Accepted Manuscript

Title: Restricted dog leucocyte antigen (DLA) class II haplotypes and

genotypes in Beagles

Author: Francesca Soutter, Lorna J. Kennedy, William E.R. Ollier, Laia

Solano-Gallego, Brian Catchpole

PII: S1090-0233(14)00530-9

DOI: http://dx.doi.org/doi: 10.1016/j.tvjl.2014.12.032

Reference: YTVJL 4380

To appear in: The Veterinary Journal

Accepted date: 29-12-2014

Please cite this article as: Francesca Soutter, Lorna J. Kennedy, William E.R. Ollier, Laia

Solano-Gallego, Brian Catchpole, Restricted dog leucocyte antigen (DLA) class II haplotypes and

genotypes in Beagles, The Veterinary Journal (2015), http://dx.doi.org/doi:

10.1016/j.tvjl.2014.12.032.

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Short Communication 1

2

Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles 3

4

5

Francesca Soutter a,*, Lorna J. Kennedy

b, William E.R. Ollier

b, Laia Solano-Gallego

c, Brian 6

Catchpole a 7

8

9

10 a

Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, 11

Hertfordshire, AL9 7TA, UK 12 b Centre for Integrated Genomic Medical Research, University of Manchester, Stopford 13

Building, Oxford Road, Manchester, M13 9PT, UK 14 c Departament de Medicina i Cirurgia Animal, Facultat de Veterinaria, Universitat Autònoma 15

de Barcelona, Barcelona, Spain 16

17

18

19

20

* Corresponding author. Tel.: +44 170 766 6801. 21

E-mail address: fsoutter@rvc.ac.uk (F. Soutter). 22

23

Page 1 of 10

Highlights 24

Dog leukocyte antigen (DLA) class II genes were examined in laboratory Beagles. 25

Laboratory Beagles represent a different immunogenetic pool compared to pet 26

Beagles. 27

Beagles do not express the full range of DLA haplotypes seen in the dog population. 28

Beagles could be suitable for vaccine studies if genetic diversity is maintained. 29

30

Abstract 31

Beagles are commonly used in vaccine trials as part of the regulatory approval 32

process. Genetic restriction within this breed and the impact this might have on vaccine 33

responses is rarely considered. This study was designed to characterise diversity of dog 34

leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose 35

offspring are used in vaccine studies. DLA haplotypes were determined by PCR and 36

sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles 37

demonstrated significantly different DLA haplotype frequencies in comparison with pet 38

Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic 39

variability within Beagles might result in selective antigen presentation and vaccine responses 40

that are not necessarily representative of those seen in other dog breeds. 41

42

Keywords: Beagle; Dog leucocyte antigen; Major histocompatibility complex 43

Page 2 of 10

Vaccine efficacy is fundamental to ensuring protection of animals against infectious 44

disease and in maintaining herd immunity. Beagles are used for vaccine trials, designed to 45

provide evidence of efficacy for regulatory approval.1 However, dog breeds, including the 46

Beagle, have been derived from at least two genetic bottlenecks (Lindblad-Toh et al., 2005), 47

which might affect their immunogenetic diversity and vaccine responses. Vaccination induced 48

antibody titres can vary with breed, although genetic factors have yet to be identified 49

(Kennedy et al., 2007b). 50

51

Dog leucocyte antigen (DLA) class II genes encode major histocompatibility complex 52

(MHC) molecules that are involved in antigen presentation to CD4+ T cells. In dogs, these 53

consist of three, highly polymorphic, loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1 54

(Wagner, 2003). There is a large degree of inter-breed variability, but often limited intra-55

breed diversity, of DLA haplotypes (Kennedy et al., 2002a). The aim of the present study was 56

to characterise DLA haplotypes in laboratory Beagle dogs and to compare their DLA 57

diversity with a pet Beagle population. 58

59

Residual blood samples were obtained from laboratory Beagles (n = 100) in a 60

commercial breeding facility, that provided puppies for vaccine trials in Europe; samples 61

were collected for another study (project number 114-C-E-01-10; date of Animal Care and 62

Ethics Committee approval 7 June 2010). Blood samples from pet Beagles (n = 47) were 63

obtained from the Royal Veterinary College genetic archive, containing residual samples 64

following completion of diagnostic testing with informed owner consent and with approval in 65

2004 from the institutional Ethics and Welfare Committee. Forty-eight pet Beagles, 66

previously genotyped at the University of Manchester (date of Animal Care and Ethics 67

1 See: http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000194.jsp&mid=WC0b01ac058002dd33

(accessed 30 September 2014).

