Prion protein gene haplotypes distribution among indigenous goat ecotypes in Tanzania

M J Kipanyula

Department of Veterinary Anatomy, Sokoine University of Agriculture,
PO Box 3016, Chuo Kikuu Morogoro, Tanzania.
kipanyula@suanet.ac.tz

Abstract

Mutations in the Prion protein gene affect the sustainability status of animals to scrapie. Genomic DNA was extracted from blood samples of 92 Small East African goat ecotypes from Morogoro, Mara, Iringa, Dodoma, Mbeya and Mtwara regions in Tanzania. The entire coding region for prion gene was amplified and then sequenced to detect the gene polymorphisms.

Two polymorphic sites with silent mutations at codons 42 (ccg→cca) and 138 ( agt→agc) were detected in 14 goats. Three distinct polymorphisms with amino acid substitutions at codons 116, 146 and 240 were also identified. These amino acid substitutions gave rise to six polymorphic alleles and genotypes. The A116V amino acid substitution was encountered in goats sampled from Iringa region. The N146S amino acid substitution was variably encountered in samples from all regions with highest frequency recorded in Mtwara region (45.5%). The P240S substitution displayed the highest variability (25%) in goats from Iringa region. The wild type (wt/S240P) was the most common genotype observed. The findings from this study suggest that the coding region of the PrP gene of the indigenous Small East African goat ecotypes in Tanzania is closer to the ‘ancestral form. Genotyping studies involving a large number of goat breeds covering a wider area in Tanzania and other African countries is highly recommended.

Key words: genotyping, neurodegeneration, PrP, scrapie

Introduction

Scrapie is among prion-borne progressive fatal and incurable transmissible neurodegenerative disease of goats and sheep. The first case of scrapie was described in England in 1759 (Hörnlimann et al 2007). Clinical manifestation of scrapie in both sheep and goats vary greatly among individual animals and develop very slowly (Ulvund 2007; 2008). The common clinical signs of scrapie in both goats and sheep include; hyperexcitability, restlessness, muscle tremor and gait abnormalities (Leontides et al 2000; Konold et al 2010). Literature describing incidences of scrapies and the susceptibility status to scrapie in the African goats and sheep is very limited (Cooper 1973; CFSPH 2007, Serano et al 2009; Kipanyula et al 2014).

Prion protein (PrP) gene polymorphism in small ruminants and its association with susceptibility to prion diseases has extensively been investigated in the past few decades. Despite the available remarkable breakthroughs in the understanding of the PrP gene polymporphisms this group of animals, the goat PrP gene has been studied less thoroughly than that of sheep. In sheep susceptibility to scrapie is primarily influenced by four polymorphic amino acid positions of the ovine PrP gene at codon 136, 141, 154 and 171, reviewed in Ulvund (2008). The presence of Adenine at codon 136 (A₁₃₆₎ and Arginine at codon 171 (R₁₇₁₎ of the PrP gene at the same time has been proven to confer resistance while concurrent presence of A₁₃₆ or Valine at codon 136 (V₁₃₆ ) and guanine at codon 171 (Q₁₇₁) renders increased susceptibility to both natural and experimental oral infections with classical scrapie (Hunter 1993; Goldmann et al 1994; Belt et al 1995; Andreoletti et al 2000; Billinis et al 2002; Gombojav et al 2003; Heaton et al 2003; Baylis et al 2004). The presence of phenylalanine (F₁₄₁), histamine (H₁₅₄) also influences the susceptibility status. Furthermore, occurrence of A₁₃₆F₁₄₁R₁₅₄Q₁₇₁/ A₁₃₆H₁₅₄Q₁₇₁ is strongly associated with susceptibility to the novel Nor98 scrapie strain (Baylis and McIntyre 2004; Moum et al 2005). In the developed countries, genotyping of corresponding PrP gene polymorphisms has frequently been applied as a reliable tool for selection decisions for breeding programmes at farm level (Hunter et al 1994; Tongue et al 2004).The genotype A₁₃₆ R₁₅₄R₁₇₁ confers resistance to scrapie and it is for that reason the European Union Decision 2003/100/EC, directed its member states to increase the frequency of the ARR allele in sheep breeding programmes.

