BUSCADOR RECOLECTA

Esta tesis plantea tres objetivos que se desarrollarán en tres capítulos independientes, correspondientes a sus respectivas publicaciones. Por un lado, se ha caracterizado la presencia y replicación de SRLV en macrófagos procedentes de granulomas inducidos tras la vacunación con formulaciones basadas en hidróxido de aluminio, el adyuvante más
común en las vacunas ovinas. En segundo lugar, se ha analizado una población de más de
1000 individuos en diferentes sistemas de producción y razas e infectados por diferentes
cepas de SRLV. Se ha realizado un estudio serológico y molecular con las herramientas
disponibles en el comercio y se ha analizado la asociación de parámetros productivos, en
granjas de carne y leche, con la presencia de infección. Por último, se han analizado
poblaciones ovinas correspondientes a cuatro razas de importancia productiva teniendo en
cuenta su estado sanitario y el genotipo de la proteína TMEM154, uno de los candidatos
más prometedores para su implantación en programas de selección genética., This thesis addresses three objectives that will be developed in three independent chapters corresponding to their respective publications. In the first one, SRLV presence and replication has been demonstrated in macrophages within granulomas induced after inoculation with aluminium-based vaccines, the most common adjuvant in ovine vaccines.
Second, a population of more than 1000 sheep belonging to different production systems,
breeds and infected by different SRLV strains has been analyzed serologically and
molecularly using commercially available tools, in association to productive traits in meat
and dairy farms. Finally, sheep corresponding to four different breeds relevant for the sheep
industry have been analyzed taking into account their infectious status and the TMEM154
(E35K) genotype, one of the most promising candidates for genetic selection programs., Proyectos de investigación ‘Adyuvantes vacunales alternativos en enfermedades infecciosas ovinas para hacer frente a los efectos adversos derivados del hidróxido de aluminio como adyuvante’, RTI2018-096-172-B-C31; ‘Inmunidad innata en procesos inmunomediados, ASIA ovino y lentivirosis animales’, MINECO AGL 2013-49137-C3-1; ‘Epidemiología molecular y control innato de las infecciones lentivirales en el ganado
Navarro’, Gobierno de Navarra. Plan de Desarrollo Rural (PDR)., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Small ruminant lentiviruses (SRLVs) are endemic in most areas of Europe, causing a
chronic infection and a multisystemic disease affecting the udder, carpal joints, lungs,
and central nervous system. Due to the lack of treatments and protective vaccination
strategies, infection control is focused on the identification of infected animals through
serological or molecular techniques. However, antigenic and genetic heterogeneity
of SRLVs represent a clear drawback for diagnosis. Infected animals may present
lower animal production parameters such as birth weight or milk production and
quality, depending on productive systems considered and, likely, to the diagnostic
method applied. In this study, four sheep flocks dedicated to dairy or meat production
were evaluated using three different ELISA and two PCR strategies to classify animal
population according to SRLV infection status. Productive parameters were recorded
along one whole lactation or reproductive period and compared between positive and
negative animals. SRLV was present in 19% of the total population, being unequally
distributed in the different flocks. Less than half of the infected animals were detected by
a single diagnostic method, highlighting the importance of combining different diagnostic
techniques. Statistical analysis employing animal classification using all the diagnostic
methods associated lambing size, lamb weight at birth, and daily weight gain with SRLV
infection status in meat flocks. Milk production, somatic cell count, fat, and protein
content in the milk were associated with SRLV infection in dairy flocks, to a greater extent
in the flock showing higher seroprevalence. A multi-platform SRLV diagnostic strategy
was useful for ensuring correct animal classification, thus validating downstream studies
investigating production traits., This research was funded by the Spanish Ministry of Science, Innovation and Universities, grant number RTI2018-096172-BC31, and by the Government of Navarra and European Union
ERDF program, grant number PDR (210160006).

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue. IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection., This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and AGL2013-49137-C3-2-R), the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-BC33), and the Recognized Research Groups of Government of Aragón (A17_17R, Animal Health and Reproduction). I.E. was a PhD student funded by the Universidad Pública de Navarra. R.d.M. was a PhD student funded by the Department of Innovation, Research and University of Aragón. J.A. and A.R.-L. were PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education).

