Genetic resistance to transmissible spongiform encephalopathies (TSE) in goats
2017; Wiley; Volume: 15; Issue: 8 Linguagem: Inglês
10.2903/j.efsa.2017.4962
ISSN1831-4732
AutoresAntonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez, Rosina Gironés, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy J. Robertson, Giuseppe Ru, Moez Sanaa, Panagiotis Skandamis, Niko Speybroeck, M. M. Simmons, Benno Ter Kuile, John Threlfall, Helene Wahlström, Pier Luigi Acutis, Olivier Andréoletti, Wilfred Goldmann, Jan Langeveld, J.J. Windig, Angel Ortiz Pelaez, Emma Snary,
Tópico(s)Trace Elements in Health
ResumoEFSA JournalVolume 15, Issue 8 e04962 Scientific OpinionOpen Access Genetic resistance to transmissible spongiform encephalopathies (TSE) in goats EFSA Panel on Biological Hazards (BIOHAZ), EFSA Panel on Biological Hazards (BIOHAZ)Search for more papers by this authorAntonia Ricci, Antonia RicciSearch for more papers by this authorAna Allende, Ana AllendeSearch for more papers by this authorDeclan Bolton, Declan BoltonSearch for more papers by this authorMarianne Chemaly, Marianne ChemalySearch for more papers by this authorRobert Davies, Robert DaviesSearch for more papers by this authorPablo Salvador Fernández Escámez, Pablo Salvador Fernández EscámezSearch for more papers by this authorRosina Gironés, Rosina GironésSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorKostas Koutsoumanis, Kostas KoutsoumanisSearch for more papers by this authorRoland Lindqvist, Roland LindqvistSearch for more papers by this authorBirgit Nørrung, Birgit NørrungSearch for more papers by this authorLucy Robertson, Lucy RobertsonSearch for more papers by this authorGiuseppe Ru, Giuseppe RuSearch for more papers by this authorMoez Sanaa, Moez SanaaSearch for more papers by this authorPanagiotis Skandamis, Panagiotis SkandamisSearch for more papers by this authorNiko Speybroeck, Niko SpeybroeckSearch for more papers by this authorMarion Simmons, Marion SimmonsSearch for more papers by this authorBenno Ter Kuile, Benno Ter KuileSearch for more papers by this authorJohn Threlfall, John ThrelfallSearch for more papers by this authorHelene Wahlström, Helene WahlströmSearch for more papers by this authorPier-Luigi Acutis, Pier-Luigi AcutisSearch for more papers by this authorOlivier Andreoletti, Olivier AndreolettiSearch for more papers by this authorWilfred Goldmann, Wilfred GoldmannSearch for more papers by this authorJan Langeveld, Jan LangeveldSearch for more papers by this authorJack J Windig, Jack J WindigSearch for more papers by this authorAngel Ortiz Pelaez, Angel Ortiz PelaezSearch for more papers by this authorEmma Snary, Emma SnarySearch for more papers by this author EFSA Panel on Biological Hazards (BIOHAZ), EFSA Panel on Biological Hazards (BIOHAZ)Search for more papers by this authorAntonia Ricci, Antonia RicciSearch for more papers by this authorAna Allende, Ana AllendeSearch for more papers by this authorDeclan Bolton, Declan BoltonSearch for more papers by this authorMarianne Chemaly, Marianne ChemalySearch for more papers by this authorRobert Davies, Robert DaviesSearch for more papers by this authorPablo Salvador Fernández Escámez, Pablo Salvador Fernández EscámezSearch for more papers by this authorRosina Gironés, Rosina GironésSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorKostas Koutsoumanis, Kostas KoutsoumanisSearch for more papers by this authorRoland Lindqvist, Roland LindqvistSearch for more papers by this authorBirgit Nørrung, Birgit NørrungSearch for more papers by this authorLucy Robertson, Lucy RobertsonSearch for more papers by this authorGiuseppe Ru, Giuseppe RuSearch for more papers by this authorMoez Sanaa, Moez SanaaSearch for more papers by this authorPanagiotis Skandamis, Panagiotis SkandamisSearch for more papers by this authorNiko Speybroeck, Niko SpeybroeckSearch for more papers by this authorMarion Simmons, Marion SimmonsSearch for more papers by this authorBenno Ter Kuile, Benno Ter KuileSearch for more papers by this authorJohn Threlfall, John ThrelfallSearch for more papers by this authorHelene Wahlström, Helene WahlströmSearch for more papers by this authorPier-Luigi Acutis, Pier-Luigi AcutisSearch for more papers by this authorOlivier Andreoletti, Olivier AndreolettiSearch for more papers by this authorWilfred Goldmann, Wilfred GoldmannSearch for more papers by this authorJan Langeveld, Jan LangeveldSearch for more papers by this authorJack J Windig, Jack J WindigSearch for more papers by this authorAngel Ortiz Pelaez, Angel Ortiz PelaezSearch for more papers by this authorEmma Snary, Emma SnarySearch for more papers by this author First published: 10 August 2017 https://doi.org/10.2903/j.efsa.2017.4962Citations: 11 Correspondence: biohaz@efsa.europa.eu Requestor: European Commission Question number: EFSA-Q-2016-00268 Panel members: Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez, Rosina Gironés, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy