Genetic susceptibility to infectious disease in East African Shorthorn Zebu: a genome-wide analysis of the effect of heterozygosity and exotic introgression

RESEARCH ARTICLE Open Access Gene ic suscep ibili y o in ec ious disease in Eas A ican Sho ho n Zebu: a genome-wide analysis o he e ec o he e ozygosi y and exo ic in og ession Gemma GR Mu ay 1,2* , Ma k EJ Woolhouse 1 , Miika Tapio 3 , Ma y N Mbole-Ka iuki 4 , Tad S Sons ega d 5 , Samuel M Thumbi 1,9 , Amy E Jennings 6 , Ilana Con adie an Wyk 7 , Ma go Chase-Topping 1 , Hen y Kia a 8 , Phil Toye 8 , Koos Coe ze 7 , Ba end M deC B ons oo 6 and Oli ie Hano e 4 Abs ac Backg ound: Posi i e mul i-locus he e ozygosi y- i ness co ela ions ha e been obse ed in a numbe o na u al popula ions. They ha e been explained by he co ela ion be ween he e ozygosi y and inb eeding, and he nega i e e ec o inb eeding on i ness (inb eeding dep ession). Exo ic in og ession in a locally adap ed popula ion has also been ound o educe i ness (ou b eeding dep ession) h ough he b eaking-up o co-adap ed genes, o he in oduc ion o non-locally adap ed gene a ian s. In his s udy we examined he in e - ela ionships be ween genome-wide he e ozygosi y, in og ession, and dea h o illness as a esul o in ec ious disease in a sample o cal es om an indigenous popula ion o Eas A ican Sho ho n Zebu (c ossb ed Bos au us xBos indicus) in wes e n Kenya. These cal es we e obse ed om bi h o one yea o age as pa o he In ec ious Disease in Eas A ican Li es ock (IDEAL) p ojec . Some o he cal es we e ound o be gene ic hyb ids, esul ing om he ecen in og ession o Eu opean ca le b eed(s) in o he indigenous popula ion. Eu opean ca le a e known o be less well adap ed o he in ec ious diseases p esen in Eas A ica. I dea h and illness as a esul o in ec ious disease ha e a gene ic basis wi hin he popula ion, we would expec bo h a nega i e associa ion o hese ou comes wi h in og ession and a posi i e associa ion wi h he e ozygosi y. Resul s: In his indigenous li es ock popula ion we obse ed nega i e associa ions be ween he e ozygosi y and bo h dea h and illness as a esul o in ec ious disease and a posi i e associa ion be ween Eu opean au ine in og ession and episodes o clinical illness. Conclusion: We obse e he e ec s o bo h inb eeding and ou b eeding dep ession in he Eas A ican Sho ho n Zebu, and he e o e ind e idence o a gene ic componen o ulne abili y o in ec ious disease. These esul s indica e ha he signi ican bu den o in ec ious disease in his popula ion could, in p inciple, be educed by al e ed b eeding p ac ices. Keywo ds: Inb eeding dep ession, He e ozygosi y, Fi ness, In ec ious disease, Exo ic in og ession, Local adap a ion, Ca le * Co espondence: [email p o ec ed] 1 Ins i u e o E olu iona y Biology, and Cen e o Immuni y, In ec ion and E olu ion, Uni e si y o Edinbu gh, Edinbu gh EH9 3JT, UK 2 Depa men o Gene ics, Uni e si y o Camb idge, Camb idge, Downing S ee , Camb idge CB2 3EH, UK Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2013 Mu ay e al.; licensee BioMed Cen al L d. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Mu ay e al. BMC E olu iona y Biology 2013, 13:246 h p://www.biomedcen al.com/1471-2148/13/246 Backg ound Posi i e mul i-locus he e ozygosi y- i ness co ela ions ha e been obse ed in a numbe o na u al popula ions [1,2]. Genome-wide he e ozygosi y is co ela ed wi h inb eeding wi hin a popula ion [3] and he e ozygosi y- i ness co ela ions ha e he e o e been associa ed wi h inb eeding dep ession [4]. Inb eeding may be de i- men al o i ness i : (i) i ness ela ed ai s a e o e - dominan , such ha he he e ozygous geno ype esul s in g ea e i ness han ei he homozygo e, o (ii) he e a e ecessi e alleles p esen in he popula ion ha e- sul in lowe i ness [5]. The ex en o which he he - e ozygosi y o gene ic ma ke s e lec s genome-wide he e ozygosi y and inb eeding is a con en ious issue, and he e ozygosi y- i ness co ela ions may esul om a ew loci o majo e ec in linkage disequilib ium wi h he gene ic ma ke s a he han he e ec o inb eeding ac oss he genome [2,3,6,7]. Howe e , when la ge numbe