Lack of relationship between Visna/maedi infection and scrapie resistance genetic markers

The relationship between Visna/maedi virus (VMV) antibody status and scrapie genetic resistance of 10,611 Rasa Aragonesa sheep from 17 flocks in Aragón (Spain) was investigated. The fifteen most common PRNP gene haplotypes and genotypes were identified and the genotypes were classified into the corresponding scrapie risk groups (groups 1 to 5). ARQ (93.3%) and ARR (31.8%) were the most common haplotypes and ARQ/ARQ (56%) and ARR/ARQ (25.6%) were the most common genotypes. The frequencies of scrapie risk groups 1, 2, 3, 4 and 5 were 3.3%, 27.3%, 63.5%, 1.2% and 4.8%, respectively. Overall Visna/maedi seroprevalence was 53% and flock seroprevalence ranged between 21-86%. A random effects logistic regression model indicated that sheep VMV serological status (outcome variable) was not associated with any particular scrapie risk group. Instead, VMV seropositivity progressively increased with age, was signif icantly greater in females compared to males and varied between flocks. The absence of a relationship between VMV infection and scrapie genotypes is important for VMV control and specifically for sheep participating in an ELISA-based Visna/maedi control program. Additional key words: sheep health; lentivirus; prion; genetic resistance; PRNP; ELISA. * Corresponding author: [email protected] Received: 19-12-13. Accepted: 27-06-14. Abbreviations used: PRNP (prion protein gene); PrPSc (prion protein scrapie); ROD (relative optical density); VM (Visna/maedi); VMV (Visna/maedi virus). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(3): 676-682 http://dx.doi.org/10.5424/sjar/2014123-5489 ISSN: 1695-971X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS Lack of relationship between Visna/maedi virus and scrapie 677 Brinkhof & van Maanen, 2007). An intensive regimen of testing at short intervals for VMV antibodies combined with strict management could result in eradication of the infection in a short period of time (Pérez et al., 2010). There are f ive different scrapie risk groups (R) named R1 to R5, that depend on the genotype of the individual: R1 includes sheep with genotype ARR/ARR that is linked to the highest resistance to scrapie whereas R5 is associated to the highest susceptibility to scrapie and includes sheep with genotypes containing the VRQ haplotype in combination with AHQ, ARH, ARQ or VRQ haplotypes. Finally, R2, R3 and R4 include sheep with genotypes with other haplotype combinations that are linked to low, intermediate or high susceptibility to scrapie (Dawson et al., 1998). In 2002, it was decided to improve the European Union (EU) sheep population genetic resistance to scrapie by implementing a compulsory prion protein gene (PRNP)based genetic selection in EU countries. All animals were genotyped and classified according to the abovementioned criteria and only R1 rams and R1, R2 and R3 ewes could be used for breeding purposes (OJ, 2001, 2003). Genetic selection favouring scrapie resistance may have unsuspected effects in other genetic traits. However, none of the health traits studied so far has demonstrated relationship with PRNP genotypes (EFSA, 2006; Sweeney & Hanrahan, 2008). Among these studies, the resistance to infection by intestinal nematodes (Gruner et al., 2004) or by Salmonella spp. (Vitezica et al., 2007), the milk somatic cell count (de Vries et al., 2005; Álvarez et al., 2006), the specific immune system responses (Eaton et al., 2007) and the blood levels of VM provirus (Harrington et al., 2009) have been investigated. In certain geographical areas, a temporal coincidence between the two control programs (against VMV and scrapie) has occurred but no scientif ic work has been performed on the possible interaction between them. The objective of this work was to assess the potential relationship between genetic susceptibility to scrapie and VMV infection. The study was conducted in more than ten thousand Rasa Aragonesa sheep in Aragón (Spain) that were enrolled in a VMV control program (Pérez et al., 2010). This work also offered the opportunity to describe the PRNP genotype frequencies in sheep from flocks enrolled in a scrapie genetic resistance selection breeding program. Material and methods Selected flocks and animals Flock selection criteria were based on: i) having at least 20% VM seroprevalence as determined in a previous study (Pérez et al., 2010), ii) being a fully scrapiegenotyped flock, and iii) having birth dates and sex records for the studied sheep. A total of 17 Rasa Aragonesa flocks distributed in Aragón (Fig. 1), were selected. The total number of animals included in the study was 10,611 sheep and the minimum, maximum, median and interquartile range flock size were 241, 2234, 593 and 281-741 sheep, respectively. Seroprevalence for VM and PRNP genotype data were obtained between years 2004 and 2008. Sheep PRNP genotypes and VMV serological antibody status Individual PRNP genotype results for codons 136, 154 and 171 were used to classify sheep in the corresponding scrapie risk groups (R1-R5; Dawson et al., 1998) (Table 1). The individual sheep VMV ELISA antibody status was known from previous analysis performed in the frame of the control and eradication program supported by the Government of Aragón and published elsewhere (Pérez et al., 2010). The test used in the previous study was the highly sensitive and specif ic recombinant protein-based ELISA “ELITESTTM” (Hyphen Biomed, France; Saman et al., 1999). Sample optical density (OD) values were divided by the ELISA plate cut-off value to obtain the relative OD (ROD), according to the manufacturer’s instructions. Statistical analysis Yates-corrected chi-square test and analysis of variance were used to compare proportions and means, respectively, using Epi Info 6.0 (CDC, Atlanta, USA). The Glimmix macro in the SAS program (SAS Inst., Cary, NC, USA) was employed to carry out random effects logistic regression analysis for investigating the relationship between sheep VMV serological status and scrapie susceptibility-related PRNP haplotypes, risk groups and genotypes (present in at least 50 sheep), taking into account age, gender and flock of 678 E. Salazar et al. / Span J Agric Res (2014) 12(3): 676-682 origin. In logistic models, VMV status was the dependent variable, gender, age and one of the previous PRNP data were included as independent f ixed categorical variables, and finally flock of origin was included as a random independent variable. Values p were from likelihood-ratio chi-square test (SAS Inst.) and alpha was 5% (two sided).