Robertson, Moez Sanaa, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, Giuseppe Ru, Marion Simmons, John Threlfall and Helene Wahlström. Declared interests: Marion Simmons and Giuseppe Ru recused themselves from the discussion during Working Group meetings that involved two documents to which they had contributed and which were submitted by the European Commission along with the mandate. Both documents are listed in the section Documentation provided to EFSA on p. 53 (numbers 1 and 2) of this Opinion. Acknowledgements: The Panel wishes to thank the members of the GoatBSE and GOAT-TSE-FREE consortia, and the Cypriot Veterinary authorities. Reproduction of the images listed below is prohibited and permission must be sought directly from the copyright holder: Figure 11 and Figures in Appendix E: © Cypriot Veterinary Services Adopted: 5 July 2017 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Breeding programmes to promote resistance to classical scrapie, similar to those for sheep in existing transmissible spongiform encephalopathies (TSE) regulations, have not been established in goats. The European Commission requested a scientific opinion from EFSA on the current knowledge of genetic resistance to TSE in goats. An evaluation tool, which considers both the weight of evidence and strength of resistance to classical scrapie of alleles in the goat PRNP gene, was developed and applied to nine selected alleles of interest. Using the tool, the quality and certainty of the field and experimental data are considered robust enough to conclude that the K222, D146 and S146 alleles both confer genetic resistance against classical scrapie strains known to occur naturally in the EU goat population, with which they have been challenged both experimentally and under field conditions. The weight of evidence for K222 is greater than that currently available for the D146 and S146 alleles and for the ARR allele in sheep in 2001. Breeding for resistance can be an effective tool for controlling classical scrapie in goats and it could be an option available to member states, both at herd and population levels. There is insufficient evidence to assess the impact of K222, D146 and S146 alleles on susceptibility to atypical scrapie and bovine spongiform encephalopathy (BSE), or on health and production traits. These alleles are heterogeneously distributed across the EU Member States and goat breeds, but often at low frequencies (< 10%). Given these low frequencies, high selection pressure may have an adverse effect on genetic diversity so any breeding for resistance programmes should be developed at Member States, rather than EU level and their impact monitored, with particular attention to the potential for any negative impact in rare or small population breeds. Summary Following a request from the European Commission, the European Food Safety Authority (EFSA) was asked to deliver a scientific opinion on genetic resistance to transmissible spongiform encephalopathies (TSEs) in goats, addressing the following terms of reference: (1) Is there sufficient scientific knowledge available to have a robust level of scientific assurance that certain polymorphisms of the prion protein gene (PRNP) present in European goat breeds confer genetic resistance to classical scrapie (i.e. to classical scrapie strains known to occur in the European Union (EU) goat population)? If this is the case, which are those polymorphisms? (2) Based on available scientific evidence, what is the frequency and distribution of PRNP polymorphisms conferring resistance to classical scrapie in European goat breeds? If possible, could EFSA produce a susceptibility ranking of goat PRNP genotypes to classical scrapie? (3) Based on available scientific evidence, what is the level of susceptibility to atypical scrapie and to BSE of the PRNP polymorphisms conferring resistance to classical scrapie? (4) What is the likely impact of measures promoting PRNP polymorphisms conferring resistance to classical scrapie in terms of susceptibility to other disease/s, of production traits and survivability, taking into account epidemiological differences between Member States (MS)? Are such polymorphisms likely to have adverse effects on genetic diversity and variability and on the maintenance of old or rare caprine breeds or those that are well-adapted to a particular region? (5) What are EFSA's recommendations concerning strategies to apply current knowledge on genetic resistance to classical scrapie in goats in order to control and/or eradicate classical scrapie in the EU goat population? A tool was developed to evaluate the genetic resistance to classical scrapie of alleles in the goat PRNP gene, which considers both the weight of evidence and strength of resistance for each allele of interest. The weight of evidence was based on a scale made of the combination of different types of studies, from the one that provides the