| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 CEA (Centro di referenza per le Encefalopatie Animali), Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154 Turin, Italy
2 Central Institute for Animal Disease Control (CIDC-Lelystad), PO Box 2004, 8203 AA Lelystad, The Netherlands
3 Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy
Correspondence
P. L. Acutis
pierluigi.acutis{at}izsto.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). PrP allelic variants valine/arginine/glutamine (VRQ) and alanine/arginine/glutamine (ARQ) at codons 136, 154 and 171, respectively, are generally associated with high susceptibility to scrapie, whereas the ARR allele has been linked to decreased susceptibility or even resistance (Belt et al., 1995; Bossers et al., 1996; Hunter et al., 1996, 1997). In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding programme to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding programme cannot yet be applied to control TSEs in goats, due to the limited knowledge of the genetics of scrapie in this species.
The polymorphisms (amino acid substitutions) described so far in caprine PrP are V21A, L23P, G37V, G49S, W102G, T110N, T110P, G127S, I142M, H143R, N146S, R154H, P168Q, R211Q, I218L, Q220H, Q222K and S240P (Goldmann et al., 1996, 1998, 2004; Wopfner et al., 1999; Billinis et al., 2002; Agrimi et al., 2003; Zhang et al., 2004; Kurosaki et al., 2005). The W102G polymorphism has been found only in combination with a variation in PrP containing only three instead of the usual five octapeptide repeats (Goldmann et al., 1998). Silent mutations have been described at codons 42 (a
g), 107 (ga), 138 (ct), 207 (ga) and 231 (ac) (Goldmann et al., 1996; Billinis et al., 2002; Zhang et al., 2004). The most common polymorphism is S240P, which has not yet been found in other species. P240 has been found in mink, ferret, the domestic dog and dingo PrP (Bartz et al., 1994; Wopfner et al., 1999). This leads to the presence of two main PrP variants in goats (S240 and P240) that can be linked to other mutations on other codons; consequently, 17 PrP alleles have been inferred so far. Based on phylogeny within goats and on sequence conservation over different species, S240 is regarded as the phylogenetic wild type for goats.
No strong association has been established between PrP polymorphisms and susceptibility to TSEs in goats. The presence of methionine at codon 142 and the three-repeat/G102 variant were associated with increased incubation periods after experimental challenge with bovine spongiform encephalopathy (BSE) and scrapie strains (Goldmann et al., 1996, 1998). Some protection offered by the R143 and H154 variants against natural scrapie infection has been suggested in Greek goats (Billinis et al., 2002) and an association between scrapie susceptibility and the distribution of genotype at codons 37, 143 and 240 was observed in the Ionica goat breed in Italy (Agrimi et al., 2003). Several factors have been suggested (Baylis & Goldmann, 2004) to explain the difficulties in associating goat PrP gene polymorphisms with prion diseases, including the low frequency of the detected mutations, the few experimental studies and the limited availability of case–control studies due to the rarity of goat scrapie in many countries.
In Italy, the first case of natural scrapie in goats was diagnosed in 1997 (Capucchio et al., 1998). Since then, 27 goat scrapie outbreaks have been reported, 11 of which were mixed flocks of sheep and goats and 16 of which were goat-only herds. A sudden rise in the incidence of scrapie, involving an exceptionally large number of goats, was reported in 1997. Implicated as a cause of the outbreak was an accidental infection from a vaccine against Mycoplasma agalactiae (Agrimi et al., 1999; Caramelli et al., 2001).
In our study, we analysed the PrP genes of goats from several Italian scrapie outbreaks to detect PrP polymorphisms and to determine PrP haplotypes by cloning. A case–control study was carried out to look for associations between PrP alleles and the occurrence of scrapie.
