Genetic diversity and flock clustering of a South African Dohne Merino flock selected for resistance to Haemonchus contortus

 

N.M. Dlamini1,2, C. Visser2, P. Soma3,  F.C. Muchadeyi1 & M.A. Snyman 4

 

1Biotechnology Platform, Agricultural Research Council, Private Bag X05, Onderstepoort, 0110, South Africa

NM Dlamini (Corresponding author) 

2Department of Animal and Wildlife Sciences, University of Pretoria, Private Bag X20, Hatfield, 0028, South Africa

3Agricultural Research Council, Animal Production Institute,  Irene 0062, South Africa

4Grootfontein Agricultural Development Institute, Private Bag X529, Middelburg (EC), 5900, South Africa 

 

 

Summary

Gastrointestinal parasitism is a major problem to livestock productivity worldwide and small ruminant production is affected the most. Expensive and unsuccessful treatment with anthelmintics, production losses and mortality due to severe infestations has a severe financial impact on sheep farmers. Resistance of gastrointestinal nematodes (GIN) to anthelmintics has become a widespread problem, with resistance of Haemonchus contortus in South Africa being one of the most severe. Farming with animals resistant to nematode infestation has been proposed as a sustainable alternative. The farm Wauldby in the Stutterheim district of South Africa has a well-documented history of heavy H. contortus challenge. In 2011, a project aimed at selection for resistance to H. contortus was implemented at Wauldby. Annually, faecal egg counts (FEC), Famacha© score (FAM) and body condition score (BCS) were recorded on all lambs from weaning in January at 4 months of age, until the end of June when the H. contortus challenge decreased. Lambs were only drenched with an anthelmintic when they had a FAM of 2.5 or higher. Breeding values for FEC were estimated for Wauldby animals born from 2011 to 2014. In this study, genome-wide SNP data generated using the Illumina® Ovine SNP50 BeadChip was used to investigate flock clustering of the Wauldby Dohne Merino flock and its association with resistance to H. contortus. A total of 192 animals were selected for inclusion in the study. Within years, animals with the highest and lowest breeding values for FEC were selected among the animals that needed drenching (cases), and those that did not need any drenching (controls). Animals from the Grootfontein Dohne merino flock, which had not been subjected to any selection for resistance, were used as a reference population. DNA obtained from blood samples were genotyped using the Illumina® Ovine SNP50 BeadChip. The principal component analysis (PCA) plot was performed using SNP & Variation Suite (SVS) from Golden Helix to illustrate the population genetic structure of animals within the Wauldby Dohne Merino and GADI Dohne Merino sheep. Four distinct genetic clusters were observed, with the GADI Dohne Merino sheep population clustering separately. The Wauldby Dohne Merino population differentiated into 3 distinct clusters. Average FEC, LFEC (log-transformed FEC), BCS and FAM recorded over the study period were compared between the different clusters for the Wauldby animals. These results indicated that it should be possible to select for resistance to H. contortus on the basis of the phenotypic traits included in the study. 

 

Key words: body condition score, gastrointestinal nematodes, Famacha©, faecal egg count

 

Introduction

Gastrointestinal parasitism is a major problem to livestock productivity worldwide and has a significant, long-term effect on small ruminant health and cause suffering and financial losses annually (Alba-Hurtado & Muñoz-Guzmán, 2012; Geurden et al., 2014). Continuous use of chemical drenches to control gastrointestinal nematodes (GIN) in sheep has led to the development of anthelmintic resistance (AR) of GIN (Riggio et al., 2013). AR has been reported in most sheep producing regions such as Australia (Falzon et al., 2014), New Zealand (Hooda et al., 1999), North, Central and South America (De Graef et al., 2013), Africa (Vatta et al., 2002) and Europe (Alba-Hurtado & Muñoz-Guzmán, 2012). Breeding for host genetic resistance is seen as a long-term strategy for controlling GIN in a sustainable way (Greer & Hamie, 2016). Haemonchus contortus is one of the most economically important gastrointestinal nematodes infecting hundreds of millions of small ruminants worldwide (Riggio et al., 2013). Genetic resistance to this worm both between and within breeds has been documented in previous studies (Hooda et al., 1999; De Souza Chagas et al., 2016). The aim of this study was to use high-throughput genome-wide SNP data generated using the Illumina® Ovine SNP50 BeadChip to investigate genetic diversity and flock clustering of a Dohne Merino flock and its association with resistance to H. contortus.

 

Materials and Methods

The farm Wauldby in the Stutterheim district in the Eastern Cape province of South Africa has a well-documented history of heavy H. contortus challenge and Haemonchus resistance to all available anthelmintics. In 2011, a Haemonchus resistant line was established in the Dohne Merino stud at Wauldby. Since 2011, faecal egg counts (FEC), body condition score (BCS) and Famacha© score (FAM) were collected and recorded annually on all the lambs from January at 4 months of age, until the end of June when the H. contortus challenge decreased. FAM was recorded weekly, while FEC and BCS scores were recorded at two-weekly intervals. Animals with FAM scores of ≥ 2.5 or BCS scores < 1.5 were subjected to anthelmintic treatment and recorded as dosed animals. Replacement lambs for the resistant line were selected from those lambs that did not receive any anthelminthic treatment. Selection was based on a selection index incorporating FEC, FAM and BCS.

