CONICET | Buscador de Institutos y Recursos Humanos

IntroductionBovine leukemia virus (BLV) shares the genomic organization structure with T-cell tropicviruses from primates (human and simian), and constitute a unique subgroup within theRetroviridae family, characterized by distinct genetic content, genomic organization, andstrategy for gene expression (Coffin 1996). BLV is associated with enzootic bovine leucosis(EBL), which is the most common neoplastic disease of cattle. Most infected cattle remainasymptomatic. By contrast, approximately 30% of cattle develop persistent lymphocytosis(PL), which is characterized as the polyclonal expansion of non-neoplastic CD5+ Blymphocytes.PL often precedes tumorigenesis but is usually regarded as clinically healthy.After a latency period of 1-8 years, less than 10% of infected cattle develop EBL, which is amalignant CD5+ B-cell lymphoma (Ferrer 1980; Gillet et al. 2007). BLV has a silentdissemination in the herd due to infected cell exchange (Hopkins and DiGiacomo 1997),thus the concentration of BLV-infected cells in blood should play a major role in thesuccess of viral transmission. Even though PL cattle are considered the most risky fortransmission, asymptomatic animals may also play a role depending on their proviral load(Juliarena et al. 2007).The major histocompatibility complex (MHC) molecules are responsible for presentationof processed antigen peptides to T cells, and thereby an adaptive immune response tospecific pathogens is trigged. The high degree of polymorphism of the MHC moleculesenables the presentation of a wide array of peptides that differ in their length andsequence. Thus, the nature and strength of the immune responses depend on the peptideand the MHC molecules, and may influence disease progression (Amills et al. 1998).Page 1 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only2Bovine leukocyte antigen (BoLA), the MHC system of cattle, is highly polymorphic.Functionally, the BoLA class II gene is classified into two groups, DR and DQ. The DRA andDRB3 gene products form the DR molecule, as DRB3 is the only functional DRB gene withinBoLA (Takeshima and Aida 2006).Genes from BoLA are remarkably attractive to animal breeders and veterinary geneticist,and polymorphism in BoLA genes has been relatively well studied. These genes areassociated with genetic resistance and susceptibility to a wide range of diseases, includingmastitis (Dietz et al. 1997; Rupp et al. 2007; Yoshida et al. 2009; Yoshida et al. 2012),dermatophilosis (Maillard et al. 2002), tick-borne diseases (Duangjinda et al. 2013),infection with Neospora caninum (Schwab et al. 2009), African trypanosome species(Karimuribo et al. 2011) and Theileria annulata (Glass et al. 2012b). Moreover, BoLAappears to influence other traits such as milk yield, growth and reproduction, andvariations in immune responses to antigens (Takeshima and Aida 2006) even vaccinepeptides (Garcia-Briones et al. 2000; Glass et al. 2012a).The greatest amount of evidence linking BoLA gene polymorphisms and disease outcomeis given by BLV infection. First evidences revealed an association between resistance orsusceptibility to PL and BoLA A class I alleles (Lewin and Bernoco 1986); however, theseassociations were relatively weak at population levels and different BoLA A alleles hadsignificant effects in different breeds (Lewin et al. 1988; Stear et al. 1988). Subsequentstudies showed that resistance and susceptibility to PL map more closely to MHC class IIBoLA DRB3.2 gene than to BoLA A locus (Esteban et al. 2009). Thus, most studies havefocused on BoLA DRB3.2 allele polymorphisms in Holstein cattle, and their relationshipPage 2 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only3with disease. Some alleles were associated with resistance to PL or to BLV-inducedlymphosarcoma, or the capability of controlling the number of BLV-infected cells, whileothers alleles were associated with susceptibility to PL (Xu et al. 1993; Zanotti et al. 1996;Mirsky et al. 1998; Panei et al. 2009; Nikbakht Brujeni et al. 2016). When BLV-infectedanimals were characterized into two profiles of infection ? high and low proviral