least evidence, the in vitro conversion studies, to the combination of all possible studies (epidemiological studies, experimental challenge in natural host using multiple isolates, bioassays in allele specific transgenic mice using multiple isolates and in vitro conversion studies). Whether the allele of interest had been experimentally investigated against multiple scrapie isolates from geographically different origins was considered. The strength of resistance conferred by an allele was defined by scoring it, in a traffic light colour system, as demonstrating one of three levels of resistance using the wild type of the goat PRNP gene as a baseline: Red: no resistance; Amber: partial resistance; Green: resistance. To assign the strength of resistance for each allele of interest information on the presence of field cases holding the alleles of interest, the attack rates and the incubation period in the natural host or in allele-specific transgenic mice were considered as indicators. A fourth score 'grey' was also given for the specific situation where there was just one study available to support the putative resistance of an allele. As a comparator, the tool was also applied to the knowledge on the role of the ovine ARR allele that was available at the time the opinion of the Scientific Steering Committee on safe sourcing of small ruminant materials was produced in 2001 (SSC, 2002). An extensive literature review was conducted to identify relevant alleles to which the tool could be applied, to determine the frequency and distribution of such alleles within different MS and goat breeds and to assess their resistance to atypical scrapie and bovine spongiform encephalopathy (BSE). For the nine selected alleles, namely S127, M142, R143, D145, D146, S146, H154, Q211 and K222, a considerable data set has been produced to assess the levels of resistance to classical scrapie, including details of the type of evidence for or against the association with resistance to TSE. A different class of haplotype in which a nonsense polymorphism (G32stop) occurs was also reviewed, but not included in the tool since this mutation fails to translate full-length PrP protein. In goats, there are no published studies on any effects of PRNP polymorphisms on traits other than resistance to classical scrapie. Therefore, the scant available literature on effects of PRNP polymorphisms in sheep on other traits was used to assess, by extrapolation, the likely impact in goats. It is concluded that the scientific knowledge related to scrapie resistance associated with goat PRNP gene polymorphisms has considerably expanded in the last 10 years. The K222, D146 and S146 alleles confer genetic resistance to classical scrapie strains known to occur in the EU goat population. The K222 polymorphism confers resistance against a variety of EU classical scrapie isolates that may reflect a variety of scrapie strains. However, there is no assurance that K222 carriers would be resistant to all TSE agent strains currently circulating in the EU goat population. The D146 and S146 alleles are associated with strong resistance against scrapie agent(s) currently circulating in Cyprus. However, data remain insufficient to assess the level of resistance that D146 and S146 might provide against other classical scrapie agents circulating in other EU goat populations. The weight of evidence for K222 is greater than that currently available for the D146 and S146 alleles and for the ARR allele in sheep at the time the 2002 SSC opinion was produced. There are limited data available on the frequency and distribution of the PRNP alleles conferring resistance to classical scrapie as they are only known for less than 10% of the breeds listed in EU MS, and only in a restricted number of MS. Cyprus is the only MS with accurate genotype data about the whole goat population: more than 50% of the goats in Cyprus have either the D146 or the S146 allele. In other MS, these alleles are represented at low frequencies, or are absent, with some exceptions. The K222 allele is not present in Cyprus, but has been observed in most of the investigated EU breeds, although generally at low levels (< 10%). In some breeds (Cashmere, Angora, Spanish autochthonous breeds), the polymorphisms K222, D146 and S146 have not been reported. Due to insufficient data, it is not possible to provide a ranking of susceptibility at genotype level. Based on a combination of the 'weight of evidence' and the 'strength of resistance', alleles can currently be ranked as follows, from high to low classical scrapie resistance: K222 > D146 = S146 >Q211 = H154 = M142 > S127 = H143 > wild type. There is extremely limited data on the susceptibility of goats to either atypical scrapie or BSE for the nine alleles of interest. For atypical scrapie, it was hypothesised that the H154 allele is likely to be associated with higher risk of developing atypical scrapie. For BSE, there are