AbstractBeagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restriction within this breed and the impact this might have on vaccine responses are rarely considered. This study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were determined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles had significantly different DLA haplotype frequencies in comparison with pet Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in selective antigen presentation and vaccine responses that are not necessarily representative of those seen in other dog breeds

Catchpole, B

Kennedy, L J

Oilier, W E R

Solano-Gallego, L

Soutter, F

English

PubMed

RVC Research Online

Short CommunicationRestricted dog leucocyte antigen (DLA) class II haplotypes andgenotypes in BeaglesFrancesca Soutter a,*, Lorna J. Kennedy b, William E.R. Ollier b, Laia Solano-Gallego c,Brian Catchpole aa Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, United Kingdomb Centre for Integrated Genomic Medical Research, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdomc Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria, Universitat Autònoma de Barcelona, Barcelona, SpainA R T I C L E I N F OArticle history:Accepted 29 December 2014Keywords:BeagleDog leucocyte antigenMajor histocompatibility complexA B S T R A C TBeagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restric-tion within this breed and the impact this might have on vaccine responses are rarely considered. Thisstudy was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breedingcolony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were deter-mined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagleshad significantly different DLA haplotype frequencies in comparison with pet Beagles and both groupsshowed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result inselective antigen presentation and vaccine responses that are not necessarily representative of those seenin other dog breeds.© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).Vaccine efficacy is fundamental to ensuring protection of animalsagainst infectious disease and inmaintaining herd immunity. Beaglesare used for vaccine trials, designed to provide evidence of effica-cy for regulatory approval. However, dog breeds, including the Beagle,have been derived from at least two genetic bottlenecks(Lindblad-Toh et al., 2005), which might affect their immunoge-netic diversity and vaccine responses. Vaccination induced antibodytitres can vary with breed, although genetic factors have yet to beidentified (Kennedy et al., 2007b).Dog leucocyte antigen (DLA) class II genes encode major histo-compatibility complex (MHC) molecules that are involved in antigenpresentation to CD4+ T cells. In dogs, these consist of three, highlypolymorphic, loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1 (Wagner,2003). There is a large degree of inter-breed variability, but oftenlimited intra-breed diversity, of DLA haplotypes (Kennedy et al.,2002a). The aim of the present study was to characterise DLAhaplotypes in laboratory Beagle dogs and to compare their DLA di-versity with a pet Beagle population.Residual blood samples were obtained from laboratory Beagles(n = 100) in a commercial breeding facility, that provided puppiesfor vaccine trials in Europe; sampleswere collected for another study(project number114-C-E-01-10; date of Animal Care andEthics Com-mittee approval 7 June2010). Blood samples frompetBeagles (n = 47)were obtained from the Royal Veterinary College genetic archive,containing residual samples followingcompletionof diagnostic testingwith informed owner consent and with approval in 2004 from theinstitutional Ethics andWelfare Committee. Forty-eight pet Beagles,previously genotyped at theUniversity ofManchester (date of AnimalCare and Ethics Committee approval April 2004)were also analysedand included in the pet Beagle group. Information on pedigrees andrelatedness of the dogs was not available.Genomic DNA was extracted using the GenElute Blood GenomicDNA Kit (Sigma-Aldrich). PCRwas performed in 25 μL reactions, with1 μL DNA as template, DLA-specific primers (2 μL at 20 pmol/μL finalconcentration; Sigma-Aldrich) (see Appendix: SupplementaryTable S1), 5 μL Hi-Spec additive, 2.5 μL ImmoBuffer, 1.25 μL MgCl2(2.5 mM final concentration), 0.25 μL deoxynucleotide triphos-phates (1mMfinal total concentration) and 0.1 μL (1.25 IU) ImmolaseDNA polymerase (Bioline). PCR was performed using a G-Storm GS1Thermal Cycler (Gene Technologies) at 95 °C for 10 min, followedby 35 cycles of 94 °C for 40 s, 55 °C for 30 s for DQA1 or 60 °C forDRB1 and DQB1, and 72 °C for 1min, then 72 °C for 10min. PCR prod-ucts were processed using the GenElute PCR Clean-up Kit (Sigma-Aldrich) and submitted for sequencing (Source Bioscience) usingprimer M13F. DLA analysis and allele assignment was performedusing SBT Engine version 2.17 (GenDx). Haplotype frequencies werecompared between groups using Fisher’s exact test (Monte Carlomethod) in SPSS (PASW Statistics 18, IBM).* Corresponding author. Tel.: +44 170 766 6801.E-mail address: fsoutter@rvc.ac.uk (F. Soutter).http://dx.doi.org/10.1016/j.tvjl.2014.12.0321090-0233/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).The Veterinary Journal 203 (2015) 345–347Contents lists available at ScienceDirectThe Veterinary Journaljournal homepage: www.elsevier.com/ locate / tv j lFourteen DLA haplotypes were identified (12 in pet Beagles andeight in laboratory Beagles). Only four haplotypes occurred at a fre-quency >0.05 in each group and three of these more frequenthaplotypes were the same in both groups (Table 1). Whilst someoverlap existed in haplotypes between the two groups, the labo-ratory Beagle group demonstrated a significant difference in DLAhaplotype frequencies compared with pet Beagles (P < 0.01). Morepet Beagles were homozygous (frequency = 0.45) than laboratoryBeagles (frequency = 0.21; χ2 = 11.93; P < 0.001). Pet Beagles werepredominantly homozygous for haplotype DLA-DRB1*001:02–DQA1*001:01–DQB1*002:01 (frequency = 0.70 of homozygous petBeagles; n = 43).These results show that the laboratory Beagles had a differentDLA profile, compared with the pet Beagles. Founder effects, selec-tive breeding within a closed gene pool and use of popular sires havehad