load(HPVL and LPVL respectively) (Juliarena et al. 2007), BoLA DRB3.2 *0902 allele was thestrongest associated with LPVL profile, while *1501 or 03 allele showed a significantassociation with HPVL in carrier animals (Juliarena et al. 2008). Miyasaka et al. alsoanalyzed the effect of BoLA DRB3.2 alleles on proviral load in Japanese cattle; they foundthat *0902 and *1101 alleles were associated with LPVL and *1601 allele with HPVL andidentified BoLA class II haplotypes that correlated with different PVL status (Miyasaka etal. 2013).Here, we analyzed whether HPVL or LPVL status in the BLV-infected animals from our herdunder study were associated with specific BoLA DRB3.2 alleles. This study aims tocontribute to the knowledge of the association between BoLA polymorphism anddevelopment of a BLV infection profile.Material and methodsSample collectionBlood samples were obtained from 107 BLV-infected Argentinean Holstein (Holando-Argentino) dairy cows belonging to a herd from the Tandil region (Provincia de BuenosAires, Argentina). All animals were healthy and milking (between second and thirdPage 3 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only4lactation period) and although the pedigree information was not complete in the 3 % ofthem, they came at least from 51 and 87 different bulls and cows, respectively. SerologicalBLV status was determined by testing plasma for anti-gp51 antibodies by ELISA 108(Gutierrez et al. 2001). For peripheral blood mononuclear cells (PBMCs) separation, 10 mlblood samples were collected in heparinized syringes by jugular venipuncture. Bloodsamples were transferred to 15 ml tubes and centrifuged for 15 min at 3800 rpm at 4 ºC.Buffy coat was mixed with 11 ml of cold ammonium chloride buffer (150 mM NH4Cl, 8 mMNa2CO3, and 6 mM EDTA) for red blood cells lysis. PBMCs were obtained by centrifugationat 3000 rpm during 10 min at 4 ºC. Cells were washed with PBS and centrifuged at 2500rpm for 7 min at 4 ºC. PBMCs pellets were stored at -20 ºC for DNA extraction.DNA extractionDNA from PBMCs was extracted using Qiagen columns (QIAamp DNA Mini Kit) accordingto the manufacturer´s protocol. DNA was eluted in 50 μl of water. The concentration andpurity of DNA were determined by the OD value at 260 nm and 260/280 coefficientrespectively in a NanoDrop 2000 Spectrophotometer (Thermo Scientific). The purifiedDNAs were stored at -20 ºC until use.Proviral load determination by real-time PCR (qPCR)The absolute quantification method by qPCR used for proviral load (PVL) determinationwas recently described (Farias et al. 2016). The standard curve was constructed using aplasmid carrying one copy of the entire BLV genome under control of its own promoterPage 4 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only5LTR (pBLV), with six 10-fold serial dilutions of pBLV, containing 1 million to 10 BLV copies.The qPCR conditions for pol amplification were previously described (Farias et al. 2016).Each reaction contained 30 ng of DNA from BLV infected animals. The standard curve andnon-template control were included in each run. Experiments were always performed intriplicate. BLV-positive animals were classified as high proviral load (HPVL) (> 1000 BLVcopies/reaction) and low proviral load (LPVL) (< 100 BLV copies/reaction). Beforeclassifying the animals into each group, the proviral load was evaluated two times at 6-month intervals, to confirm their PVL. Eighty-eight out of 107 animals were HPVL, whilethe remaining 19 proved to be LPVL.BoLA DRB3.2 genotypingPCR reactions were performed in a final volume of 15 μl, where the volume mixturecontained: 6 ng of genomic DNA, 0.03 X PCR buffer (Mg2+ plus), 0.3 μM of each primer(HL030; 5?-ATCCTCTCTCTGCAGCACATTTCC-3?, and HL031; 5?