s o ma ke s a e used, es ima es o genome-wide he e ozy- gosi y a e g ea ly imp o ed, a e mo e likely o co ela e wi h inb eeding, and a e less likely o co ela e wi h he i ness e ec s o a ew loci [2,3]. Inb eeding has been obse ed o ha e a nega i e e ec on i ness- ela ed ai s in many popula ions [8], e.g. [9,10], including some Eu opean ca le popula ions [11,12]. In a numbe o na u al popula ions suscep ibil- i y o in ec ious disease has been ound o be associa ed wi h he e ozygosi y e.g. [13-15] and inb eeding e.g. [16]. In ec ious disease suscep ibili y in humans has been ound o ha e a s ong gene ic componen in nume ous s udies e iewed in [17], wi h many genes and gene ic pa hways associa ed wi h suscep ibili y o di e en dis- eases. The majo his ocompa ibili y complex (MHC) is conside ed o play a signi ican ole in de e mining sus- cep ibili y o a ious pa hogens [18], and MHC- a ge ed b eeding p og ammes ha e been sugges ed o cap i e popula ions [19,20]. In one na u al popula ion i was ound ha he e ec o a single locus linked o he MHC exceeded he e ec o genome-wide he e ozygosi y on i ness [21]. Exo ic in og ession in a locally adap ed popula ion may also educe i ness. A numbe o s udies ha e obse ed a nega i e e ec o in og ession on i ness (ou b eeding dep ession) [22,23]. These may esul om ei he (i) he b eak-up o co-adap ed epis a ic in e ac ion be ween genes, o (ii) he in oduc ion o non-locally adap ed alleles [24]. In his s udy, we es ed o associa ions o he e ozygosi y and exo ic Eu opean au ine b eed in og ession wi h dea h and illness due o in ec ious disease du ing he i s yea o li e, in a popula ion o Eas A ican sho ho n zebu (EASZ) ca le (c ossb ed Bos au us xBos indicus) om wes e n Kenya. Mo e han 500 cal es we e ollowed o he i s yea o hei li e as pa o he In ec ious Disease in Eas A ican Li es ock (IDEAL) p ojec [25]. The cal es we e closely moni o ed h oughou ; dea hs and episodes o clinical illness we e epo ed and ollowed up by ained e e ina y s a . Du ing he s udy, mo e han 15% o he cal es died, wi h he majo i y o dea hs due o in ec ious diseases, mos commonly Eas Coas Fe e , helmin h in- ec ions, hea wa e and ypanosomiasis [26]. Gi en he hea y in ec ious disease bu den on hese ca le coupled wi h minimal disease con ol o ea men , an abili y o su i e in ec ion is likely o be signi ican ly associa ed wi h i ness. Fu he mo e, mo e han hal he su i ing cal es we e epo ed o ha e a leas one episode o clinical ill- ness du ing he s udy pe iod, and almos all hese episodes we e a ibu ed o in ec ious disease (see Me hods). We p opose ha clinical illness is also indica i e o a ulne - abili y o in ec ious disease ha could a ec i ness in his en i onmen . All cal es in he IDEAL popula ion we e geno yped using he 50K Illumina® Bo ineSNP50 beadchip . 1 (55,777 SNPs be o e quali y con ol). These SNPs a e ela i ely e enly dis ibu ed ac oss he genome, wi h an a e age o 1,895 on each au osome ( anging om 1,009 o 3,553) and 1,362 on he X ch omosome (102 emain unassigned). The s udy popula ion was ound o be pa ly in o- g essed by one o possibly mo e Eu opean au ine b eeds [27,28] (de e mined om analysis o genome-wide SNP da a using STRUCTURE [29]). This e lec s he e ec s o a subs an ial b eed imp o emen p og amme, in ol ing he c ossing o local ca le wi h Eu opean b eeds, ha ook place in he mid-1990s wi h in e mi en c oss-b eeding he ea e . As a esul , app oxima ely 20% o he cal es show le els o in og ession consis en wi h c ossing wi h Eu opean b eeds ≤5 gene a ions ago ( ac ion Eu opean au ine > 2 -6 ) [27]. We would also expec some inb eeding o occu in his popula ion, since ma ing is la gely unmanaged, he e is a ela i ely low numbe o b eeding bulls, and ca le a e gene ally only anspo ed o e sho dis ances (only a small ac ion o ca le a e aded and hese o e dis- ances ypically jus 20-30 km). Also, his o ically, his e- gion has expe ienced majo inde pes epidemics, which ha e educed he e ec i e popula ion size o he EASZ [28]. In his s udy we calcula e he genome-wide he e o- zygosi y o he cal es in he IDEAL popula ion using he same SNP da a se as was used o calcula e exo ic in og ession [28]. In his s udy we aim o de e mine whe he inb eeding (as es ima ed by SNP he e ozygosi y) and ou b eeding (as es ima ed by he es ima ed p opo ion o gene ic ma ke s ha descend om a Eu opean au ine b eed) a ec he likelihood o dea h o illness due o in ec ious disease and he e o e whe he he e is e idence o a gen- e ic basis o ulne abili y o in ec ious disease in EASZ. Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 2 o 8 h p://www.biomedcen al.com/1471-2148/13/246 We use esul s om ea lie s udies ha ha e de e mined he deg ee o Eu opean au ine in og ession [28] and desc ibe and analyse he causes o ins ances o dea h and illness o all he cal es in he IDEAL p ojec [25,26]. Th ough analysis o hese da a we ind associa ions o bo h he e ozygosi y and Eu opean au ine in og ession wi h ulne abili y o in ec ious disease du ing he i s yea o li e in his popula ion o EASZ. Resul s The sample size a ailable o analysis was 518 cal es (see Me hods o inclusion/exclusion c i e ia), o which 68 (13%) died o in ec ion- ela ed disease wi hin one yea o bi h (see Me hods o mo e de ails). O he su i o s, episodes o clinical illness (almos always ela ed o in- ec ions) we e epo ed in 243 (54%) (Table 1, Addi ional ile 1: Table S1). The dis ibu ions o obse ed he e ozygosi y alues o non-in og essed (de ined as < 2 -6 Eu opean au ine) and in og essed cal es (> 2 -6 Eu opean au ine) a e shown in Figu e 1. The mean he e ozygosi y o non-in og essed cal es is 0.28 (wi h a s anda d de ia ion (SD) o 0.01). This is no an unusual le el o he e ozygosi y o a ca le popu- la ion. Hols ein-F iesians (Eu opean au ine) ha e been ound o ha e a e age he e ozygosi y o 0.33 (SD = 0.01), Je sey (Eu opean au ine) ha e an a e age he e ozygosi y o 0.25 (SD = 0.03), N’dama (A ican au ine) ha e an a e - age he e ozygosi y o 0.17 (SD = 0.03) and E hiopian Sheko (admixed A ican au ine and Asian zebu) ha e an a e age he e ozygosi y o 0.26 (SD = 0.0003) [27]. Ino de o es whe he o no ou es ima eso he e ozygosi y a e likely o e lec ue genome-wide he e ozygosi y,we andomlysampledou SNPswi h- ou eplacemen 100 imes and de e mined he deg ee o co ela ion in he he e ozygosi y es ima es o cal es based on one hal o he ma ke s and he es ima es based on he o he hal [2,6]. We ound a consis en ly s ong co ela ion be ween he es ima es (mean Pea son’s = 0.98, SD = 0.0012, ange = 0.977-0.982). Since some SNPs may be in linkage disequilib ium wi h one ano he , we epea ed he analysis wi h wo smalle subse s (each ep esen ing a di e en andom sample, wi hou e- placemen , o one 10 h o he o al SNPs). E en wi h his much smalle se o ma ke s, a s ong co ela ion be ween he es ima es was ound (mean Pea son’s =0.91, SD = 0.0053, ange = 0.89-0.92). The non-in og essed cal es show a skewed dis ibu ion o he e ozygosi y alues, wi h he majo i y (93%) con- ained wi hin a nea -symme ical dis ibu ion ± 0.010 om a mode o 0.284 (Figu e 1). Howe e , he e is a long ail o 28 cal es wi h lowe alues. We ca ego ise cal es wi h alues < 0.274 as “low”he e ozygosi y, assumed o e- lec a highe deg ee o inb eeding han he popula ion no m. In og essed cal es show a di e en dis ibu ion o he e ozygosi y alues ha a e gene ally highe han hose o non-in og essed cal es (Figu e 1) (Mann Whi ney U es , P<0.001). The ela ionship be ween he e ozygosi y and in og es- sion is shown in Figu e 2. As expec ed, he e is a clea in- c ease in he e ozygosi y wi h inc easing in og ession. Howe e , in addi ion o he 28 non-in og essed cal es wi h low he e ozygosi y, he e a e se e al in og essed cal es wi h he e ozygosi y alues ha appea low ela i e o he amoun o in og ession. In o de o in es iga e he e ec o a ia ion in he e ozygosi y on he i ness o in o- g essed cal es independen ly o he e ec o in og ession we de ine a g oup o cal es wi h low he e ozygosi y ela- i e o hei deg ee o in og ession. We de ine his g oup by inding he bes i linea eg ession and i e a i ely ex- cluding cal es wi h he e ozygosi y alues > 0.01 below hei expec ed alue. This p ocedu e esul s in