an impact on genetic diversity within pedigree breeds(Lindblad-Toh et al., 2005; Calboli et al., 2008). The differencebetween these populations could be the result of selective breed-ing practices, since the groups have been bred for different purposesand likely represent subpopulations from historical Beagle stock withdistinct founders (Kennedy et al., 2002b).Neither group represents the DLA diversity in the dog popula-tion as a whole, where some breeds (e.g. Jack Russell terrier) expressa more varied DLA profile, whereas others (e.g. Rottweiler) are evenmore restricted (Kennedy et al., 2007a). The limited diversity of DLAtypes could influence vaccine responses in Beagles and a producttested in Beagles will not necessarily perform similarly in other dogbreeds.Both groups were relatively restricted in DLA haplotype diver-sity. Whilst this restriction is not as marked as for some breeds, suchas the Rottweiler, where only four haplotypes have been identi-fied (Kennedy et al., 2002a), it is likely to affect the repertoire ofpeptide epitopes presented to CD4+ T cells upon antigenic stimu-lation. Since some haplotypes are only represented in a small numberof Beagles, there is a risk that their frequency may diminish in sub-sequent generations if breeding strategy is not considered withina closed colony.There was a significant difference in DLA homozygosity betweengroups. Without pedigrees or breeding strategy information, we canonly hypothesise that this high frequency of homozygosity withinthe pet Beagles might indicate a higher degree of inbreeding and/or a potential dominant sire effect. Homozygosity appears to varybetween dog breeds, depending on the size of the population base;a previous study found 28.4% homozygosity across breeds (Kennedyet al., 2002a).Laboratory Beagles represent a different immunogenetic poolcomparedwith pet Beagles. Theymay be a suitable choice for vaccinestudies if genetic diversity and heterozygosity can be maintainedby strategic breeding. However, laboratory Beagles do not expressthe full range of DLA haplotypes and thus are not representativeof the diversity seen within the pet dog population as a whole. Thisposes potential problems for the use of any one breed in vaccinestudies, since DLA type could influence responses to antigenic chal-lenge. MHC class II heterozygosity in humans has been associatedwith enhanced resistance and more effective clearance of patho-gens (Thursz et al., 1997; Carrington et al., 1999). Further studiesare needed to investigate the impact of DLA genotype on suscep-tibility to infectious disease and response to vaccination in dogs.Conflict of interest statementNone of the authors of this paper has a financial or personal re-lationship with other people or organisations that couldinappropriately influence or bias the content of the paper.AcknowledgementsThis study was supported by a Biotechnology and Biological Sci-ences Research Council (BBSRC) Collaborative Awards in Science andEngineering (CASE) studentship (BB/I015655/1) in partnership withZoetis. We wish to thank Susanna Martorell for sample collectionand Angela Holder for technical assistance. Preliminary results werepresented as an Abstract at the Worldleish 5 Conference, Porto deGalinhas, Brazil, 13–17 May 2013.Appendix: Supplementary materialSupplementary data to this article can be found online atdoi:10.1016/j.tvjl.2014.12.032.ReferencesCalboli, F.C., Sampson, J., Fretwell, N., Balding, D.J., 2008. Population structure andinbreeding from pedigree analysis of purebred dogs. Genetics 179, 593–601.Carrington, M., Nelson, G.W., Martin, M.P., Kissner, T., Vlahov, D., Goedert, J.J., Kaslow,R., Buchbinder, S., Hoots, K., O’Brien, S.J., 1999. HLA and HIV-1: Heterozygoteadvantage and B*35-Cw*04 disadvantage. Science 283, 1748–1752.Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Bennett, D., Angles, J.M., Day, M.J.,Carmichael, N., Innes, J.F., Isherwood, D., et al., 2002a. Extensive interbreed, butminimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. TissueAntigens 59, 194–204.Table 1Frequencies of three DLA locus haplotypes in pet and laboratory Beagles.Haplotype Pet Beagles (n = 95) Laboratory Beagles (n = 100) Fisher’s exact test PvalueDRB1 DQA1 DQB1 Number ofhomozygous dogsTotal number ofhaplotypesHaplotypefrequency (%)Number ofhomozygous dogsTotal number ofhaplotypesHaplotypefrequency (%)001:02 001:01 002:01 30 87 45.8 2 34 17 <0.01015:01 009:01 001:01 6 36 18.9 5 50 25 NS001:01 001:01 002:01 2 21 11 8 56 28 <0.01002:01 009:01 001:01 1 17 8.9 0 7 3.5 <0.05008:01 003:01 004:01 1 9 4.7 0 0 0 <0.01006:01 005:01 007:01 1 8 4.2 6 46 23 <0.01001:02 009:01 001:01 1 5 2.6 0 0 0 <0.05006:01 004:01 013:03 1 2 1 0 0 0 NS015:01 006:01 003:01 0 2 1 0 0 0 NS002:01 009:01 002:01 0 1 0.5 0 2 1 NS008:02 001:01 002:01 0 1 0.5 0 0 0 NS020:01 004:01 013:03 0 1 0.5 0 0 0 NS015:01 006:01 020:02 0 0 0 0 4 2 NS048:01 004:02 023:01 0 0 0 0 1 0.5 NSTotal 43 190 100 21 200 100NS, not significant (P ≥ 0.05).346 F. Soutter et al./The Veterinary Journal 203 (2015) 345–347Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Courtenay, O., Carter, S.D., Ollier,W.E., Thomson, W., 2002b. Evidence for extensive DLA polymorphism in differentdog populations. Tissue Antigens 60, 43–52.Kennedy, L.J., Barnes, A., Short, A., Brown, J.J., Lester, S., Seddon, J., Fleeman, L.,Francino, O., Brkljacic, M., Knyazev, S., et al., 2007a. Canine DLA diversity: 1. Newalleles and haplotypes. Tissue Antigens 69 (Suppl. 1), 272–288.Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L., Fooks, A.R., Baxter, D.N., Ollier, W.E.,2007b. Factors influencing the antibody response of dogs vaccinated againstrabies. Vaccine 25, 8500–8507.Lindblad-Toh, K., Wade, C.M., Mikkelsen, T.S., Karlsson, E.K., Jaffe, D.B., Kamal, M.,Clamp, M., Chang, J.L., Kulbokas, E.J., 3rd, Lander, E.S., 2005. Genome sequence,comparative analysis and haplotype structure of the domestic dog. Nature 438,803–819.Thursz, M.R., Thomas, H.C., Greenwood, B.M., Hill, A.V., 1997. Heterozygote advan-tage for HLA class-II type in hepatitis B virus infection. Nature Genetics 17,11–12.Wagner, J.L., 2003. Molecular organisation of the canine major histocompatibilitycomplex. The Journal of Heredity 94, 23–26.347F. Soutter et al./The Veterinary Journal 203 (2015) 345–347

Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles

Francesca Soutter

Lorna J. Kennedy

William E.R. Ollier

Laia Solano-Gallego

Brian Catchpole

Crossref

The Veterinary Journal

Soutter, Francesca

Kennedy, Lorna J.

Ollier, William E.R.

Solano-Gallego, Laia

Catchpole, Brian

Elsevier - Publisher Connector 

Short Communication
Restricted dog leucocyte antigen (DLA) class II haplotypes and
genotypes in Beagles
Francesca Soutter a,*, Lorna J. Kennedy b, William E.R. Ollier b, Laia Solano-Gallego c,
Brian Catchpole a
a Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, United Kingdom
b Centre for Integrated Genomic Medical Research, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom
c Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria, Universitat Autònoma de Barcelona, Barcelona, Spain
A R T I C L E I N F O
Article history:
Accepted 29 December 2014
Keywords:
Beagle
Dog leucocyte antigen
Major histocompatibility complex
A B S T R A C T
Beagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restric-
tion within this breed and the impact this might have on vaccine responses are rarely considered. This
study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding
colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were deter-
mined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles
had signiﬁcantly different DLA haplotype frequencies in comparison with pet Beagles and both groups
showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in
selective antigen presentation and vaccine responses that are not necessarily representative of those seen
in other dog breeds.
© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Vaccine eﬃcacy is fundamental to ensuring protection of animals
against infectious disease and inmaintaining herd immunity. Beagles
are used for vaccine trials, designed to provide evidence of eﬃca-
cy for regulatory approval. However, dog breeds, including the Beagle,
have been derived from at least two genetic bottlenecks
(Lindblad-Toh et al., 2005), which might affect their immunoge-
netic diversity and vaccine responses. Vaccination induced antibody
titres can vary with breed, although genetic factors have yet to be
identiﬁed (Kennedy et al., 2007b).
Dog leucocyte antigen (DLA) class II genes encode major histo-
compatibility complex (MHC) molecules that are involved in antigen
presentation to CD4+ T cells. In dogs, these consist of three, highly
polymorphic, loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1 (Wagner,
2003). There is a large degree of inter-breed variability, but often
limited intra-breed diversity, of DLA haplotypes (Kennedy et al.,
2002a). The aim of the present study was to characterise DLA
haplotypes in laboratory Beagle dogs and to compare their DLA di-
versity with a pet Beagle population.
Residual blood samples were obtained from laboratory Beagles
(n = 100) in a commercial breeding facility, that provided puppies
for vaccine trials in Europe; sampleswere collected for another study
(project number114-C-E-01-10; date of Animal Care andEthics Com-
mittee approval 7 June2010). Blood samples frompetBeagles (n = 47)
were obtained from the Royal Veterinary College genetic archive,
containing residual samples followingcompletionof diagnostic testing
with informed owner consent and with approval in 2004 from the
institutional Ethics andWelfare Committee. Forty-eight pet Beagles,
previously genotyped at theUniversity ofManchester (date of Animal
Care and Ethics Committee approval April 2004)were also analysed
and included in the pet Beagle group. Information on pedigrees and
relatedness of the dogs was not available.
Genomic DNA was extracted using the GenElute Blood Genomic
DNA Kit (Sigma-Aldrich). PCRwas performed in 25 μL reactions, with
1 μL DNA as template, DLA-speciﬁc primers (2 μL at 20 pmol/μL ﬁnal
concentration; Sigma-Aldrich) (see Appendix: Supplementary
Table S1), 5 μL Hi-Spec additive, 2.5 μL ImmoBuffer, 1.25 μL MgCl2
(2.5 mM ﬁnal concentration), 0.25 μL deoxynucleotide triphos-
phates (1mMﬁnal total concentration) and 0.1 μL (1.25 IU) Immolase