Selection programmes in goats based on PrP gene polymorphism have not yet been established, despite high number of polymorphisms discovered during the last 20 years of research. Our understanding of the association of the reported goat PrP gene polymorphisms with scrapie susceptibility or resistance is very limited. To date there are about 43 reported goat PrP gene amino acid polymorphisms: 18(W→R), 21(V→A), 22(G→C), 23(L→P), 32(G→Stop), 37(G→V), 39(S→R), 49(G→S), 63(P→L), 74(G→D), 101(Q→R), 102(W→G), 110(T→N), 110(T→P), 116 (A→V), 112(M→T), 127(G→S), 133 (L→Q), 137 (M→I), 139(R→S), 141(L→F), 142(I→M), 143(H→R), 145(G→D), 146(N→S), 146(N→D), 151(R→H), 154(R→H), 163(Q→Stop), 168(P→Q), 185(I→F), 194(T→P), 201(F→L), 208(I→T), 211(R→Q), 211(R→G), 215(Q→R), 218(I→L), 219 (T→I), 220(Q→H), 222(Q→K), 232 (M→R), 240(P→S) (Goldmann et al 1996, 1998, 2004, 2011; Wopfner et al 1999; Billinis et al 2002; Agrimi et al 2003; Zhang et al 2004; Kurosaki et al 2005; Acutis et al 2006; 2010; Papasavva-Stylianou et al 2007; 2011; Zhou et al 2008; Vaccari et al 2009; Serrano et al 2009; Bouzalas et al 2010; White et al 2012; Benestad et al 2012; Acı´n et al 2013; Kipanyula et al 2014). Furthermore, the following silent mutation involving different nucleotides substitutions have also been reported at codons: 42 (g→a), 83(c→a), 102 (c→t), 107 (g→a), 122 (a→g), 125 (a→g),138 (t→c), 179 (g→t), 181 (c→t), 201 (c→t), 202 (c→t), 207 (g→a), 219 (c→t), 231 (a→c), 232 (g→a), 237(a→g), (Billinis et al 2002; Vaccari et al 2006; Acutis et al 2006; Papasavva-Stylianou et al 2007;2011; Babar et al 2008 Zhou et al 2008; Acı´n et al 2013).

Despite, a wealth of literature on goat PrP gene polymorphism, limited documentation is available on PrP gene haplotypes and their distribution in the indigenous African goats. Information about the PrP gene polymorphisms and their distribution in the SEA ecotypes is also limited. The aim of this study was therefore to investigate on the distribution of the PrP gene polymorphisms in the ecotypes of SEA goats from different geographical locations in Tanzania.

Materials and methods

Animals and blood sample collection

A total of 92 SEA goats were randomly sampled from eight different geographical locations in Tanzania: South western highlands (Mbeya and Iringa), Southern part (Mtwara), Northern part (Tarime and Serengeti, Mara), Central part (Dodoma), and Eastern part (Morogoro). Only genetically unrelated animals were included in the study. Blood sample was obtained from the ear vein by a needle prick and immediately applied onto FTA® elute micro cards (Whatman, USA). The FTA® elute cards were air dried for about an hour at around 25^°C, packed and transported for further processing at the Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences, Section of Small Ruminant Research and Herd Health.

Genetic analysis

The genomic DNA was extracted by the FTA® elute card standard protocol. The entire coding region (CDS) in exon 3 of the caprine PrP gene was amplified by polymerase chain reaction (PCR) in a Peltier Thermal Cycler, PTC 200 (MJ Research Inc, Massachusetts USA). The outer forward and reverse primers used respectively were PRNP oa-ch F (5'-gACTTTAAg TgATTTTTACgTgg-3') and PRNP oa-ch R (5'-TggCAAAgATTAAgAA gATAATg-3') for bidirectional amplification of the gene as previously described (Babar et al 2008; Kipanyula et al 2014). Additional primers; inner forward primer PrP-L2F (5’-ggAACAAgCCCAgTAAgCCAAA-3’) and the reverse primer PrP7-U-R (5’-AggAAggCCCCCTACCACTgCT-3’) TIB MOLBIOL (GmbH, Germany), were employed to countercheck the observed PrP gene polymorphisms.

The PCR reaction was performed in 10µl reaction volume containing 25 – 50ng genomic DNA, 2 mM MgCl₂, 0,2 mM dNTPs (Illustra PCR Nucleotide mixture, GE Healthcare, UK) 0.6µM each primers and 0.5 units Ampli Taq Gold in Buffer Gold (Applied Biosystems, Foster City, CA). The temperature profile for all PCR reactions were; denaturation at 95°C for 7 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds and extension at 72°C for 60 seconds. A step of final elongation was performed at 72°C for 60 seconds and the holding temperature was 4°C. The PCR products were then verified by electrophoresis using a 2% agarose gel.

For Sequencing, 5µl of the PCR products was purified by using 2µl of illustra Exo ProStar (1-Step enzyme PCR and sequence reaction) according to the manufacturer’s instructions (GE Healthcare, UK). Sequencing reaction was carried out with ABI PRISM BigDye Terminator v.1.1 Ready Reaction Mixture (Applied Biosystems, USA) as per manufacturer’s instruction. The PCR products were precipitated with 60% ethanol (final concentration), then washed in 70% ethanol and sequenced by capillary electrophoresis using an ABI PRISM^TM310 Genetic Analyzer (Applied Biosystems, USA).