he i ed eg ession line shown in Figu e 2 (wi h he -0.01 h eshold also indica ed). Using his c i e ion, 9 o he 113 in o- g essed cal es a e ca ego ised as low he e ozygosi y. Two o hese 9 died and 6 o he s expe ienced a leas one clin- ical episode (see Table 1). We compa ed bo h cal es ha died and hose ha su i ed bu expe ienced any episode o clinical illness o a e e ence ca ego y o heal hy cal es ( hose ha su - i ed and had no epo ed episode o clinical illness). We conside ed wo ca ego ical p edic o s o hese ou - comes, low he e ozygosi y and being in og essed, using a mul inomial logis ic eg ession (desc ibed in Me hods; esul s in Table 2). We used hese ca ego ical p edic- o s, a he han analysing he es ima es di ec ly, due o he non-no mali y o he da a ( he dis ibu ion o alues o bo h he e ozygosi y and in og ession a e highly skewed) and o allow us o obus ly accoun o he Table 1 Numbe s o cal es ha all in o he di e en ca ego ies used o he s a is ical analysis o he e ec s o he e ozygosi y and in og ession on likelihood o dea h and clinical illness Low he e ozygosi y High he e ozygosi y In og essed Non-in og essed In og essed Non-in og essed Died 2 111144 Su i ed wi h clinical episode 6 13 64 160 Su i ed wi hou clinical episode 1 4 29 173 Cu -o s o high/low he e ozygosi y and in og essed/non-in og essed a e de ined in Me hods. Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 3 o 8 h p://www.biomedcen al.com/1471-2148/13/246 ela ionship be ween he e ozygosi y and in og ession (Figu es 1 and 2, Addi ional ile 1: Table S1). We i s conside he associa ion be ween p edic o s and cal dea h due o in ec ious disease. Fo non-in og essed cal es ( ac ion Eu opean au ine < 2 -6 ) only he e is s ong e idence o a ela ionship be ween dea h and low he e ozy- gosi y: odds a io (OR) = 10.8 (95% CIs 3.0-48.1); P< 0.001 (Table 3). Fo all cal es, his ela ionship emains when bo h low he e ozygosi y and in og ession a e included as p edic o s: OR = 9.5 (3.5-27.9); P<0.001(Table2).The eis no e idence o an in e ac ion be ween low he e ozygosi y and in og ession (P> 0.05) and he e is no e idence o an associa ion be ween dea h and deg ee o in og ession (OR = 1.4 [0.7-2.9]; P= 0.39) (Table 2). No signi ican di e ences we e ound in he na u e o in ec ious disease- ela ed dea hs among he low he e ozygosi y cal es. These esul s a e obus o a ia ion in he cu -o o in o- g essed cal es (see Me hods). Clinical illness is a less well de ined pheno ype han dea h, since i includes a wide ange o disease signs and i canno be unequi ocally de e mined whe he o no an in- s ance o clinical illness was he esul o in ec ious disease o no (al hough i is likely ha he o e whelming majo i y we e). Despi e his, clinical illness and dea h a e clea ly ela ed ou comes. Mo eo e , because many mo e cal es expe ienced clinical illness du ing hei i s yea han died, his ou come may p o ide inc eased powe o de- ec small e ec s. Fo non-in og essed cal es he e is Figu e 1 His og am o equency o he e ozygosi y alues o Eu opean au ine in og essed ( ed) and non-in og essed (blue) Eas A ican Sho ho n Zebu cal es. Figu e 2 Plo o he e ozygosi y agains p opo ion Eu opean in og ession o all cal es in ou analysis. Cal es ha died a e highligh ed ( ed symbols). The bes i ela ionship be ween he e ozygosi y (H) and in og ession (I) o non-inb ed cal es is shown ( hick line: eg ession equa ion H = 0.284 (95% con idence in e als ±0.0003) + I*0.312 (±0.006), R 2 = 0.972). The cu -o o de ine ‘low’he e ozygosi y cal es a ies wi h in og ession, since he expec ed o mean he e ozygosi y a ies wi h in og ession. Cal es ha a e mo e ha 0.01 below he expec ed he e ozygosi y (blue dashed line) o hei amoun o in og ession a e de ined as ‘low’he e ozygosi y (see Resul s and Me hods). The o ange dashed line indica es he cu -o o in og essed cal es (2 -6 %). Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 4 o 8 h p://www.biomedcen al.com/1471-2148/13/246 e idence o a ela ionship be ween illness and low he - e ozygosi y: OR = 3.5 (95% CIs 1.05-15.1); P= 0.026 (Table 3). Fo all cal es, his ela ionship emains when bo h low he e ozygosi y and in og ession a e included as p edic o s: OR = 3.4 [1.3-9.3]; P= 0.018 (Table 2). Howe e , o his pheno ype he e is also s ong e idence o an associa ion wi h in og ession, wi h in og essed cal es being mo e likely o expe ience illness (OR = 2.4 [1.5-3.8]; P< 0.001). Again he e is no e idence o any in e ac ion be ween low he e ozygosi y and in og ession (P> 0.05). These esul s a e obus o a ia ion in he cu - o o in og essed cal es (see Me hods). Discussion Ou esul s indica e ha he e is a gene ic basis o ulne - abili y o in ec ious disease in Eas A ican Sho ho n Zebu. We ind a posi i e associa ion be ween low he e o- zygosi y and bo h dea h and illness. Since we use a eason- able p oxy o genome-wide he e ozygosi y (40,457 SNPs ac oss all au osomes), and he es ima es o genome-wide he e ozygosi y a e highly co ela ed o di e en subse s o SNPs, we conside i likely ha ou esul s a e indica- i eo hep esenceo inb eedingdep essionin he s udy popula ion, a he han being a consequence o localised associa ions be ween i ness and loci o la ge e ec [6,7,17]. Ou esul s indica e he p esence o i ness- ela ed geno- ypes ha a e ei he o e -dominan o ha e ecessi e dele- e ious alleles in he popula ion. Al hough he p opo ion o cal es ha we classi ied as “low”he e ozygosi y was small (app oxima ely 7%), he e ec on in ec ious disease- ela ed mo ali y was la ge (OR ~ 9) and he e was also an e ec on clinical illness (OR ~ 3). Ou esul s gi e popula- ion a ibu able ac ions ( o he non-in og essed popu- la ion) due o low he e ozygosi y o 14% o mo ali y and 4% o clinical illness [30]. In p inciple, he e o e, cases o illness and dea hs could be educed by be e managemen p ac ices (e.g. bull o a ion). We ound e idence ha Eu opean au ine in og ession a ec ed heal h as i was a signi ican p edic o o clinical illness (Table 2). As he e we e only 13 dea hs among hose wi h ac ional in og ession g ea e han 2 -6 we had e y li le powe o de ec any e ec on dea h alone. None- heless, ou esul s o hese wo pheno ypes a e no in- consis en , as he con idence in e als o he odds a ios o e lap (see Table 2). We sugges ha clinical illness is a ma ke o ulne abili y in his popula ion and conse- quen ly ha Eu opean au ine in og essed cal es a e mo e ulne able in his sense. We could ind no non-gene ic con ounding ac o s ha could be d i ing he obse ed associa ions be ween in og ession and ei he dea h o illness. The in o- g essed cal es (e en hose wi h high in og ession) could no be dis inguished om non-in og essed cal es by he ield eam [25]; exo ic in og ession was only dis- co e ed upon gene ic analysis o he cal es. And, al hough he in og essed cal es we e no ound uni o mly dis ib- u ed among he 20 sub-loca ions o he s udy [28], he e is no e idence o geog aphic a ia ion in he a e o mo al- i y o o clinical illness. Since he EASZ ances o s o he s udy popula ion ha e been p esen in Eas A ica o many gene a ions [28], i migh be expec ed ha hey may ha e in some way adap ed o he speci ic challenges posed by ha en i on- men , in pa icula o he in ec ious diseases p esen in ha egion. In ec ious disease was he p ima y cause o i s yea mo ali y and illness in his popula ion, and hus he abili y o wi hs and in ec ion is likely o be o signi ican selec i e ad an age. On he o he hand, he in og essed animals descend, in pa , om Eu opean ca le, which ha e no had he same his o y o selec i e p essu es. I has been ound ha Eu opean b eeds a e mo e suscep ible han local b eeds o some in ec ious diseases ha a e p e alen in A ica, such as Eas Coas Fe e [31,32], bo ine ube culosis [33,34], and ypano- somiasis [35,36]. Thus, he Eu opean b eeds a e, in his impo an espec , less well adap ed o he Eas A ican Table 2 Summa y o esul s o a mul inomial logis ic eg ession o he e ec s o genome-wide he e ozygosi y and in og ession (as wo le el ac o s) on he likelihood o dea h and he likelihood o clinical illness Pheno ype Dead cal es (n = 68) Ill cal es (n = 243) Fac o s Es ima e (SE) OR (95% CI) P- alue Es ima e (SE) OR (95% CI) P- alue Low he e ozygosi y 