DNA polymerase (Bioline). PCR was performed using a G-Storm GS1
Thermal Cycler (Gene Technologies) at 95 °C for 10 min, followed
by 35 cycles of 94 °C for 40 s, 55 °C for 30 s for DQA1 or 60 °C for
DRB1 and DQB1, and 72 °C for 1min, then 72 °C for 10min. PCR prod-
ucts were processed using the GenElute PCR Clean-up Kit (Sigma-
Aldrich) and submitted for sequencing (Source Bioscience) using
primer M13F. DLA analysis and allele assignment was performed
using SBT Engine version 2.17 (GenDx). Haplotype frequencies were
compared between groups using Fisher’s exact test (Monte Carlo
method) in SPSS (PASW Statistics 18, IBM).
* Corresponding author. Tel.: +44 170 766 6801.
E-mail address: fsoutter@rvc.ac.uk (F. Soutter).
http://dx.doi.org/10.1016/j.tvjl.2014.12.032
1090-0233/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
The Veterinary Journal 203 (2015) 345–347
Contents lists available at ScienceDirect
The Veterinary Journal
journal homepage: www.elsevier.com/ locate / tv j l
Fourteen DLA haplotypes were identiﬁed (12 in pet Beagles and
eight in laboratory Beagles). Only four haplotypes occurred at a fre-
quency >0.05 in each group and three of these more frequent
haplotypes were the same in both groups (Table 1). Whilst some
overlap existed in haplotypes between the two groups, the labo-
ratory Beagle group demonstrated a signiﬁcant difference in DLA
haplotype frequencies compared with pet Beagles (P < 0.01). More
pet Beagles were homozygous (frequency = 0.45) than laboratory
Beagles (frequency = 0.21; χ2 = 11.93; P < 0.001). Pet Beagles were
predominantly homozygous for haplotype DLA-DRB1*001:02–
DQA1*001:01–DQB1*002:01 (frequency = 0.70 of homozygous pet
Beagles; n = 43).
These results show that the laboratory Beagles had a different
DLA proﬁle, compared with the pet Beagles. Founder effects, selec-
tive breeding within a closed gene pool and use of popular sires have
had an impact on genetic diversity within pedigree breeds
(Lindblad-Toh et al., 2005; Calboli et al., 2008). The difference
between these populations could be the result of selective breed-
ing practices, since the groups have been bred for different purposes
and likely represent subpopulations from historical Beagle stock with
distinct founders (Kennedy et al., 2002b).
Neither group represents the DLA diversity in the dog popula-
tion as a whole, where some breeds (e.g. Jack Russell terrier) express
a more varied DLA proﬁle, whereas others (e.g. Rottweiler) are even
more restricted (Kennedy et al., 2007a). The limited diversity of DLA
types could inﬂuence vaccine responses in Beagles and a product
tested in Beagles will not necessarily perform similarly in other dog
breeds.
Both groups were relatively restricted in DLA haplotype diver-
sity. Whilst this restriction is not as marked as for some breeds, such
as the Rottweiler, where only four haplotypes have been identi-
ﬁed (Kennedy et al., 2002a), it is likely to affect the repertoire of
peptide epitopes presented to CD4+ T cells upon antigenic stimu-
lation. Since some haplotypes are only represented in a small number
of Beagles, there is a risk that their frequency may diminish in sub-
sequent generations if breeding strategy is not considered within
a closed colony.
There was a signiﬁcant difference in DLA homozygosity between
groups. Without pedigrees or breeding strategy information, we can
only hypothesise that this high frequency of homozygosity within
the pet Beagles might indicate a higher degree of inbreeding and/
or a potential dominant sire effect. Homozygosity appears to vary
between dog breeds, depending on the size of the population base;
a previous study found 28.4% homozygosity across breeds (Kennedy
et al., 2002a).
Laboratory Beagles represent a different immunogenetic pool
comparedwith pet Beagles. Theymay be a suitable choice for vaccine
studies if genetic diversity and heterozygosity can be maintained
by strategic breeding. However, laboratory Beagles do not express
the full range of DLA haplotypes and thus are not representative
of the diversity seen within the pet dog population as a whole. This
poses potential problems for the use of any one breed in vaccine