Material for the study was available from 177 goats taken from six herds (herd 1, 64 animals; herd 2, 11; herd 3, 150; herd 4, 172; herd 5, 246; herd 6, 75) that had scrapie outbreaks between 1998 and 2003. In four of these herds (nos 1, 3, 4 and 5), the vaccine against M. agalactiae had been administered. The animals were primarily of the Maltese, Camosciata and crossed breeds. Their age ranged from 1 to 10 years. Twenty-five scrapie-positive cases (age range, 4·5–9 years) were present and distributed among the six different outbreaks as follows: herd 1, 2/61; herd 2, 1/11; herd 3, 0/35; herd 4, 13/14; herd 5, 7/39; herd 6, 2/17 (cases/sample size). Material from the one positive case of the third outbreak was unavailable. For scrapie diagnosis, the obex region was examined by histopathology, immunohistochemistry and/or Western blotting.
Genomic DNA was isolated from 108 frozen brain tissue and 69 EDTA-treated blood samples by using manual Qiagen kits or Thermo Labsystems KingFisher kits, respectively. PCR amplification of the entire open reading frame of the PrP gene was performed according to a protocol described previously (Acutis et al., 2004), using the primers p8(+) (5'-CAGGTTAACGATGGTGAAAAGCCACATAGG-3') and p9(–) (5'-GGAATTCTATCCTACTATGAGAAAAATGAGG-3') (Bossers et al., 1996). PrP polymorphisms were detected by direct DNA sequencing on both strands of the PCR products by using dye terminator cycle sequencing and an ABI Prism 310 Genetic Analyser (Applied Biosystems). Sequencing primers were p8(+), p61(+) (5'-AACCAACATGAAGCATGTGG-3'), p60(–) (5'-GATAGTAACGGTCCTCATAG-3') and p9(–) (Belt et al., 1995). The primers hybridized to the target PrP DNA at codons 1–7, 109–116, 147–154 and 249–257, respectively. To link the detected polymorphisms into an allele sequence (haplotype), PCR-amplified products of selected samples were cloned in a TA cloning vector (Invitrogen). At least five clones each were analysed by sequencing to identify the polymorphisms per allele. Several polymorphism combinations (haplotypes) were checked from different animals to exclude potential heterogeneous coupling.
A 
2 test was performed to look for associations between each allele and scrapie status. This was done by comparing the frequencies of genotypes with and without an allele between cases and controls: heterozygotes and homozygotes for the same allele were combined in a single group. When data were sufficient for multivariate analysis, a mixed logistic regression model with a binomially distributed error term was fitted to the outcome variable (i.e. the scrapie status). Age and vaccination were included in the model as covariates, whereas ‘herd’ was included as a random effect to control for the effect of clustering of goats within each outbreak. All descriptive statistics and data manipulation were performed by using Stata Statistical Software version 9 (Stata Corporation); the Stata macro gllamm was used to fit the mixed logistic model.
Eleven polymorphisms were identified (Table 1). Two of these polymorphisms had not been reported previously: at codon 133, a ctgcag substitution caused an amino acid change of LQ and at codon 137, an atgata substitution led to the amino acid change MI. As reported previously by Goldmann et al. (1996), silent mutations were also found at codon 42 (ccaccg) (134 goats, 55 of which were homozygotes) and at codon 138 (agcagt in 142 goats, 57 of which were homozygotes), and a new silent nucleotide change was detected at codon 202 (accact) (four heterozygous goats). The three-octarepeat variant was not found in any of the examined animals. Twelve alleles were determined (Table 1) by single-allele sequencing (by cloning), which were found combined in 37 different genotypes (Table 2). The most frequent alleles were 1 (corresponding to the phylogenetic wild type of sheep) and 2, which were distinguished only by the codon 240 S/P dimorphism. Predominant genotypes were 1/2 and 2/2. According to Goldmann et al. (1996), silent mutations at codons 42 and 138 were found in linkage with the dimorphism at codon 240: 42a and 138c were linked to codon S240, whereas 42g and 138t were linked to codon P240. Similarly, the novel ct substitution at codon 202 was found solely in linkage with P240.