currently no data available on the resistance/susceptibility to BSE infection in D146 and S146 allele carriers, but an oral challenge study indicated incomplete resistance to BSE in K222 allele carriers. There are no data available on the susceptibility of goats with the H154 allele to BSE infection. Information on the relationship of PRNP alleles with other traits is lacking in goats and therefore the likely impact of measures promoting selection for resistance on other traits has been inferred from studies in sheep. From this literature, it is inferred that a direct effect of PRNP alleles on health and production traits is unlikely. However, in breeds with low frequencies of the favourable allele(s), and breeds with small effective population size, selection could affect other traits, with the direction of change being unpredictable. Given the low frequencies of favourable alleles in most breeds, including breeds with large populations such as Alpine and Saanen and old breeds that are well adapted to particular regions, high selection pressure is likely to have an adverse effect on genetic diversity. The following recommendations concerning the strategies of breeding for resistance were made: (a); genetic resistance can be considered to be an effective tool to control classical scrapie in goats and may be offered as an option for MS to control classical scrapie in goats; (b) outbreak management for classical scrapie in goat herds could be based on the selection of genetically resistant animals, as defined for sheep in the Regulation 999/2001 (EC); (c) breeding for resistance programmes should be designed to take into account the starting allele frequencies with the view to preventing loss of genetic diversity and they should be developed and managed at MS level, with the impact of their implementation monitored, and with consideration being given to derogations if implementation is compulsory; (d) before developing any breeding for resistance programme, baseline surveys are needed to establish the resistant-allele frequencies in the relevant goat populations at both MS and breed level. Consideration was given to other aspects such as (a) informing relevant stakeholders, especially breeders, about genetic resistance to classical scrapie in goats; (b) producing guidance on how to disseminate resistant alleles in goat breeds; (c) establishing a central database to know where and how many resistant animals are available for breeding purposes at MS level; (d) encouraging the creation of a pool of resistant animals, semen, embryos and ova for dissemination of the resistant alleles in the population; (e) investigating the potential association of alleles conferring resistance to scrapie with other traits; this may be achieved through ad hoc studies or monitored during the breeding programmes. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor As regards ovine animals, the former Scientific Steering Committee (SSC) highlighted the resistance to transmissible spongiform encephalopathies (TSE) of ARR/ARR sheep in its 2002 Scientific Opinion on safe sourcing of small ruminant materials,1 which was based on its 1999 Scientific Opinion on the policy of breeding and genotyping of sheep.2 These scientific opinions were the basis for the adoption of Regulation (EC) No 260/20033, which revised the requirements for eradication measures in case of the detection of TSE in a holding by allowing the destruction of susceptible ovine animals only and requiring the implementation of measures aimed at increasing sheep's resistance to TSEs in the infected holding. In addition, these scientific opinions were the basis for the adoption of Commission Decision 2003/100/EC4, which laid down requirements for the establishments of breeding programmes for resistance to TSE in sheep. The breeding programmes requirements were then integrated into Regulation (EC) No 999/2001 by Commission Regulation (EC) No 1923/20065 and Commission Regulation (EC) No 727/20076. In 2006, in its Opinion on the breeding programme for TSE resistance in sheep', the Scientific Panel on Biological Hazard of the European Food Safety Authority (EFSA) confirmed the appropriateness of such a strategy (EFSA, 2006). As regards caprine animals, until recently, there was not enough scientific knowledge on resistance to TSE in goats to adopt similar measures. However, based on the development of scientific evidence, EFSA, in its 2014 Scientific Opinion on the scrapie situation in the European Union (EU) after 10 years of monitoring and control in sheep and goats (EFSA BIOHAZ Panel, 2014), recommended that selection activities and dissemination of resistant bucks should be promoted and that formal breeding for resistance programmes, similar to those already implemented for sheep, should be initiated for goats. In recent months, Cyprus and Italy have, respectively, submitted the attached scientific reports (see Documentation provided to