studies, since DLA type could inﬂuence responses to antigenic chal-
lenge. MHC class II heterozygosity in humans has been associated
with enhanced resistance and more effective clearance of patho-
gens (Thursz et al., 1997; Carrington et al., 1999). Further studies
are needed to investigate the impact of DLA genotype on suscep-
tibility to infectious disease and response to vaccination in dogs.
Conﬂict of interest statement
None of the authors of this paper has a ﬁnancial or personal re-
lationship with other people or organisations that could
inappropriately inﬂuence or bias the content of the paper.
Acknowledgements
This study was supported by a Biotechnology and Biological Sci-
ences Research Council (BBSRC) Collaborative Awards in Science and
Engineering (CASE) studentship (BB/I015655/1) in partnership with
Zoetis. We wish to thank Susanna Martorell for sample collection
and Angela Holder for technical assistance. Preliminary results were
presented as an Abstract at the Worldleish 5 Conference, Porto de
Galinhas, Brazil, 13–17 May 2013.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.tvjl.2014.12.032.
References
Calboli, F.C., Sampson, J., Fretwell, N., Balding, D.J., 2008. Population structure and
inbreeding from pedigree analysis of purebred dogs. Genetics 179, 593–601.
Carrington, M., Nelson, G.W., Martin, M.P., Kissner, T., Vlahov, D., Goedert, J.J., Kaslow,
R., Buchbinder, S., Hoots, K., O’Brien, S.J., 1999. HLA and HIV-1: Heterozygote
advantage and B*35-Cw*04 disadvantage. Science 283, 1748–1752.
Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Bennett, D., Angles, J.M., Day, M.J.,
Carmichael, N., Innes, J.F., Isherwood, D., et al., 2002a. Extensive interbreed, but
minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. Tissue
Antigens 59, 194–204.
Table 1
Frequencies of three DLA locus haplotypes in pet and laboratory Beagles.
Haplotype Pet Beagles (n = 95) Laboratory Beagles (n = 100) Fisher’s exact test P
value
DRB1 DQA1 DQB1 Number of
homozygous dogs
Total number of
haplotypes
Haplotype
frequency (%)
Number of
homozygous dogs
Total number of
haplotypes
Haplotype
frequency (%)
001:02 001:01 002:01 30 87 45.8 2 34 17 <0.01
015:01 009:01 001:01 6 36 18.9 5 50 25 NS
001:01 001:01 002:01 2 21 11 8 56 28 <0.01
002:01 009:01 001:01 1 17 8.9 0 7 3.5 <0.05
008:01 003:01 004:01 1 9 4.7 0 0 0 <0.01
006:01 005:01 007:01 1 8 4.2 6 46 23 <0.01
001:02 009:01 001:01 1 5 2.6 0 0 0 <0.05
006:01 004:01 013:03 1 2 1 0 0 0 NS
015:01 006:01 003:01 0 2 1 0 0 0 NS
002:01 009:01 002:01 0 1 0.5 0 2 1 NS
008:02 001:01 002:01 0 1 0.5 0 0 0 NS
020:01 004:01 013:03 0 1 0.5 0 0 0 NS
015:01 006:01 020:02 0 0 0 0 4 2 NS
048:01 004:02 023:01 0 0 0 0 1 0.5 NS
Total 43 190 100 21 200 100
NS, not signiﬁcant (P ≥ 0.05).
346 F. Soutter et al./The Veterinary Journal 203 (2015) 345–347
Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Courtenay, O., Carter, S.D., Ollier,
W.E., Thomson, W., 2002b. Evidence for extensive DLA polymorphism in different
dog populations. Tissue Antigens 60, 43–52.
Kennedy, L.J., Barnes, A., Short, A., Brown, J.J., Lester, S., Seddon, J., Fleeman, L.,
Francino, O., Brkljacic, M., Knyazev, S., et al., 2007a. Canine DLA diversity: 1. New
alleles and haplotypes. Tissue Antigens 69 (Suppl. 1), 272–288.
Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L., Fooks, A.R., Baxter, D.N., Ollier, W.E.,
2007b. Factors inﬂuencing the antibody response of dogs vaccinated against
rabies. Vaccine 25, 8500–8507.
Lindblad-Toh, K., Wade, C.M., Mikkelsen, T.S., Karlsson, E.K., Jaffe, D.B., Kamal, M.,
Clamp, M., Chang, J.L., Kulbokas, E.J., 3rd, Lander, E.S., 2005. Genome sequence,
comparative analysis and haplotype structure of the domestic dog. Nature 438,
803–819.
Thursz, M.R., Thomas, H.C., Greenwood, B.M., Hill, A.V., 1997. Heterozygote advan-
tage for HLA class-II type in hepatitis B virus infection. Nature Genetics 17,
11–12.
Wagner, J.L., 2003. Molecular organisation of the canine major histocompatibility
complex. The Journal of Heredity 94, 23–26.
347F. Soutter et al./The Veterinary Journal 203 (2015) 345–347


Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles 

Beagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restriction within this breed and the impact this might have on vaccine responses are rarely considered. This study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were determined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles had significantly different DLA haplotype frequencies in comparison with pet Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in selective antigen presentation and vaccine responses that are not necessarily representative of those seen in other dog breeds

Ollier, William E. R.

Solano Gallego, Laia

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Short CommunicationRestricted dog leucocyte antigen (DLA) class II haplotypes andgenotypes in BeaglesFrancesca Soutter a,*, Lorna J. Kennedy b, William E.R. Ollier b, Laia Solano-Gallego c,Brian Catchpole aa Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, United Kingdomb Centre for Integrated Genomic Medical Research, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdomc Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria, Universitat Autònoma de Barcelona, Barcelona, SpainA R T I C L E I N F OArticle history:Accepted 29 December 2014Keywords:BeagleDog leucocyte antigenMajor histocompatibility complexA B S T R A C TBeagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restric-tion within this breed and the impact this might have on vaccine responses are rarely considered. Thisstudy was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breedingcolony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were deter-mined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagleshad signiﬁcantly different DLA haplotype frequencies in comparison with pet Beagles and both groupsshowed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result inselective antigen presentation and vaccine responses that are not necessarily representative of those seenin other dog breeds.© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).Vaccine eﬃcacy is fundamental to ensuring protection of animalsagainst infectious disease and inmaintaining herd immunity. Beaglesare used for vaccine trials, designed to provide evidence of eﬃca-cy for regulatory approval. However, dog breeds, including the Beagle,have been derived from at least two genetic bottlenecks(Lindblad-Toh et al., 2005), which might affect their immunoge-netic diversity and vaccine responses. Vaccination induced antibodytitres can vary with breed, although genetic factors have yet to beidentiﬁed (Kennedy et al., 2007b).Dog leucocyte antigen (DLA) class II genes encode major histo-compatibility complex (MHC) molecules that are involved in antigenpresentation to CD4+ T cells. In dogs, these consist of three, highlypolymorphic, loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1 (Wagner,2003). There is a large degree of inter-breed variability, but oftenlimited intra-breed diversity, of DLA haplotypes (Kennedy et al.,2002a). The aim of the present study was to characterise DLAhaplotypes in laboratory Beagle dogs and to compare their DLA di-versity with a pet Beagle population.Residual blood samples were obtained from laboratory Beagles(n = 100) in a commercial breeding facility, that provided puppiesfor vaccine trials in Europe; sampleswere collected for another study(project number114-C-E-01-10; date of Animal Care andEthics Com-mittee approval 7 June2010). Blood samples frompetBeagles (n = 47)were obtained from the Royal Veterinary College genetic archive,containing residual samples followingcompletionof diagnostic testingwith informed owner consent and with approval in 2004 from theinstitutional Ethics andWelfare Committee. Forty-eight pet Beagles,previously genotyped at theUniversity ofManchester (date of AnimalCare and Ethics Committee approval April 2004)were also analysedand included in the pet Beagle group. Information on pedigrees andrelatedness of the dogs was not available.Genomic DNA was extracted using the GenElute Blood GenomicDNA Kit (Sigma-Aldrich). PCRwas performed in 25 μL reactions, with1 μL DNA as template, DLA-speciﬁc primers (2 μL at 20 pmol/μL ﬁnalconcentration; Sigma-Aldrich) (see Appendix: SupplementaryTable S1), 5 μL Hi-Spec additive, 2.5 μL ImmoBuffer, 