Small ruminant lentiviruses (SRLVs) are widely spread in the ovine and caprine populations,
causing an incurable disease aecting animal health and production. Vaccine development is hindered
owing to the high genetic heterogeneity of lentiviruses and the selection of T-cell and antibody escape
mutants, requiring antigen delivery optimization. Sendai virus (SeV) is a respiratory paramyxovirus
in mice that has been recognized as a potent inducer of innate immune responses in several species,
including mouse and human. The aim of this study was to stimulate an innate antiviral response in
ovine cells and evaluate the potential inhibitory eect upon small ruminant lentivirus (SRLV) infections.
Ovine alveolar macrophages (AMs), blood-derived macrophages (BDMs), and skin fibroblasts (OSFs)
were stimulated through infection with SeV encoding green fluorescent protein (GFP). SeV eciently
infected ovine cells, inducing an antiviral state in AM from SRLV naturally-infected animals,
as well as in in vitro SRLV-infected BDM and OSF from non-infected animals. Supernatants from
SeV-infected AM induced an antiviral state when transferred to fresh cells challenged with SRLV.
Similar to SRLV, infectivity of an HIV-1-GFP lentiviral vector was also restricted in ovine cells infected
with SeV. In myeloid cells, an M1-like proinflammatory polarization was observed together with
an APOBEC3Z1 induction, among other lentiviral restriction factors. Our observations may boost
new approximations in ameliorating the SRLV burden by stimulation of the innate immune response
using SeV-based vaccine vectors., This research was funded by Spanish Ministry of Science, Innovation, and Universities, grant number
RTI2018-096172-B-C31; Consejo Superior de Investigaciones Científicas, i-Coop and EMHE Program; and by
Government of Navarra (CONECTIM) and by Project NIETO-CM B2017/BMD-3731 to E.N.-V. The APC was
funded by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for
Research (URICI).

[EN] Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation, SI, This research was funded by Spanish Ministry of Science, Innovation, and Universities, grant number RTI2018-096172-B-C31. The APC was funded by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). I.E. was funded by Universidad Pública de Navarra. Hugo Ramírez was financially supported by the “Programa de apoyos para la superación del personal académico (PASPA)” scholarship from DGAPA-UNAM

The use of vaccines containing aluminum (Al) adjuvants is widespread in ovine production. Al adjuvants induce an effective immune-response but lead to the formation of post-vaccination granulomas from which Al can disseminate. This work aims to study the accumulation of Al in the central nervous system of sheep subcutaneously inoculated with Al-hydroxide containing products. Lumbar spinal cord and parietal lobe from 21 animals inoculated with 19 doses of Vaccine (n = 7), Adjuvant-only (n = 7) or phosphate-buffered saline as Control (n = 7) were analyzed with transversely heated graphite furnace atomic absorption spectroscopy and lumogallion staining for Al analytical measurements and Al tisular localization respectively. In the lumbar spinal cord, Al median content was higher in both the Adjuvant-only and Vaccine group (p = .001) compared with the Control group. Animals of the Adjuvant-only group showed the higher individual measurements in the lumbar spinal cord (14.36 μg/g and 7.83 μg/g). In the parietal lobe, Al median content tended to be higher in the Adjuvant-only group compared with Control group (p = .074). Except for three replicates of the Adjuvant-only group, Al content was always below 1 μg/g. In the lumbar spinal cord, lumogallion-reactive Al deposits were more abundant in the gray matter than in the white matter in both Vaccine (p = .034) and Adjuvant-only groups (p = .017) and Al deposits were mostly associated with glial-like cells (p = .042). In the parietal lobe, few Al deposits, which were sometimes related to blood vessels, were found. In sheep, Al-hydroxide adjuvants inoculated in the subcutaneous tissue selectively accumulate in the lumbar spinal cord., RM is a PhD student funded by the Department of Innovation, Research and University of Aragon, Spain. JA and ARL are PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education). This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and RTI2018-096172-B-C33), the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-B-C33) and the Government of Aragón (A17_17R, Animal Health and Reproduction).