|
|
shows the 2 comparisons. Only two alleles (2 and 12) showed a significant association with scrapie status. Allele 12 was not present in any scrapie case, thus suggesting a potential protective effect. The complete absence of cases with this allele precluded any further statistical analysis. Univariate analysis showed that the presence of allele 2 was associated with an increased risk of scrapie [odds ratio (OR), 6·4; 95 % confidence interval (CI), 1·8–22·4]. This positive effect was still evident after adjusting for age and vaccine (OR, 8·6; 95 % CI, 1·8–40·2), but lost its statistical significance when the mixed logistic model included the herd as a random effect (OR, 2·4; 95 % CI, 0·4–15·2).
|
Our case–control study included a relatively high number of scrapie outbreaks and animals; however, a possible selection bias cannot be ruled out, as case and control recruitment was restricted to the limited material available. Even so, taking into account data from other studies, several suggestions can be made. Both alleles 1 (S240) and 2 (P240) were found in scrapie-positive animals; the significant positive association between allele 2 and scrapie positivity revealed by univariate analysis was unconfirmed by multivariate analysis. An association between the 240 polymorphism and scrapie has been excluded by some authors (Goldmann et al., 1996; Billinis et al., 2002), who hypothesized that this codon is probably eliminated during post-translational processing of the caprine PrP. It could well be, however, that the polymorphisms at codon 240 modulate disease susceptibility by interfering with mRNA stability or they may be linked to another quantitative trait of the animal.
Allele 3 (37V) did not appear to be associated with scrapie status. Alleles 4 (P110), 5 (S127), 6 (Q133) and 7 (I137) were found only in healthy animals, but at a frequency too low to establish an association. Allele 5 was found only in the Camosciata breed; the same amino acid change was also found in Mongolian sheep (Gombojav et al., 2003) with an unknown association with scrapie. In Dutch Swifter and Icelandic sheep, a mutation at the same amino acid position of allele 7 (137) was found, but with a change from M to T instead of M to I (Bossers et al., 1996; Thorgeirsdottir et al., 1999). No association between this codon and scrapie in sheep has been established, although the M137T polymorphism also seems to modulate sheep PrP conversion (Bossers et al., 2000). Allele 8 (M142) has been shown to prolong incubation periods in goats challenged experimentally with scrapie or BSE (Goldmann et al., 1996), thus suggesting that this allele may confer partial resistance to the disease. In our study, we were unable to assess this association because of the low frequency of this allele in general and because no scrapie-positive goats with allele 8 were present in our sample: the frequency in our Italian goats (2 %) was much lower than that reported by Goldmann et al. (2004) for the UK (28 %). Should more extensive studies confirm this frequency, allele 8 might not be a practical target for genetic selection in goats in Italy. Alleles 9 (R143) and 10 (H154) are thought to offer some protection against scrapie infection in Greek goats (Billinis et al., 2002). Our results differed in that the two alleles were found to occur at similar frequencies in both scrapie-affected and healthy animals. Furthermore, the age of the positive animals carrying these alleles was no higher than that of the other cases (data not shown), which suggests no alteration in the incubation period. The reasons for these differences may depend on the small sample size, the different susceptibility profiles in the goat populations by other breed- or country-specific factors or the presence of a different isolate/strain of scrapie agent. In sheep, the H154 polymorphism seems to have different effects. It is associated with resistance in some breeds (Dawson et al., 1998; Thorgeirsdottir et al., 1999), but with susceptibility in other breeds and countries (Dawson et al., 1998; Acutis et al., 2004; Vascellari et al., 2005).
Allele 11 is characterized by a polymorphism at codon 168 (PQ), which is also polymorphic in sheep, with a change from P to L. In sheep, this mutation appears to prolong the incubation period of the disease (Baylis & Goldmann, 2004). What is remarkable is that, despite its very low frequency in the study sample, allele 11 was found in one scrapie-positive goat. The animal was 8 years old, but was not the oldest case. This result suggests that amino acid changes at codon 168 have a different effect on susceptibility to scrapie in sheep and in goats, as has been demonstrated, for instance, in sheep having codon A136 or V136, that results in different disease susceptibility.