1.25 μL MgCl2(2.5 mM ﬁnal concentration), 0.25 μL deoxynucleotide triphos-phates (1mMﬁnal total concentration) and 0.1 μL (1.25 IU) ImmolaseDNA polymerase (Bioline). PCR was performed using a G-Storm GS1Thermal Cycler (Gene Technologies) at 95 °C for 10 min, followedby 35 cycles of 94 °C for 40 s, 55 °C for 30 s for DQA1 or 60 °C forDRB1 and DQB1, and 72 °C for 1min, then 72 °C for 10min. PCR prod-ucts were processed using the GenElute PCR Clean-up Kit (Sigma-Aldrich) and submitted for sequencing (Source Bioscience) usingprimer M13F. DLA analysis and allele assignment was performedusing SBT Engine version 2.17 (GenDx). Haplotype frequencies werecompared between groups using Fisher’s exact test (Monte Carlomethod) in SPSS (PASW Statistics 18, IBM).* Corresponding author. Tel.: +44 170 766 6801.E-mail address: fsoutter@rvc.ac.uk (F. Soutter).http://dx.doi.org/10.1016/j.tvjl.2014.12.0321090-0233/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).The Veterinary Journal 203 (2015) 345–347Contents lists available at ScienceDirectThe Veterinary Journaljournal homepage: www.elsevier.com/ locate / tv j lFourteen DLA haplotypes were identiﬁed (12 in pet Beagles andeight in laboratory Beagles). Only four haplotypes occurred at a fre-quency >0.05 in each group and three of these more frequenthaplotypes were the same in both groups (Table 1). Whilst someoverlap existed in haplotypes between the two groups, the labo-ratory Beagle group demonstrated a signiﬁcant difference in DLAhaplotype frequencies compared with pet Beagles (P < 0.01). Morepet Beagles were homozygous (frequency = 0.45) than laboratoryBeagles (frequency = 0.21; χ2 = 11.93; P < 0.001). Pet Beagles werepredominantly homozygous for haplotype DLA-DRB1*001:02–DQA1*001:01–DQB1*002:01 (frequency = 0.70 of homozygous petBeagles; n = 43).These results show that the laboratory Beagles had a differentDLA proﬁle, compared with the pet Beagles. Founder effects, selec-tive breeding within a closed gene pool and use of popular sires havehad an impact on genetic diversity within pedigree breeds(Lindblad-Toh et al., 2005; Calboli et al., 2008). The differencebetween these populations could be the result of selective breed-ing practices, since the groups have been bred for different purposesand likely represent subpopulations from historical Beagle stock withdistinct founders (Kennedy et al., 2002b).Neither group represents the DLA diversity in the dog popula-tion as a whole, where some breeds (e.g. Jack Russell terrier) expressa more varied DLA proﬁle, whereas others (e.g. Rottweiler) are evenmore restricted (Kennedy et al., 2007a). The limited diversity of DLAtypes could inﬂuence vaccine responses in Beagles and a producttested in Beagles will not necessarily perform similarly in other dogbreeds.Both groups were relatively restricted in DLA haplotype diver-sity. Whilst this restriction is not as marked as for some breeds, suchas the Rottweiler, where only four haplotypes have been identi-ﬁed (Kennedy et al., 2002a), it is likely to affect the repertoire ofpeptide epitopes presented to CD4+ T cells upon antigenic stimu-lation. Since some haplotypes are only represented in a small numberof Beagles, there is a risk that their frequency may diminish in sub-sequent generations if breeding strategy is not considered withina closed colony.There was a signiﬁcant difference in DLA homozygosity betweengroups. Without pedigrees or breeding strategy information, we canonly hypothesise that this high frequency of homozygosity withinthe pet Beagles might indicate a higher degree of inbreeding and/or a potential dominant sire effect. Homozygosity appears to varybetween dog breeds, depending on the size of the population base;a previous study found 28.4% homozygosity across breeds (Kennedyet al., 2002a).Laboratory Beagles represent a different immunogenetic poolcomparedwith pet Beagles. Theymay be a suitable choice for vaccinestudies if genetic diversity and heterozygosity can be maintainedby strategic breeding. However, laboratory Beagles do not expressthe full range of DLA haplotypes and thus are not representativeof the diversity seen within the pet dog population as a whole. Thisposes potential problems for the use of any one breed in vaccinestudies, since DLA type could inﬂuence responses to