-TTTAAATTCGCGCTCACCTCGCCGCT-3´), 0.2 mM of dNTP and 0.75 U Paq5000® DNAPolymerase (Agilent Technologies, Stratagene) and water up to a total volume of 15 μl.Conditions for amplification were 94 °C for 3 min, followed by 35 cycles of 20 s at 94 °C, 20s at 60 °C (annealing temperature), and 1 min at 72 °C. Reactions were finished with a 72°C, 5-min extension. PCR reactions were performed in a Mastercycler (Eppendorf®)thermocycler. Amplified fragments from DNA were visualized in a 1% agarose gel andsequenced using BigDye® chemistry on an ABI3130xl sequencer (Applied Biosystems)following the manufacturer?s protocol. Sequencing reactions were performed in bothPage 5 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only6senses using the same primers as for PCR reactions, in a total volume of 10 μl containing a1:10 dilution of the PCR product as template and 0.1 μM of primer, in addition to the mixand buffer 5X of BigDye® Terminator kit. The sequence analysis and obtaining genotypeswas performed using Haplofinder (Baxter et al. 2008).Statistics analysisAssociation between different BoLA DRB3.2 alleles and proviral load was estimated by Chi2test or exact Fisher test, as appropriate, implemented by PROC FREQ (SAS v9.3, InstituteInc., Cary, NC, USA). With genes that were significant to the bivariate analysis, logisticregression was performed to identify genes that best explain the CPV, using the PROCLOGISTIC (SAS v9.3).ResultsAnalysis of BoLA DRB3.2 genotypingAfter genotyping, a total of 18 BoLA DRB3.2 alleles defined according to the ISAG BoLANomenclature Committee were identified in these Argentinean Holstein cattle. The fivemost frequently alleles were BoLA DRB3.2*1001 (17.29%), *1501 (16.36%), *1101(14.95%), *0101 (12.15%) and *1201 (11.21%) (Table 1). We found 47 genotypes, whereBoLA DRB3.2*1001 + BoLA-DRB3.2*1501 (9 animals, 18.75%), BoLA DRB3.2*1101 + BoLADRB3.2*1501 (8 animals, 16.67%) and BoLA DRB3.2*1001 + BoLA DRB3.2*1201 (6 animals,12.5%) were the most frequent in the population (data not shown).Page 6 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only7Association between BoLA DRB3.2 alleles and proviral load (PVL).BLV-positive cattle (N=107) were categorized into 2 groups according to their PVL byqPCR: 88 had HPVL and 19 had LPVL. The presence or absence of each BoLA DRB3.2 allelein this cattle population was analyzed in association with PVL (Table 2). No significantassociation was observed between PVL and alleles having frequency lower than 10%.Among alleles with higher frequency in the population, *1501 and *1201 weresignificantly associated with HPVL (p=0.0230 and p=0.0111 respectively), while allele*0201 was significantly associated with LPVL (p=0.0030) (Table 2).Distribution of BoLA DRB3.2 alleles significantly associated with PVL.BoLA DRB3.2*1501, *1201 and *0201 were significantly associated with PVL. Contrastswere estimated in order to compare absence and presence of each one in homozygousand heterozygous form.BoLA DRB3.2*1501 allele is present in heterozygosis in 94% of HPVL animals; animals thatcarry it are about 5 times more likely to have HPLP (Chi2 test: p=0.0230) (Table 3). BoLADRB3.2*1201 allele is present only in HPVL animals (Fisher exact test: p=0.0187) (Table 4).BoLA DRB3.2*0201 allele is present in heterozygous in 50% of HPVL animals and 50% ofLPVL animals; among animals that do not possess this allele, 87% have HPVL (Fisher exacttest: p=0.0030) (Table 5). Thus, animals that have *0201 allele would be protected todevelop HPVL.A logistic regression model was run with the BoLA DRB3.2*0201 and *1501 alleles as theindependent variables, and the HPVL profile as the dependent variable. This analysisPage 7 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only8indicated that *0201 allele is a protective factor (p=0.0024) while *1501 allele is a riskfactor (p=0.0341) for HPVL profile (Table 6). Thus, genes that best explains the PVL areBoLA DRB3.2*0201 (as a protection factor) and *1501 (as a risk factor), with no significantinteraction estimated between them (p=0.957).DiscussionIn this study, we analyzed the distribution of BoLA DRB3.2 alleles in an ArgentineanHolstein herd and its association with the BLV proviral load. In this group of animals(N=107), we identified 18 alleles and BoLA DRB3.2*1001 had the highest allelic frequency(17.29%). There is not too much information about the allelic diversity of BoLA DRB3 classII gene in the different cattle breeds. As far as we know, only one study described thisdiversity in South American Holstein populations (Takeshima et al. 2015). These authorsfound