1.13 (0.27) 9.5 (3.5-27.9) < 0.001 0.61 (0.26) 3.4 (1.2-9.3) 0.018 In og essed 0.16 (0.19) 1.4 (0.7-2.9) 0.39 0.43 (0.12) 2.4 (1.5-3.8) < 0.001 Cu -o s o high/low he e ozygosi y and in og essed/non-in og essed a e de ined in Me hods. Heal hy cal es we e he e e ence ca ego y. CI = con idence in e al. SE = s anda d e o . OR = odds a io. P- alues in bold ype a e s a is ically signi ican . Table 3 Summa y o esul s o Fishe ’s exac es s o he e ec s o low genome-wide he e ozygosi y (as a wo-le el ac o ) on he likelihood o dea h and he likelihood o clinical illness o he sub-popula ions o non-in og essed and in og essed cal es Pheno ypes Dead s heal hy cal es Ill s heal hy cal es Subpopula ion OR 95% CI P- alue OR 95% CI P- alue Non-in og essed 10.8 3.0-48.1 < 0.001 3.5 1.05-15.1 0.026 In og essed 5.3 0.24-321 0.213 2.72 0.31-129 0.671 Cu -o s o high/low he e ozygosi y and in og essed/non-in og essed a e de ined in Me hods. OR = odds a io. CI = con idence in e al. P- alues in bold ype a e s a is ically signi ican . Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 5 o 8 h p://www.biomedcen al.com/1471-2148/13/246 en i onmen , and in og essed animals, while mo e he e ozygous due o ou b eeding, a e also less locally adap ed. While inc eased he e ozygosi y is likely o be bene icial i i ness ela ed ai s a e o e -dominan o he e a e ecessi e dele e ious alleles in he popula ion, he b eaking up o o loss o locally adap ed geno ypes is likely o be de imen al o i ness. Bo h hese compe ing o ces appea o be a wo k in his popula ion o Eas A ican Sho ho n Zebu cal es in wes e n Kenya. Conclusions In conclusion, in his indigenous ca le popula ion om he opics we ind s ong associa ions be ween genome- wide he e ozygosi y and bo h dea h and clinical illness due o in ec ious diseases. The e is e idence ha in og ession o Eu opean au ine b eeds also inc eases ulne abili y o in ec ious diseases. We he e o e belie e ha we ha e ob- se ed he e ec o bo h inb eeding and ou b eeding de- p ession, and ha e ound e idence o a gene ic componen o ulne abili y o in ec ious disease and o adap a ion in he Eas A ican Sho ho ned Zebu popula ion in wes e n Kenya. This implies ha he signi ican bu den o in ec- ious disease in his popula ion could, in p inciple, be educed by be e b eeding p ac ices. Me hods Da a se The IDEAL s udy popula ion consis ed o 548 cal es, which we e moni o ed closely h oughou hei i s yea o li e [25]. Th ee o hese cal es we e excluded om his analysis because hey ecei ed ea men o disease. The cal es we e sampled om a egion in wes e n Kenya (o app oxima ely 45 × 90 km) (co e ing some o he Busia, Teso, Siaya, Bu e e/Mumias and Bungoma dis ic s). This egion a e sed ou di e en ypes o ag o-ecological zones. The egion was di ided in o sub- loca ions o app oxima ely 10 × 10 km, o which 20 we e andomly selec ed (wi hin each ag o-ecological zone) o s udy [25]. Small-scale ca le a ming is common in his egion and he sub-loca ions con ain app oxima ely 80 o 90 households, o which app oxima ely 60% own ca le (gene ally anging om 1-10 ca le pe household). New bo n cal es we e ec ui ed in o he s udy om hese 20 sub-loca ions o e a pe iod o 2 yea s (2007-2009). Cal es ha showed isible signs o exo ic b eed in og ession we e excluded, and only cal es om small a ms, whe e he ca le spen a leas pa o hei ime ou side, exposed o in ec ious disease, we e ec ui ed [25]. Gene ic da a All cal es in he IDEAL popula ion we e geno yped using he 50K Illumina® Bo ineSNP50 beadchip . 