antigenic chal-lenge. MHC class II heterozygosity in humans has been associatedwith enhanced resistance and more effective clearance of patho-gens (Thursz et al., 1997; Carrington et al., 1999). Further studiesare needed to investigate the impact of DLA genotype on suscep-tibility to infectious disease and response to vaccination in dogs.Conﬂict of interest statementNone of the authors of this paper has a ﬁnancial or personal re-lationship with other people or organisations that couldinappropriately inﬂuence or bias the content of the paper.AcknowledgementsThis study was supported by a Biotechnology and Biological Sci-ences Research Council (BBSRC) Collaborative Awards in Science andEngineering (CASE) studentship (BB/I015655/1) in partnership withZoetis. We wish to thank Susanna Martorell for sample collectionand Angela Holder for technical assistance. Preliminary results werepresented as an Abstract at the Worldleish 5 Conference, Porto deGalinhas, Brazil, 13–17 May 2013.Appendix: Supplementary materialSupplementary data to this article can be found online atdoi:10.1016/j.tvjl.2014.12.032.ReferencesCalboli, F.C., Sampson, J., Fretwell, N., Balding, D.J., 2008. Population structure andinbreeding from pedigree analysis of purebred dogs. Genetics 179, 593–601.Carrington, M., Nelson, G.W., Martin, M.P., Kissner, T., Vlahov, D., Goedert, J.J., Kaslow,R., Buchbinder, S., Hoots, K., O’Brien, S.J., 1999. HLA and HIV-1: Heterozygoteadvantage and B*35-Cw*04 disadvantage. Science 283, 1748–1752.Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Bennett, D., Angles, J.M., Day, M.J.,Carmichael, N., Innes, J.F., Isherwood, D., et al., 2002a. Extensive interbreed, butminimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. TissueAntigens 59, 194–204.Table 1Frequencies of three DLA locus haplotypes in pet and laboratory Beagles.Haplotype Pet Beagles (n = 95) Laboratory Beagles (n = 100) Fisher’s exact test PvalueDRB1 DQA1 DQB1 Number ofhomozygous dogsTotal number ofhaplotypesHaplotypefrequency (%)Number ofhomozygous dogsTotal number ofhaplotypesHaplotypefrequency (%)001:02 001:01 002:01 30 87 45.8 2 34 17 <0.01015:01 009:01 001:01 6 36 18.9 5 50 25 NS001:01 001:01 002:01 2 21 11 8 56 28 <0.01002:01 009:01 001:01 1 17 8.9 0 7 3.5 <0.05008:01 003:01 004:01 1 9 4.7 0 0 0 <0.01006:01 005:01 007:01 1 8 4.2 6 46 23 <0.01001:02 009:01 001:01 1 5 2.6 0 0 0 <0.05006:01 004:01 013:03 1 2 1 0 0 0 NS015:01 006:01 003:01 0 2 1 0 0 0 NS002:01 009:01 002:01 0 1 0.5 0 2 1 NS008:02 001:01 002:01 0 1 0.5 0 0 0 NS020:01 004:01 013:03 0 1 0.5 0 0 0 NS015:01 006:01 020:02 0 0 0 0 4 2 NS048:01 004:02 023:01 0 0 0 0 1 0.5 NSTotal 43 190 100 21 200 100NS, not signiﬁcant (P ≥ 0.05).346 F. Soutter et al./The Veterinary Journal 203 (2015) 345–347Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Courtenay, O., Carter, S.D., Ollier,W.E., Thomson, W., 2002b. Evidence for extensive DLA polymorphism in differentdog populations. Tissue Antigens 60, 43–52.Kennedy, L.J., Barnes, A., Short, A., Brown, J.J., Lester, S., Seddon, J., Fleeman, L.,Francino, O., Brkljacic, M., Knyazev, S., et al., 2007a. Canine DLA diversity: 1. Newalleles and haplotypes. Tissue Antigens 69 (Suppl. 1), 272–288.Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L., Fooks, A.R., Baxter, D.N., Ollier, W.E.,2007b. Factors inﬂuencing the antibody response of dogs vaccinated againstrabies. Vaccine 25, 8500–8507.Lindblad-Toh, K., Wade, C.M., Mikkelsen, T.S., Karlsson, E.K., Jaffe, D.B., Kamal, M.,Clamp, M., Chang, J.L., Kulbokas, E.J., 3rd, Lander, E.S., 2005. Genome sequence,comparative analysis and haplotype structure of the domestic dog. Nature 438,803–819.Thursz, M.R., Thomas, H.C., Greenwood, B.M., Hill, A.V., 1997. Heterozygote advan-tage for HLA class-II type in hepatitis B virus infection. Nature Genetics 17,11–12.Wagner, J.L., 2003. Molecular organisation of the canine major histocompatibilitycomplex. The Journal of Heredity 94, 23–26.347F. Soutter et al./The Veterinary Journal 203 (2015) 345–347

Kennedy, LJ

Ollier, W E R

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Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles.

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Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles

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