Page 3 of 10

Committee approval April 2004) were also analysed and included in the pet Beagle group. 68

Information on pedigrees and relatedness of the dogs was not available. 69

70

Genomic DNA was extracted using the GenElute Blood Genomic DNA Kit (Sigma-71

Aldrich). PCR was performed in 25 L reactions, with 1 L DNA as template, DLA-specific 72

primers (2 L at 20 pmol/L final concentration; Sigma-Aldrich) (see Appendix: 73

Supplementary Table 1), 5 L Hi-Spec additive, 2.5 L ImmoBuffer, 1.25 L MgCl2 (2.5mM 74

final concentration), 0.25 L deoxynucleotide triphosphates (1 mM final total concentration) 75

and 0.1 L (1.25 IU) Immolase DNA polymerase (Bioline). PCR was performed using a G-76

Storm GS1 Thermal Cycler (Gene Technologies) at 95 °C for 10 min, followed by 35 cycles 77

of 94 °C for 40 s, 55 °C for 30 s for DQA1 or 60 °C for DRB1 and DQB1, and 72 °C for 1 78

min, then 72 °C for 10 min. PCR products were processed using the GenElute PCR Clean-up 79

Kit (Sigma-Aldrich) and submitted for sequencing (Source Bioscience) using primer M13F. 80

DLA analysis and allele assignment was performed using SBT Engine version 2.17 (GenDx). 81

Haplotype frequencies were compared between groups using Fisher’s exact test (Monte Carlo 82

method) in SPSS (PASW Statistics 18, IBM). 83

84

Fourteen DLA haplotypes were identified (12 in pet Beagles and eight in laboratory 85

Beagles). Only four haplotypes occurred at a frequency >0.05 in each group and three of these 86

more frequent haplotypes were the same in both groups (Table 1). Whilst some overlap 87

existed in haplotypes between the two groups, the laboratory Beagle group demonstrated a 88

significant difference in DLA haplotype frequencies compared to pet Beagles (P <0.01). More 89

pet Beagles were homozygous (frequency = 0.45) than laboratory Beagles (frequency = 0.21; 90

2 = 11.93; P <0.001). Pet Beagles were predominantly homozygous for haplotype DLA-91

Page 4 of 10

DRB1*001:02--DQA1*001:01--DQB1*002:01 (frequency = 0.70 of homozygous pet 92

Beagles; n = 43). 93

94

These results show that the laboratory Beagles had a different DLA profile, compared 95

with the pet Beagles. Founder effects, selective breeding within a closed gene pool and use of 96

popular sires has had an impact on genetic diversity within pedigree breeds (Lindblad-Toh et 97

al., 2005; Calboli et al., 2008). The difference between these populations could be the result 98

of selective breeding practices, since the groups have been bred for different purposes and 99

likely represent subpopulations from historical Beagle stock with distinct founders (Kennedy 100

et al., 2002b). 101

102

Neither group represents the DLA diversity in the dog population as a whole, where 103

some breeds (e.g. Jack Russell terrier) express a more varied DLA profile, whereas others 104

(e.g. Rottweiler) are even more restricted (Kennedy et al., 2007a). The limited diversity of 105

DLA-types could influence vaccine responses in Beagles such a product tested in Beagles will 106

not necessarily perform similarly in other dog breeds. 107

108

Both groups were relatively restricted in DLA haplotype diversity. Whilst this 109

restriction is not as marked as for some breeds, such as the Rottweiler, where only four 110

haplotypes have been identified (Kennedy et al., 2002a), it is likely to affect the repertoire of 111

peptide epitopes presented to CD4+ T cells upon antigenic stimulation. Since some haplotypes 112

are only represented in a small number of Beagles, there is a risk that their frequency may 113

diminish in subsequent generations if breeding strategy is not considered within a closed 114

colony. 115

116

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There was a significant difference in DLA homozygosity between groups. Without 117

pedigrees or breeding strategy information, we can only hypothesise that this high frequency 118

of homozygosity within the pet Beagles might indicate a higher degree of inbreeding and/or a 119

potential dominant sire effect. Homozygosity appears to vary between dog breeds, depending 120

on the size of the population base; a previous study found 28.4% homozygosity across breeds 121

(Kennedy et al., 2002a.). 122

123

Laboratory Beagles represent a different immunogenetic pool compared to pet 124

Beagles. They may be a suitable choice for vaccine studies if genetic diversity and 125

heterozygosity can be maintained by strategic breeding. However, laboratory Beagles do not 126

express the full range of DLA haplotypes and thus are not representative of the diversity seen 127

within the pet dog population as a whole. This poses potential problems for the use of any one 128

breed in vaccine studies, since DLA type could influence responses to antigenic challenge. 129