1 in- cluding 55,777 SNPs be o e quali y con ol. S anda d quali y con ol p ocedu es we e applied o he da a p io o analysis e iewed in [37]. This was implemen ed using GenABEL. A mino allele equency o SNPs cu -o o 1% was applied and a SNP call- a e cu -o o 90%. A cu -o o call a e o an animal o 90% was applied. And an iden i y by s a e (IBS) h eshold o 90% and a cu -o odds o 1000-1 was applied o decide whe he a cal should be excluded based on sex/X-linked ma ke da a inconsis ency (which bo h elimina e e o s due o sample mis-iden i ica ion). These quali y con ol h esh- olds le a o al o 40,457 au osomal SNPs o subsequen analysis. A Ha dy-Weinbe g equilib ium (HWE) cu -o was no applied, since a di e gence om HWE can esul om ac o s o he han geno yping e o (in his case in og ession). As a esul o quali y con ol, 9 cal es and 13,856 o he 54,313 ini ial au osomal SNPs had o be excluded om he 50K SNP chip da a se due o unsuccess ul geno yping o mis-iden i ica ion o indi iduals. This e- duced he geno yping da a se o his s udy o 536 cal es and 40,457 au osomal SNPs. Fi ness pheno ype –dea h The bes p oxy o i ness in his s udy is su i al o i s yea . Eigh y-eigh o he o iginal 548 cal es died du ing he s udy, and all bu nine o hese we e subjec o a de- ailed pos -mo em examina ion [25,26]. In mos cases he cause o dea h was ound o be in ec ious disease. These esul s a e desc ibed in de ail elsewhe e [25,26], bu he mos equen causes o dea h we e Eas Coas Fe e (caused by he ick-bo ne p o ozoan Theile ia pa a), hea wa e (caused by he ick-bo ne icke sia Eh lichia uminan ium) and helmin hosis (especially associa ed wi h he nema ode Haemonchus placei). O he 88 dea hs, 9 we e ound o ha e causes unlikely o be associa ed wi h any he i able i ness, such as auma and s a a ion, and 10 had an unde e mined cause o dea h. These cal es we e excluded om he analysis, so as o make he pheno ype mo e likely o ha e a consis - en (al hough likely complex) gene ic basis. This esul ed in a da a se , a e quali y con ol, o 68 cal es ha died due o in ec ious disease, and 450 cal es ha su i ed hei i s yea (Table 1, Addi ional ile 1: Table S1). Follow-up s udies indica ed ha he dea h a e o e he nex 1-2 yea s was much lowe han du ing he i s yea . Fi ness pheno ype –clinical illness All cal es we e moni o ed o signs o clinical illness h oughou he s udy. Episodes o clinical illness we e e- po ed by he cal ’s owne , isi ing animal heal h wo ke s o he p ojec s a and we e subjec o de ailed in es iga- ion using a s anda dised p o ocol and, whe e necessa y, ollowed up by he p ojec ’s e e ina y s a [25]. O he su i ing cal es in he s udy, 243 expe ienced a leas one Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 6 o 8 h p://www.biomedcen al.com/1471-2148/13/246 epo ed episode o clinical illness. Clinical signs anged om mild o se e e. T ea men was p o ided i he cal was in dis ess and i s keepe ag eed; ea ed cal es (n = 3) we e subsequen ly excluded om he s udy. In a ew in- s ances a se e ely ill cal was eu hanized; his was ega ded as equi alen o a dea h. Almos all episodes o clinical ill- ness could be a ibu ed o in ec ions, howe e causal links a e di icul o de ini i ely es ablish and so episodes canno be unequi ocally di ided in o hose ha we e caused by in ec ious disease and hose ha we e no . Fo his eason all episodes o clinical illness we e used o dis inguish be- ween heal hy and unheal hy cal es (whe e his di e ence in heal h can gene ally be a ibu ed o a ia ion in sus- cep ibili y o in ec ious disease). This di ision esul s in 207 cal es ha nei he died no expe ienced any e- po ed episodes o clinical illness up o one yea old. Eu opean au ine in og ession Using a cu -o o ac ion 2 -6 in og ession we iden i- ied 113 in og essed cal es, wi h a maximum o 36.1% Eu opean au ine in og ession. The in og essed cal es we e no ound uni o mly dis ibu ed among he 20 sub-loca ions o he s udy: hey a e ound mainly in 12 sub-loca ions in he no he n and cen al egions o he s udya ea[28].Themos likelyo igino hisEu opean au ine in og ession in he s udy a ea is a numbe o ecen and con inuing b eed imp o emen p og ammes ha use exo ic animals and semen, and ca le ma ke s in he s udy a ea con inue o sell c ossb ed ca le [28]. Themos ac i ep og amme ookplacein hemid 1990s, equi alen o app oxima ely 5 EASZ gene a ions be o e ou s udy ook place. This is e lec ed in ou cu - o o ac ion2 -6 in og ession, co esponding o he expec ed le el o in og ession a e 5 gene a ions o back-c ossing wi h indigenous ca le since he F1 gene - a ion. Howe e , o con i m he obus ness o he p ecise alue o he cu -o , we epea ed all analyses using al e na- i e cu -o alues bu ob