that BoLA DRB3*1501 appeared to be widely spread in the cattle of countries ofSouth America: 18.2% in Bolivia, 17.7% in Paraguay, 21.4% in Peru, 21,7% in Chile and14.7% in Argentina. We found a slight higher frequency of this allele in our population(16.36%). BoLA DRB3*0101 and *1101 were also in high frequency in these Holsteinpopulation of South American countries. Even though BoLA DRB3*0101 was the mostfrequently found in Argentina (17.7%), it was present one third less in our population(12.15%). The greater differences in allelic frequency between Takeshima?s and our studywere in BoLA DRB3*1201 (7.3% vs 11.21%), *1001 (5% vs 17.29%) and *0201 (3.2% vs6.54%). The higher frequency of the analyzed alleles found in this research could be aresult of the selection for trait parameters applied in our herd. And this fact might havePage 8 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only9implications on susceptibility or resistance to diseases, immunological and productiontrails, and vaccine responses.Related to the infection with BLV in Holstein breed, several studies shown that thesusceptibility to PL was associated with the presence of BoLA DRB3*0101, *1501 or 03,*1101 or 02 and *1201 (Xu et al. 1993; Zanotti et al. 1996; Mirsky et al. 1998; Juliarena etal. 2008; Panei et al. 2009; Nikbakht Brujeni et al. 2016). In our study, BoLA DRB3*1201was the most significantly associated with HPVL (p=0.0111), and this might be due to thefact that its frequency in our population was about one third higher than in otherArgentinean Holstein populations (11.21% vs 7.3%, respectively) (Takeshima et al. 2015).We also found a strong association between HPVL and the presence of BoLA DRB3*1501(p=0.0230). These two alleles are the most frequently associated with susceptibility to PLor HPVL in BLV-infected animals from different cattle breeds (Xu et al. 1993; Sulimova etal. 1995; Zanotti et al. 1996; Udina et al. 2003; Juliarena et al. 2008). BoLA DRB3*1001 hadthe highest frequency in our herd, and in the literature it was is referred as a susceptible(Xu et al. 1993; Udina et al. 2003) or a neutral allele (Xu et al. 1993) for PL development.However, no association with BLV status could be established in BoLA DRB3*1001 carrieranimals from our study. In Japanese cattle, Miyasaka et al. found that *1601 allele wasassociated with HPVL (Miyasaka et al. 2013), but we could not identify animals carryingthis allele in our herd.Resistance to PL or a LPVL profile was associated with alleles that are generally lessdistributed in cattle population. We found a strong association between DRB3.2*0201 andLPVL (p=0.030). We can speculate that the absence of this allele could be an indicative ofPage 9 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only10a putative development of HPVL (Table 5). Panei et al. studied a herd of 81 ArgentineanHolstein animals by PCR-RFLP and mentioned BoLA DRB3.2*11, *23, *28, *25 and *40(ISAG *0902, *2703, *0701 or 03, and non-classified respectively) as associated with PLresistance (Panei et al. 2009); but we could not find the same results in our population.Even though several studies cited *1101 linked to PL susceptibility in different cattlebreeds (Xu et al. 1993; Sulimova et al. 1995; Udina et al. 2003; Panei et al. 2009; NikbakhtBrujeni et al. 2016), it was associated with LPVL in Japanese cattle (Miyasaka et al. 2013).We found a significant number of animals carrying this allele in our herd, compared to thepopulation of Holstein in Argentina (Takeshima et al. 2015); however no association couldbe established between allele BoLA DRB3.2*1101 and BLV PVL. In the literature, BoLADRB3.2*0902 (or *11 determined by PCR-RFLP) allele was the strongest associated with PLresistance or LPVL profile in Holstein cattle (Xu et al. 1993; Zanotti et al. 1996; Mirsky etal. 1998; Juliarena et al. 2008; Panei et al. 2009; Miyasaka et al. 2013). In our study, we didnot found this association; it could be due to a lower frequency of presentation of *0902allele in this herd in comparison with the Argentinean Holstein populations (4.21% vs6.4%, respectively) (Takeshima et al. 2015).Genetic selection for BoLA