The Grootfontein Dohne Merino stud is kept at Grootfontein Agricultural Development Institute (GADI) near Middelburg in the Eastern Cape Province under veld conditions. The GADI Dohne Merino sheep were used as a reference flock for the Wauldby animals, as no specific selection for helminth resistance was done in the GADI flock

Breeding values for FEC were estimated for the data available on the Wauldby animals from 2011 to 2014. Within years, animals with the highest and lowest EBV for FEC were selected among the dosed (n=48, Low EBV FEC; n= 48, High EBV FEC), as well as the not dosed (n=52, Low EBV FEC; n=48, High EBV FEC) groups. Animals were selected within years to account for any possible genetic trends. In the case of the Grootfontein Dohne Merino animals, FEC data for the 2014 and 2016 born lambs were available. Animals with the highest and lowest FEC within each year were selected for genotyping (n=25/year).

DNA was isolated using the DNA isolation NucleoMag® VET kit (NucleoMag - MACHEREY-NAGEL GmbH & Co KG, Düren, Germany). DNA with a concentration of ≥25ng/ul were genotyped at the Agricultural Research Council, Biotechnology Platform (ARC-BTP) using the Illumina® Ovine SNP50 BeadChip (Illumina Inc., San Diego, CA).

The SNP genotype data were subjected to quality control measures using PLINK v1.07 software (Purcell et al., 2007). After data quality control, 47518 SNPs with an average genotyping rate of 0.94 were available for further analyses.

The principal component analysis (PCA) plot was performed using SNP & Variation Suite (SVS) from Golden Helix on all animals of the Wauldby Dohne Merino and GADI Dohne Merino sheep populations. Average FEC, LFEC (log-transformed FEC), BCS and FAM recorded over the study period were compared between the different genetic clusters for the Wauldby animals.

 

Results and discussion

The principal component analysis (PCA1) plot is depicted in Figure 1. Four distinct clusters were observed, with the GADI Dohne Merino sheep population clustering on its own (Cluster 1). Three distinct clusters were observed for the Wauldby Dohne Merinos (Clusters 2 to 4) consisting of a mixture of animals from the case and control groups.  

  

Figure 1. PCA based clustering of Wauldby farm Dohne Merino and GADI Dohne Merino sheep populations. Cluster 1 consisted of Dohne Merino Sheep from GADI whilst Clusters 2-4 were from the Wauldby Dohne Merino flock.

 

The averages for the various individual resistance traits or combinations of traits are summarised in Table 1 and Appendix Table A1 for the three genetic clusters of the Wauldby Dohne Merino sheep. From Tables 1 and 2 it can be seen that animals in Cluster 3 had lower FEC, lower FAM, higher BCS and higher selection index values than the animals in Clusters 2 and 4. The majority of the sires (88%) of the animals in Cluster 3 was selected for the resistant line, while only 4.0% and 7.8% of the sires in Clusters 2 and 4 respectively, were selected sires. 

 

Trait

 

Genetic clusters

 

2

3

4

 

 

 

 

FECA

7249a ± 770

3927b ± 840

5321b ± 697

FEC179

4853a ± 995

1554b ± 1030

4012a ± 975

BCSA

2.19 ± 0.08

2.31a ± 0.09

2.18b ± 0.08

BCS179

2.16a ± 0.06

2.30b ± 0.06

2.19a ± 0.06

SI179

7.68a ± 0.27

8.36b ± 0.28

7.74a ± 0.27

EBV-FEC

115a ± 98

-629 ± 84b

-2 ± 45a

EBV-FAM

-0.029a ± 0.012

-0.024a ± 0.010

0.01 5b ± 0.005

EBV-BCS

-0.024a ± 0.009

0.058b ± 0.008

0.005c ± 0.004

a,b,c  Values with different superscripts differ significantly (P <0.05) between clusters within rows; FECA/BCSA = Faecal egg count / Body condition score averaged over all recordings per year; FEC179/BCS179= Average faecal egg / body condition score count for the 1st, 7th and 9th recordings; Selection index = Body condition score – Log-transformed FEC – Famacha© score; EBV-FEC = Estimated breeding value for faecal egg count, etc.  

 

Conclusion

The Wauldby Dohne Merino and GADI Dohne Merino populations are two genetically different populations. The results indicated that selection for resistance has resulted in genetic differentiation between animals, and the establishment of a more resistant line of animals. It should be possible to select for resistance to H. contortus on the basis of the phenotypic traits included in the study. 

Table 1. Averages for resistance traits for the different genetic clusters of the Wauldby Dohne Merino sheep.

 

 

List of references

Alba-Hurtado, F. & Muñoz-Guzmán, M.A., 2012. Immune responses associated with resistance to haemonchosis in sheep. BioMed Res. Int. 2013.

