(0.42–1.91) 0.781 72 29 0.437 1.15
(0.84–1.56)
0.384 3 14 10.95
(0.71–1.27) 0.747 48 16 0.770 1.12
(0.45–2.76) 0.810
Posi i e 27 6 59 31 12 45 13 5
Pa hogens 2021,10, 83 16 o 16
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