DRB3.2*0902 allele has been proposed to breed cattle that areresistant to HPVL development (Juliarena et al. 2016). Evidences from the literature revealthat the polymorphism in the BoLA class II gene influences immune response by peptidebinding, antigen presentation, T-cell repertoire and cytokine networks (Takeshima andAida 2006). Thus, allelic differences in BLV infected animals may play an important role inthe development of effective immune responses. However, BLV-resistance might not bePage 10 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only11only related to a single allele of the DRB3.2 gene. Indeed, about 19% of cattle carrying theBoLA DRB3.2*0902 allele develop HPVL (Juliarena et al. 2008). It must be considered thatexpression of BoLA DRB3 might be influenced by polymorphisms in other surroundinggenes. An important gene of the immune system that has been linked to the BoLA regionis the promoter region of tumor necrosis factor-alpha (TNF-α); both genes are located inthe q22 arm of the bovine chromosome 23 and in linkage disequilibrium (Lendez et al.2015). Concerning BLV infection, TNF-α is closely related with the immune responseagainst viral dissemination, stimulating the elimination of infected lymphocytes (Kabeya etal. 1999). Konnai et al. described that genotype G/G in position -824 of the promoterregion of TNF-α was associated with low transcriptional activity of the promoter region.The frequency of this genotype G/G was higher in individuals with BLV-induced lymphomathan in asymptomatic carrier individuals. Moreover, a tendency toward an increased BLVprovirus load in cattle with the TNF- α ?824 G/G homozygote, compared to the A/Ahomozygote or genotype A/G, was observed (Konnai et al. 2006). Recent results from ourgroup, analyzing a large population of cattle with HPVL or LPVL, revealed a significantassociation between LPVL and a low frequency of the G/G genotype at position -824(Lendez et al. 2015). That means that an animal carrying this polymorphism most probablybelong to the HPVL group. In the present study, we also analyzed the polymorphism in thepromoter region of TNF-α; however, no statistical differences were found betweenanimals with HPVL or LPVL (data not shown), probably due to the small number of animalswith LPVL. Taking together, it seems that several polymorphisms in different genes maycontribute to develop a resistance profile in BLV-infected cattle.Page 11 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production ScienceFor Review Only12On the other hand, other issues like production traits improvement, resistance to mastitispathogens, somatic cell counts and milk yield have also been related to specific BoLAalleles (Dietz et al. 1997; Rupp et al. 2007; Yoshida et al. 2009; Baltian et al. 2012; Yoshidaet al. 2012). However, some controversies exist between different studies. Recently,Abdalla et al. communicate a genome-wide association mapping in order to analyze BLVincidence in US Holstein cattle population, together with milk yield and somatic cell score,combining pedigree and molecular marker information, for the detection of genomicregions and gene pathways associated with the disease. The study revealed BLV incidenceas a complex trait, possibly modulated by several genes of small effects (Abdalla et al.2016).Our study aims to contribute to the knowledge of the association between BoLApolymorphism and development of a BLV infection profile. Genes that best explains thePVL in this population resulted BoLA DRB3.2*0201 (as a protection factor) and *1501 (as arisk factor). Allelic differences may play an important role in the development of effectiveimmune responses, against BLV and other pathogens. Thus, genetic selection based onspecific BoLA alleles should be implemented with great caution considering the variableeffects of these genes on a wide range of diseases and production traits. A betterunderstanding of how BoLA polymorphism contributes to the development of effectiveimmune response and the establishment of a BLV status is desirable to schedule andevaluate control measures.Page 12 of 25http://www.publish.csiro.au/nid/72.htmAnimal Production Science