A noteworthy result of our study is the significant association between allele 12 (K222) and healthy animals. No goats carrying this allele were found to be scrapie-positive, even though the frequency was not low. In contrast, all other alleles with similar frequencies were also actually present in the group of cases. Moreover, allele 12 had a relatively high frequency in all but one herd, whether vaccinated or not (4·9 % in herd 1; 9 % in herd 2; 7·14 % in herd 3; 0 % in herd 4; 11·5 % in herd 5; 8·8 % in herd 6). A multivariate analysis to assess further the possible protection given by allele 12 in goats could not be done precisely because of the absence of scrapie-affected animals, so potential confounders on 2 analysis could not be explored. It is noteworthy that a similar change is found in human PrP (Q219K) (Shibuya et al., 1998), where it serves as a unique protective factor against sporadic Creutzfeldt–Jakob disease. This could support our hypothesis for a protective role of lysine at codon 222 in goats, assuming that the same single amino acid change seems to have the same effect in different species. In vitro experiments to study the efficacy of this polymorphism in converting normal PrP into pathological PrP are now under way. More genetic data on goats in scrapie outbreaks will need to be collected to confirm whether K222 can really be a practical target for breeding towards scrapie resistance. The possibility that a genetic influence on TSE susceptibility may also be present in the goat, but conferred by alleles different from those of sheep, may also be useful generally for TSE research in other species (with similar or even identical alleles) in the same way that sheep genetics originally revealed disease associations and potential mechanisms of disease.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Agrimi, U., Ru, G., Cardone, F., Pocchiari, M. & Caramelli, M. (1999). Epidemic of transmissible spongiform encephalopathy in sheep and goats in Italy. Lancet 353, 560–561.[Medline]
Agrimi, U., Conte, M., Morelli, L. & 9 other authors (2003). Animal transmissible spongiform encephalopathies and genetics. Vet Res Commun 27 (Suppl. 1), 31–38.
Bartz, J. C., McKenzie, D. I., Bessen, R. A., Marsh, R. F. & Aiken, J. M. (1994). Transmissible mink encephalopathy species barrier effect between ferret and mink: PrP gene and protein analysis. J Gen Virol 75, 2947–2953.
Baylis, M. & Goldmann, W. (2004). The genetics of scrapie in sheep and goats. Curr Mol Med 4, 385–396.[CrossRef][Medline]
Belt, P. B. G. M., Muileman, I. H., Schreuder, B. E. C., Bos-de Ruijter, J., Gielkens, A. L. J. & Smits, M. A. (1995). Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 76, 509–517.
Billinis, C., Panagiotidis, C. H., Psychas, V., Argyroudis, S., Nicolaou, A., Leontides, S., Papadopoulos, O. & Sklaviadis, T. (2002). Prion protein gene polymorphisms in natural goat scrapie. J Gen Virol 83, 713–721.
Bossers, A., Schreuder, B. E. C., Muileman, I. H., Belt, P. B. G. M. & Smits, M. A. (1996). PrP genotype contributes to determining survival times of sheep with natural scrapie. J Gen Virol 77, 2669–2673.
Bossers, A., de Vries, R. & Smits, M. A. (2000). Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. J Virol 74, 1407–1414.
Capucchio, M. T., Guarda, F., Isaia, M. C., Caracappa, S. & Di Marco, V. (1998). Natural occurrence of scrapie in goats in Italy. Vet Rec 143, 452–453.[Medline]
Caramelli, M., Ru, G., Casalone, C., Bozzetta, E., Acutis, P. L., Calella, A. & Forloni, G. (2001). Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae. Vet Rec 148, 531–536.
Dawson, M., Hoinville, L. J., Hosie, B. D. & Hunter, N. (1998). Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Scrapie Information Group. Vet Rec 142, 623–625.[Medline]
Goldmann, W., Martin, T., Foster, J., Hughes, S., Smith, G., Hughes, K., Dawson, M. & Hunter, N. (1996). Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. J Gen Virol 77, 2885–2891.