ained e y simila esul s. He e ozygosi y Indi idual mean genome-wide he e ozygosi y was com- pu ed as he p opo ion o all au osomal geno yped loci ha passed quali y con ol (40,457 SNPs) ha we e he - e ozygous. This was implemen ed using GenABEL [38]. To es o he whe he his is likely o be a good es ima e o genome-wide he e ozygosi y, we andomly sampled SNPs wi hou eplacemen 100 imes and calcula ed he Spea man co ela ion coe icien o he he e ozygosi y es- ima es o cal es based on one hal o he ma ke s and he es ima es based on he o he hal [2,6]. Since SNPs may in linkage disequilib ium wi h one ano he we e- pea ed he analysis compa ing smalle subse s. S a is ical me hods The compa isons be ween heal hy cal es (n = 217) and hose ha died as a esul o in ec ious disease (n = 68) o su i ed bu expe ienced clinical illness (n = 243) was in es iga ed using a 3-le el mul inomial logis ic eg ession analysis using P oc Logis ic SAS e sion 9.3 (SAS Ins i u e Inc., Ca y, NC). Bo h he e ozygosi y and in og ession we e included as p edic o s and we e ca ego ised o he pu poses o analysis. Using a cu -o o ac ion 2 -6 in og ession we iden i ied 113 in og essed cal es. We epea ed all analyses using al e na i e cu -o s o 2 -7 ,2 -5 and 2 -4 (6, 4 and 3 gene a ions o c ossing espec i ely). We ca ego ised non-in og essed cal es wi h he e ozy- gosi y < 0.274 (mo e han 0.10 below he mean o his g oup) as low he e ozygosi y, assumed o e lec a highe deg ee o inb eeding han he popula ion no m (28 cal es). We de ined low he e ozygosi y cal es in he in og essed g oup by inding he bes i linea eg ession o he e ozy- gosi y agains in og ession and i e a i ely excluding cal es wi h he e ozygosi y alues > 0.01 below hei expec ed alue ( esul ing in 9 o he 113 in og essed cal es being ca ego ised as low he e ozygosi y). We conduc ed con i ma o y uni a ia e analyses using Fishe ’s exac es (S a Xac e sion 8, Cy el So wa e Co p, Camb idge, MA, USA) o he non-in og essed and in og essed ca ego ies, allowing us o in es iga e he e ec o he e ozygosi y o hese sepa a ely. A ailabili y o suppo ing da a Geno yping da a can be ound a D yad (h p://da ad yad. o g/ esou ce/doi:10.5061/d yad.bc598). Addi ional ile Addi ional ile 1: Table S1. Table desc ibing he e ozygosi y (He ), p opo ion Eu opean au ine (%ET), whe he hey died du ing he s udy (and whe he his is known o ha e been he esul o an in ec ious disease) and whe he hey expe ienced one o mo e clinical episodes, o all o he cal es included in his analysis. Compe ing in e es s The au ho s decla ed ha hey ha e no compe ing in e es s. Au ho s’con ibu ions MW, OH, HK, PT, KC and MB designed he IDEAL p ojec . GM, MW and OH d a ed he manusc ip . GM calcula ed es ima es o he e ozygosi y. MM and MT calcula ed es ima es o in og ession. TS comple ed he SNP geno yping and SNP ma ke sco ing. MCT, MW and GM did he s a is ical analysis. ST, AJ and IC did ieldwo k o he IDEAL p ojec , pe o med au opsies and diagnos ic es s. All au ho s ead and app o ed he inal manusc ip . Acknowledgemen s The IDEAL p ojec was unded by he Wellcome T us (g an e e ence WT079445MF). USDA-ARS bo ine unc ional labo a o y and Geneseek e e ina y diagnos ics p o ided in aluable echnical assis ance o he geno yping o he samples. This p ojec was also pa ially suppo ed by USDA Ag icul u al Resea ch Se ice p ojec 1265-31000-104-00D. Men ion o ade names o comme cial p oduc s in his a icle is solely o he pu pose o p o iding speci ic Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 7 o 8 h p://www.biomedcen al.com/1471-2148/13/246 in o ma ion and does no imply ecommenda ion o endo semen by he U.S. Depa men o Ag icul u e. 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Bioin o ma ics 2007, 23:1294–1296. doi:10.1186/1471-2148-13-246 Ci e his a icle as: Mu ay e al.:Gene ic suscep ibili y o in ec ious disease in Eas A ican Sho ho n Zebu: a genome-wide analysis o he e ec o he e ozygosi y and exo ic in og ession. BMC E olu iona y Biology 2013 13:246. Mu ay e al. BMC E olu iona y Biology 2013, 13:246 Page 8 o 8 h p://www.biomedcen al.com/1471-2148/13/246