Statistical analysis

All sequences were analysed using the Geneious version R 6.1.5 created by Biomatters (available from http://www.geneious.com) previously described by Drummond et al (2010). All sequences were aligned with a reference goat PrP gene (Accession Number: DQ345068) to compare the complete coding region. The summarized data were then processed in Microsoft Excel spreadsheet and subsequently imported to Epi Info^TM software version 7 (Centre for Disease Control and Prevention 2010) for further analyses. Genotypes and polymorphic alleles, together with the associated amino acids encoded for were identified and their respective frequencies were computed. StatCalc 2 x 2 contingency tables (Epi Info^TM) using χ²-square test were used for comparison of the frequencies within and between different locations from which SEA ecotypes were sampled with p value set at <0.05 considered as statistically significant.

Results

General PrP gene variability in the studied SEA ecotypes

In Tanzania the SEA comprise of a large group of indigenous breeds of goats consisting of different ecotypes and mainly identified according to localities within country and other parts of East Africa (Chenyambuga et al 2004). The goat PrP gene polymorphisms and their distribution in indigenous SEA goats from Morogoro, Mara, Iringa, Dodoma, Mbeya and Mtwara regions in Tanzania were determined. Two polymorphic sites with silent mutations at codons 42 (ccg→cca) and 138 (agt→agc) were detected in 14 goats out of the 92 goats (Table 1). Three distinct polymorphisms with amino acid substitutions at codon 116, 146 and 240 were identified in 37 goats sampled from different regions (Table 1). These amino acid substitutions gave rise to a total of 6 polymorphic alleles and genotypes of the indigenous goats in the study areas. Furthermore, the PrP ORFs of all goats analyzed in the present study had five peptide repeats (2 nona- and 3 octa-) from codon 54 to 102 and with a homozygous A₁₃₆R ₁₅₄Q₁₇₁.

PrP gene polymorphism in SEA goat ecotypes in the selected region of Tanzania

Considering individual mutations observed in this study, the variability differed from one location to another. The A116V amino acid substitution was detected in goats sampled from Iringa region only (Table 2). The N146S amino acid substitution was variably encountered in samples from all regions. The highest frequency for this mutation was recorded in Mtwara region (45.5%), while Iringa and Mbeya ranked second each (30%). The lowest variability for the N146S was encountered in goats sampled from Dodoma region. The P240S substitution displayed the highest variability (25%) in samples collected from goats in Iringa region (Table 2). Table 3 show the allelic frequencies in PrP gene of indigenous SEA from Morogoro, Mara, Iringa, Dodoma, Mbeya and Mtwara regions in Tanzania. The wild type (WT) allele is the allele 1, and most of the SEA ecotypes analyzed in this study had high frequency of this allele. Other alleles (2-6) occurred at different frequencies and distribution among different regions as summarized in Table 3.

The wild type (wt/S240P) was the most common genotype encountered in goats from the studied regions (Table 4). The wt/s240P genotype was observed in the following order in the studied areas: Mara (80%) > Dodoma (73%) > Mbeya (70) > Morogoro (68%) > Mtwara (50%) > Iringa (45%). However, differences between localities were not statistically significant. The genotype P240S/A116V and S240P/A146S were only observed in goats from Iringa (5%) and Dodoma (9.1%) regions respectively. Other genotypes were variably distributed with different genotypic frequency as presented in Table 4.

Discussion

This study gave insights on the goat PrP gene polymorphisms and their distribution among indigenous SEA goat ecotypes in Tanzania. Certainly, susceptibility status of small ruminants depends very much on the PrP gene profile. Studies in sheep have established that the PrP gene polymorphisms define the susceptibility status to scrapie. While occurrence of certain sheep PrP gene polymorphisms confers high susceptibility to scrapie, in the contrary others confer resistance to the disease. Evidence of similar associations of the goat PrP gene polymorphisms with susceptibility status to scrapie have not been exemplarily demonstrated despite a huge wealth of data generated during the past few years. Although the goat PrP gene profiles from different goat keeping countries worldwide have been studied extensively, the sub-saharan Africa is still lagging behind.  The SEA goat breed, which is the main indigenous strain in Tanzania, has not been well characterized genetically. Instead, it consists mainly of ecotypes named depending on the geographical locations such as Maasai, Ugogo, Ujiji, Sukuma and Newala. These names are often used to identify various SEA goats in different localities (Chenyambuga et al 2004). .