EFSA), in order to substantiate the scientific basis of measures that they propose to promote resistance to TSEs in goats. Given the long-term effect of measures of selection and dissemination of animals with a certain genotype, it is necessary to have a solid level of scientific certainty concerning the resistance to TSE of the genotype(s) to be selected and the impact that such measures are likely to have. EFSA is therefore requested to provide a scientific opinion on the following questions: Is there sufficient scientific knowledge available to have a robust level of scientific assurance that certain polymorphisms of the prion protein gene (PRNP) present in European goat breeds confer genetic resistance to classical scrapie (i.e. to classical scrapie strains known to occur in the EU goat population)? If this is the case, which are those polymorphisms? Based on available scientific evidence, what is the frequency and distribution of PRNP polymorphisms conferring resistance to classical scrapie in European goat breeds? If possible, could EFSA produce a susceptibility ranking of goat PRNP genotypes to classical scrapie? Based on available scientific evidence, what is the level of susceptibility to atypical scrapie and to BSE of the PRNP polymorphisms conferring resistance to classical scrapie? What is the likely impact of measures promoting PRNP polymorphisms conferring resistance to classical scrapie in terms of susceptibility to other disease/s, of production traits and survivability, taking into account epidemiological differences between Member States (MS)? Are such polymorphisms likely to have adverse effects on genetic diversity and variability and on the maintenance of old or rare caprine breeds or those that are well-adapted to a particular region? What are EFSA's recommendations concerning strategies to apply current knowledge on genetic resistance to classical scrapie in goats in order to control and/or eradicate classical scrapie in the EU goat population? 1.2 Interpretation of the Terms of Reference With regard to certain points of the Terms of Reference (ToR), these are the interpretations of the Working Group (WG): The WG will address the genetic resistance at breed level whenever possible and at population level when there is insufficient genetic uniformity in groups of goats that may constitute a breed from the legal point of view. The definition of 'strong genetic resistance' proposed by the European Commission included that an animal holding a polymorphism conferring strong resistance 'do not transmit classical scrapie during the productive life'. The WG will provide an answer on resistance to disease in individual animals, according to available knowledge considering all uncertainties and with the data available. The impact of breeding for resistance on small populations will be assessed without referring to any particular population (ToR4). 2 Data and methodologies 2.1 Data Scrapie data were obtained from the EU TSE database, which collects standardised surveillance data on all testing activities in all MS. The overall caseload is based on cases detected through passive and active surveillance, and during the application of eradication measures. Due to the biased and variable nature of passive surveillance, data from clinical cases were excluded from the analysis, and only the more unbiased active surveillance data, namely the animals slaughtered for human consumption (SHC) and the animals not slaughtered for human consumption (NSHC), were used for describing national trends in scrapie prevalence and for describing the occurrence by EU MS. Information was obtained from Eurostat to describe goat demographics. In particular, the number of goats, number of breeding females, number of holdings and number of female breeding holdings were recorded for each MS in both 2005 and 2013. Eurostat was also used to obtain data on the amount of goat meat produced at slaughterhouses in the EU (2006–2015).7 The amount of goat meat produced at slaughterhouses in the EU for the period 2006–2015 was extracted from the EU statistics website, as part of the annual statistical surveys.8 Goat breed data was obtained from the EFABIS database.9 Data on the consumption of goat meat in the EU were obtained from EFSA's Comprehensive European Food Consumption database. The EFSA Comprehensive European Food Consumption Database (Comprehensive Database)10 provides a compilation of national information on food consumption at individual level. It was first built in 2010 (EFSA, 2011a; Huybrechts et al., 2011; Merten et al., 2011). Details on how the Comprehensive Database is used are published in the Guidance of EFSA (EFSA, 2011b). EFSA used its food classification system 'FoodEx' to categorise all foods and beverages included in the Comprehensive Database (EFSA, 2011a). Available data on goat meat consumption in the EU was extracted and summarised. Data on the movements of live goats for