De Graef, J., Claerebout, E. & Geldhof, P., 2013. Anthelmintic resistance of gastrointestinal cattle nematodes. Vlaams Diergen Tijds. 82, 113-23.

de Souza Chagas, A.C., Domingues, L.F., Gaínza, Y.A., Barioni-Júnior, W., Esteves, S.N. & Niciura, S.C.M., 2016. Target selected treatment with levamisole to control the development of anthelmintic resistance in a sheep flock. Parasitol. Res. 115, 1131-9.

Falzon, L.C., O’Neill, T., Menzies, P., Peregrine, A., Jones-Bitton, A. & Mederos, A., 2014. A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep. Prev. Vet. Med. 117, 388-402.

Geurden, T., Hoste, H., Jacquiet, P., Traversa, D., Sotiraki, S., di Regalbono, A.F., Tzanidakis, N., Kostopoulou, D., Gaillac, C. & Privat, S., 2014. Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy. Vet. Parasitol. 201, 59-66.

Greer, A.W. & Hamie, J.C., 2016. Relative maturity and the development of immunity to gastrointestinal nematodes in sheep: an overlooked paradigm? Parasite immunol. 38, 263-72.

Hooda V., Yadav C., Chaudhri S. & Rajpurohit B. (1999) Variation in resistance to haemonchosis: selection of female sheep resistant to Haemonchus contortus. J. helminthol. 73, 137-42.

Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A.R., Bender, D., Maller, J., Sklar, P., De Bakker, P.I.W., Daly, M.J. & Sham, P.C., 2007. PLINK: a toolset for whole-genome association and population-based linkage analysis. Am. J. Human Gen. 81.

Riggio, V., Matika, O., Pong-Wong, R., Stear, M. & Bishop, S., 2013. Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs. Hered. 110, 420-9.

Vatta, A., Krecek, R., Van der Linde, M., Motswatswe, P., Grimbeek, R., Van Wijk, E. & Hansen, J., 2002. Haemonchus spp. in sheep farmed under resource-poor conditions in South Africa-effect on haematocrit, conjunctival mucous membrane colour and body condition. J S Afr Vet Assoc. 73, 119-23.


 

APPENDIX A

Table A1. Averages for resistance traits for the different genetic clusters of the Wauldby Dohne Merino sheep.

Trait

 

Genetic clusters

 

2

3

4

 

 

 

 

FEC1

7249a ± 770

3927b ± 840

5320b ± 700

FEC7

3993a ± 1245

97b ± 1358

3513a ± 1126

FEC8

4238a ± 988

379b ± 1077

3404a ± 894

FEC9

2722a ± 778

53b ± 848

2581 a ± 704

FEC11

2068a ± 464

771b ± 506

1983a ± 420

FECA

7249a ± 770

3927b ± 840

5321b ± 697

FEC179

4853a ± 995

1554b ± 1030

4012a ± 975

FAM9

0.94 ± 0.13

0.79a ± 0.13

0.97b ± 0.13

FAM10

1.06a ± 0.11

0.87b ± 0.11

1.02a ± 0.10

FAM179

1.70 ± 0.19

1.91 ± 0.20

1.93 ± 0.19

BCS7

2.12a ± 0.07

2.31b ± 0.08

2.21 ± 0.07

BCS8

2.14a ± 0.07

2.32b ± 0.07

2.24b ± 0.07

BCS9

2.19 ± 0.06

2.26 ± 0.07

2.18 ± 0.06

BCS10

2.23a ± 0.06

2.35b ± 0.07

2.32b ± 0.06

BCSA

2.19 ± 0.08

2.31a ± 0.09

2.18b ± 0.08

BCS179

2.16a ± 0.06

2.30b ± 0.06

2.19a ± 0.06

SI179

7.68a ± 0.27

8.36b ± 0.28

7.74a ± 0.27

EBV-FEC

115 ± 98a

-629 ± 84b

-2 ± 45a

EBV-LFEC

0.067 ± 0.026a

-0.209 ± 0.022b

0.005 ± 0.012a

EBV-FAM

-0.029 ± 0.012a

-0.024 ± 0.010a

0.015 ± 0.005b

EBV-BCS

-0.024 ± 0.009a

0.058 ± 0.008b

0.005 ± 0.004c

a,b,c  Values with different superscripts differ significantly (P <0.05) between clusters within rows

FEC1 = Faecal egg count of 1st recording, etc.; FECA = Faecal egg count averaged over all recordings per year; FEC169 = Average faecal egg count for the 1st, 6th and 9th recordings, etc; FAM9 = Famacha© score for the 9th recording; FAM179 = Average Famacha© score for the 1st, 7th and 9th recordings; BCS7 = Body condition score for the 7th recording; BCSA = Body condition score averaged over all recordings per year; BCS179 = Average body condition score for the 1st, 7th and 9th recordings; Selection index = Body condition score – Log-transformed FEC – Famacha© score; EBV-FEC = Estimated breeding value for faecal egg count, etc.  

 

 

Published

Proc. 11th Wrld. Congr. Gen. Appl. Livest. Prod., Auckland, New Zealand, 11-16 February