Scrapie is one of oldest neurodegenerative diseases to be described under the group of transmissible spongiform encephalopathies (TSEs). In developing countries more so in sub-Saharan Africa, this disease is highly neglected by animal health authorities and non-governmental organizations dealing with animal health. One of the reasons behind such a wide neglect is the lack of information about the disease from both active and passive surveillances targeting TSEs. Thus, it is necessary to develop investigations on several aspects of this group of diseases in sub-Saharan African countries, with special emphasis on studies focused at determining the susceptibility status of the indigenous African small ruminant populations, in the absence of strict animal’s movement and importation policies.

Some of the PrP gene alleles discovered from previous studies, have been implicated to confer relatively increased resistance to scrapie development when compared to wild-type alleles (Papasavva-Stylianou et al 2007; White et al 2012; Acutis et al 2012; Acín et al 2013). Notably, a limited number of goat polymorphisms have been linked with increased susceptibility to the disease (Bouzalas et al 2010; White et al 2012). Nonetheless, limited data is available on the PrP gene profile and its distribution among indigenous Tanzanian goat ecotypes. Even the few studies which have attempted to analyze the PrP gene in African goats have reported highly variable distribution of the discovered polymorphisms (Serrano et al 2009; Vaccari et al 2009). Overall the number of mutations encountered in the goat PrP gene in the indigenous SEA ecotypes studied was relatively small compared to other similar studies carried out in other goat breeds elsewhere (Goldmannn et al 2004; Vaccari et al 2009). The findings from this study suggest that majority of the indigenous SEA still have the ancestral forms of the PrP gene and more so in remote areas where cross breeding has not been carried extensively.

Of the three PrP gene polymorphisms observed in the Tanzanian SEA ecotypes, N146S and P240S/S240P were the most prominent in the studied goat population. However, there were no differences in the frequencies of these polymorphisms between goats from the various parts of the country although the means were similar to populations in other countries (Babar et al 2009; White et al 2012; Acín et al 2013). This observation suggests that the mutation is highly conserved among the SEA ecotypes. The high frequency of the polymorphism N146S and P240S/S240P have been associated with increased resistance to scrapie in different goat breeds (Papasavva-Stylianou et al 2007; 2011; Goldmann et al 2011; White et al 2012) . Although a mechanistic explanation for the ability of N146S to confer some degree of resistance to the disease lacks, it is tempting to suggest that the SEA ecotypes are likely to be on the advantageous side in case of an outbreak. Furthermore, our results suggest that the SEA ecotypes in Tanzania are likely to be closer to each other than they are other breeds characteristics. None of the alleles, which are proposed to increase susceptibility to scrapie, were observed in any of the SEA ecotypes studied.

Overall, goats sampled in Iringa, Morogoro and Mtwara had relatively high number polymorphisms. The reason against the observed trend could be attributed to uncontrolled crossbreeding carried by farmers with some exotic goat breeds. Several small ruminants crossbreeding programmes in Tanzania were started in late 1960’s with the primary aim of improving productivity of the SEA goats. Certainly, such programmes involved introduction of exotic dairy goat breeds such as the Alpine, Toggenburg, Anglo-Nubian, Saanen and white Norwegian goats into the country to improve milk production in local SEA goats (Das and Sendalo1989). Allele 1(WT) and genotype S240P were most prevalent in Mara region (Northern part of the country) where in our knowledge a limited number of local goat breeding programmes have been implemented so far. Certainly, some of the mutations observed in the SEA ecotypes are likely to have been introduced by crossbreeding with exotic goat breeds.

Conclusions

• The comparisons of distribution of the indigenous goat PrP gene polymorphisms between different locations in Tanzania show differences in distribution as well as their frequencies.
  
  
• The coding region of the goat PrP gene of the indigenous SEA goat ecotypes is closer to ‘ancestral form.
  
  
• Only the polymorphisms associated with resistance to scrapie were detected.
  
  
• Introduction of exotic breeds of goats for breeding purpose should be done cautiously to minimize introduction of exotic PrP gene haplotypes, especially those associated with susceptibility to scrapie and other TSEs.

Conflict of interest

The author declares that he has no financial or personal relationships which may have inappropriately influenced the writing of this article.

Acknowledgements

This study was funded by the Program for Agriculture and Natural Resource Transformation for Improved Livelihood (PANTIL), in collaboration with the Section of Small Ruminant Research of the former Norwegian School of Veterinary Science (NVH), from 2014 reorganized within the Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences. The technical support received from Professor Martha Ulvund and Eli Brundtland during design and implementation of this study is highly acknowledged.

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Received 18 September 2015; Accepted 22 December 2015; Published 1 February 2016

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