breeding in 2014, 2015 and 2016 were extracted from the TRACES database.11 2.2 Methodologies In order to address the ToR as in the mandate, and particularly provide recommendations concerning strategies for a breeding programme to control and/or eradicate classical scrapie in goats (ToR5), it is important to give an overview of the goat sector in the EU in order to put the answers into context. As a consequence of this, Section 3.1 considers information for the EU on the goat demographics in MS, goat meat production, consumption patterns, movement of live goats for breeding within the EU, their genetic diversity, and the scrapie situation in the EU. 2.2.1 Literature reviews An evaluation of the PRNP polymorphisms associated with genetic resistance in goats (ToR 1, 2, 3) was carried out through an extensive literature review. To retrieve data on the PRNP polymorphisms associated with genetic resistance in goats, a literature search in the PubMed database was undertaken. The publication of the first scientific paper describing polymorphisms in the PRNP gene in goats (Goldmann et al., 1996) was used as the starting date of the literature search, with a buffer of one extra year. No language restrictions were applied for the literature search (all languages included in the PubMed search engine were included: 56). The literature search was conducted on 20 July 2016. The following search string was applied: (goat* OR caprine*) AND (TSE* OR BSE OR scrapie OR PrP* OR PRNP OR prion* OR allel* OR gen*) AND (polymorph* OR breed* OR resist* OR susceptib*). These terms were searched in the titles and abstracts of books and documents, case reports, classical articles, clinical trials, comments, comparative studies, data sets, editorials, electronic supplementary materials, English abstracts, introductory journal articles, journal articles, news, newspaper articles, randomised controlled trials, reviews, scientific integrity reviews, systematic reviews, technical reports and validation studies. A total of 946 references were retrieved and a double screening (two pairs of reviewers each independently screened half of the full list) looking for potentially relevant references was conducted. Discrepancies were discussed between the two reviewers until a final shortlist of references was agreed. A subset of 50 references was selected and considered in this assessment by reviewing in full. A further 12 references were shortlisted for their relevance to other ToR during the screening process. To retrieve data on the doppel protein (Dpl), a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene, PRNP, in human and mice, a literature search in the PubMed database was undertaken. The time of publication was restricted to the period 1/1/1995–31/12/2016. No language restrictions were applied for the literature search (all languages included in the PubMed search engine were included: 56). The literature search was conducted on 15 December 2016. The following search string was applied: (sheep OR goat*) AND (doppel OR Doppel OR sprn OR SPRN). These terms were searched in the titles and abstracts of books and documents, case reports, classical article, clinical trial, comment, comparative study, data set, editorial, electronic supplementary materials, English abstract, introductory journal article, journal article, news, newspaper article, randomised controlled trial, review, scientific integrity review, systematic reviews, technical report and validation studies. A total of 27 references were retrieved and all of them were considered in this assessment by reviewing the full papers. 2.2.2 Analysis of surveillance data Analysis of scrapie surveillance data was conducted separately by disease: classical scrapie (CS) vs atypical scrapie (AS). In each individual subset, descriptive frequency tables were produced showing the breakdown of animals tested and cases by country, year, and surveillance stream: SHC and NSHC. The potential for a confounding effect of stream in the case of CS became evident after comparing the stream-specific prevalence and the different distribution of the number of tests carried out in each stream by country or by year. A spatial description of the presence of the scrapie types was carried out by producing two sets of maps: the occurrence of CS and AS for the period 2002–2015 by MS; surveillance stream-adjusted for CS and crude for AS prevalence rates for the period 2002–2015 by MS, produced through proportional symbol mapping. The adjustment on surveillance stream was carried out by means of a direct standardisation using the proportion of tests carried out in the MS in the NSHC vs SHC, in sheep and goats, respectively. Negative binomial models were used to fit 'count of cases detected' and 'year' to estimate the country-specific and stream-adjusted annual prevalence ratios (PR). Significance levels of the slope of the linear funct
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