MHC class II heterozygosity in humans has been associated with enhanced resistance and 130

more effective clearance of pathogens (Thursz et al., 1997; Carrington et al., 1999). Further 131

studies are needed to investigate the impact of DLA genotype on susceptibility to infectious 132

disease and response to vaccination in dogs. 133

134

Conflict of interest statement 135

None of the authors of this paper has a financial or personal relationship with other 136

people or organisations that could inappropriately influence or bias the content of the paper. 137

138

Acknowledgments 139

This study was supported by a Biotechnology and Biological Sciences Research 140

Council (BBSRC) Collaborative Awards in Science and Engineering (CASE) studentship 141

Page 6 of 10

(BB/I015655/1) in partnership with Zoetis. We wish to thank Suzanna Martorell for sample 142

collection and Angela Holder for technical assistance. Preliminary results were presented as 143

an Abstract at the Worldleish 5 Conference, Porto de Galinhas, Brazil, 13-17 May 2013. 144

145

Appendix: Supplementary material 146

Supplementary data associated with this article can be found, in the online version, at 147

doi … [Note from Editor to Editorial Production Department: Please insert digital object 148

identifier.] 149

150

References 151

Calboli, F.C., Sampson, J., Fretwell, N., Balding, D.J., 2008. Population structure and 152

inbreeding from pedigree analysis of purebred dogs. Genetics 179, 593-601. 153

154

Carrington, M., Nelson, G.W., Martin, M.P., Kissner, T., Vlahov, D., Goedert, J.J., Kaslow, 155

R., Buchbinder, S., Hoots, K., O’Brien, S.J., 1999. HLA and HIV-1: Heterozygote 156

advantage and B*35-Cw*04 disadvantage. Science 283, 1748-1752. 157

158

Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Bennett, D., Angles, J.M., Day, M.J., 159

Carmichael, N., Innes, J.F., Isherwood, D., et al., 2002a. Extensive interbreed, but 160

minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. Tissue 161

Antigens 59, 194-204. 162

163

Kennedy, L.J., Barnes, A., Happ, G.M., Quinnell, R.J., Courtenay, O., Carter, S.D., Ollier, 164

W.E., Thomson, W., 2002b. Evidence for extensive DLA polymorphism in different 165

dog populations. Tissue Antigens 60, 43-52. 166

167

Kennedy, L.J., Barnes, A., Short, A., Brown, J.J., Lester, S., Seddon, J., Fleeman, L., 168

Francino, O., Brkljacic, M., Knyazev, S., et al., 2007a. Canine DLA diversity: 1. 169

New alleles and haplotypes. Tissue Antigens 69 (Suppl. 1), 272-288. 170

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Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L., Fooks, A.R., Baxter, D.N., Ollier, W.E., 172

2007b. Factors influencing the antibody response of dogs vaccinated against rabies. 173

Vaccine 25, 8500-8507. 174

175

Lindblad-Toh, K., Wade, C.M., Mikkelsen, T.S., Karlsson, E.K., Jaffe, D.B., Kamal, M., 176

Clamp, M., Chang, J.L., Kulbokas, E.J., 3rd, Lander, E.S., 2005. Genome sequence, 177

comparative analysis and haplotype structure of the domestic dog. Nature 438, 803-178

819. 179

180

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Thursz, M.R., Thomas, H.C., Greenwood, B.M., Hill, A.V., 1997. Heterozygote advantage for 181

HLA class-II type in hepatitis B virus infection. Nature Genetics 17, 11-12. 182

183

Wagner, J.L., 2003. Molecular organisation of the canine major histocompatibility complex. 184

Journal of Heredity 94, 23-26. 185

186

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Table 1 187

Frequencies of three DLA locus haplotypes in pet and laboratory Beagles. 188

189

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 00:901 002:01 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

190

NS, not significant (P ≥0.05). 191

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Appendix: Supplementary Table 1 192

Oligonucleotide primers used in the study. 193

194

Primer Direction Primer sequence Amplicon size

(base pairs)

DLA-DRB1 Forward 5’-CCGTCCCCACCAGCACATTTC-3’ 270

Reverse 5’-TGTAAAACGACGGCCAGTGTCACACACCTCAGCACCA-3’

DLA-DQA1 Forward 5’-TGTAAAACGACGGCCAGTCTCAGCTGACCATGTTGC-3’ 243

Reverse 5’-GGACAGATTCAGTGAAGAGAG-3’

DLA-DQB1 Forward 5’-TGTAAAACGACGGCCAGTCTCACTGGCCCGGCCTGTCTC-3’ 267

Reverse 5’-CACCTCGCCGCTGAACGTG-3’

M13F Forward 5’-TGTAAAACGACGGCCAGT-3’

195

The M13F primer-binding region used in the sequencing reaction is underlined. 196

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