Goldmann, W., Chong, A., Foster, J., Hope, J. & Hunter, N. (1998). The shortest known prion protein gene allele occurs in goats, has only three octapeptide repeats and is non-pathogenic. J Gen Virol 79, 3173–3176.[Abstract]
Goldmann, W., Perucchini, M., Smith, A. & Hunter, N. (2004). Genetic variability of the PrP gene in a goat herd in the UK. Vet Rec 155, 177–178.
Gombojav, A., Ishiguro, N., Horiuchi, M., Serjmyadag, D., Byambaa, B. & Shinagawa, M. (2003). Amino acid polymorphisms of PrP gene in Mongolian sheep. J Vet Med Sci 65, 75–81.[CrossRef][Medline]
Hunter, N., Foster, J. D., Goldmann, W., Stear, M. J., Hope, J. & Bostock, C. (1996). Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch Virol 141, 809–824.[CrossRef][Medline]
Hunter, N., Goldmann, W., Foster, J. D., Cairns, D. & Smith, G. (1997). Natural scrapie and PrP genotype: case-control studies in British sheep. Vet Rec 141, 137–140.
Kurosaki, Y., Ishiguro, N., Horiuchi, M. & Shinagawa, M. (2005). Polymorphisms of caprine PrP gene detected in Japan. J Vet Med Sci 67, 321–323.[Medline]
Shibuya, S., Higuchi, J., Shin, R. W., Tateishi, J. & Kitamoto, T. (1998). Codon 219 Lys allele of PRNP is not found in sporadic Creutzfeldt–Jakob disease. Ann Neurol 43, 826–828.[CrossRef][Medline]
Thorgeirsdottir, S., Sigurdarson, S., Thorisson, H. M., Georgsson, G. & Palsdottir, A. (1999). PrP gene polymorphism and natural scrapie in Icelandic sheep. J Gen Virol 80, 2527–2534.
Vascellari, M., Aufiero, G. M., Nonno, R. & 7 other authors (2005). Diagnosis and PrP genotype target of scrapie in clinically healthy sheep of Massese breed in the framework of a scrapie eradication programme. Arch Virol 150, 1959–1976.[CrossRef][Medline]
Wopfner, F., Weidenhöfer, G., Schneider, R., von Brunn, A., Gilch, S., Schwarz, T. F., Werner, T. & Schätzl, H. M. (1999). Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. J Mol Biol 289, 1163–1178.[CrossRef][Medline]
Zhang, L., Li, N., Fan, B., Fang, M. & Xu, W. (2004). PRNP polymorphisms in Chinese ovine, caprine and bovine breeds. Anim Genet 35, 457–461.[Medline]
Received 16 August 2005;
accepted 14 December 2005.
This article has been cited by other articles:
|
|
 |
|
  S. Colussi, G. Vaccari, C. Maurella, C. Bona, R. Lorenzetti, P. Troiano, F. Casalinuovo, A. Di Sarno, M. G. Maniaci, F. Zuccon, et al. Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats J. Gen. Virol., December 1, 2008; 89(12): 3173 - 3176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Rongyan, L. Xianglong, L. Lanhui, L. Xiangyun, and F. Fujun Evolution and Differentiation of the Prion Protein Gene (PRNP) among Species J. Hered., November 1, 2008; 99(6): 647 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vaccari, C. D'Agostino, R. Nonno, F. Rosone, M. Conte, M. A. Di Bari, B. Chiappini, E. Esposito, L. De Grossi, F. Giordani, et al. Prion Protein Alleles Showing a Protective Effect on the Susceptibility of Sheep to Scrapie and Bovine Spongiform Encephalopathy J. Virol., July 1, 2007; 81(13): 7306 - 7309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Goldmann, F. Houston, P. Stewart, M. Perucchini, J. Foster, and N. Hunter Ovine prion protein variant A136R154L168Q171 increases resistance to experimental challenge with bovine spongiform encephalopathy agent J. Gen. Virol., December 1, 2006; 87(12): 3741 - 3745. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