Small ruminant lentiviruses (SRLVs) are widely spread in the ovine and caprine populations, causing an incurable disease affecting animal health and production. Vaccine development is hindered owing to the high genetic heterogeneity of lentiviruses and the selection of T-cell and antibody escape mutants, requiring antigen delivery optimization. Sendai virus (SeV) is a respiratory paramyxovirus in mice that has been recognized as a potent inducer of innate immune responses in several species, including mouse and human. The aim of this study was to stimulate an innate antiviral response in ovine cells and evaluate the potential inhibitory effect upon small ruminant lentivirus (SRLV) infections. Ovine alveolar macrophages (AMs), blood-derived macrophages (BDMs), and skin fibroblasts (OSFs) were stimulated through infection with SeV encoding green fluorescent protein (GFP). SeV efficiently infected ovine cells, inducing an antiviral state in AM from SRLV naturally-infected animals, as well as in in vitro SRLV-infected BDM and OSF from non-infected animals. Supernatants from SeV-infected AM induced an antiviral state when transferred to fresh cells challenged with SRLV. Similar to SRLV, infectivity of an HIV-1-GFP lentiviral vector was also restricted in ovine cells infected with SeV. In myeloid cells, an M1-like proinflammatory polarization was observed together with an APOBEC3Z1 induction, among other lentiviral restriction factors. Our observations may boost new approximations in ameliorating the SRLV burden by stimulation of the innate immune response using SeV-based vaccine vectors., This research was funded by Spanish Ministry of Science, Innovation, and Universities, grant number
RTI2018-096172-B-C31; Consejo Superior de Investigaciones Científicas, i-Coop and EMHE Program; and by Government of Navarra (CONECTIM) and by Project NIETO-CM B2017/BMD-3731 to E.N.-V. “The APC was funded by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).” L.d.P.-M. and I.E. were funded by Universidad Pública de Navarra. S.R.-R. was funded by an FPI fellowship granted by Universidad San Pablo CEU. R.R. was supported by the Spanish Ministry of Science and Innovation “Ramón y Cajal” contract., Peer reviewed

Sheep health management strategies often include the use of aluminum (Al)-containing vaccines. These products were associated with the appearance of the ovine autoimmune/inflammatory syndrome induced by adjuvants (ASIA syndrome), which included an array of ethological changes in the affected animals. The aim of this pilot study was to investigate cognitive and behavioral changes in sheep subjected to a protocol of repetitive inoculation with Al-containing products. Twenty-one lambs were assigned to three groups (n = 7 each): Control, Adjuvant-only, and Vaccine. Vaccine group was inoculated with commercial Al- hydroxide containing vaccines; Adjuvant-only group received the equivalent dose of Al only (Alhydrogel®), and Control group received Phosphate-buffered saline. Sixteen inoculations were administered within a 349-day period. Ethological changes were studied in late summer (7 inoculations) and mid-winter (16 inoculations). Animals in Vaccine and Adjuvant-only groups exhibited individual and social behavioral changes. Affiliative interactions were significantly reduced, and aggressive interactions and stereotypies increased significantly. They also exhibited a significant increase in excitatory behavior and compulsive eating. There were increased levels of stress biomarkers in these two groups. In general, changes were more pronounced in the Vaccine group than they were in the Adjuvant-only group. Some changes were already significant in summer, after seven inoculations only. This study is the first to describe behavioral changes in sheep after having received repetitive injections of Al-containing products, and may explain some of the clinical signs observed in ovine ASIA syndrome., J. Asín was a PhD student funded by the Ministry of Education, Culture and Sports (MECD). R. Reina was supported by a “Ramón y Cajal” contract from the Spanish Ministry of Economy, Industry and Competitiveness. This work was funded by several grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and AGL2013-49137-C3-2-R) and the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-B-C33).

© 2021 by the authors., Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation., This research was funded by Spanish Ministry of Science, Innovation, and Universities,
grant number RTI2018-096172-B-C31. The APC was funded by the CSIC Open Access Publication
Support Initiative through its Unit of Information Resources for Research (URICI). I.E. was funded
by Universidad Pública de Navarra. Hugo Ramírez was financially supported by the “Programa de
apoyos para la superación del personal académico (PASPA)” scholarship from DGAPA-UNAM., Peer reviewed

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue. IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection., This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (AGL2013-49137-C3-1-R and AGL2013-49137-C3-2-R), the Ministry of Science, Innovation and Universities (RTI2018-096172-B-C31 and RTI2018-096172-B-C33), and the Recognized Research Groups of Government of Aragón (A17_17R, Animal Health and Reproduction). I.E. was a PhD student funded by the Universidad Pública de Navarra. R.d.M. was a PhD student funded by the Department of Innovation, Research and University of Aragón. J.A. and A.R.-L. were PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education)., Peer reviewed

Small ruminant lentiviruses (SRLVs) are widely spread in the ovine and caprine populations, causing an incurable disease affecting animal health and production. Vaccine development is hindered owing to the high genetic heterogeneity of lentiviruses and the selection of T-cell and antibody escape mutants, requiring antigen delivery optimization. Sendai virus (SeV) is a respiratory paramyxovirus in mice that has been recognized as a potent inducer of innate immune responses in several species, including mouse and human. The aim of this study was to stimulate an innate antiviral response in ovine cells and evaluate the potential inhibitory effect upon small ruminant lentivirus (SRLV) infections. Ovine alveolar macrophages (AMs), blood-derived macrophages (BDMs), and skin fibroblasts (OSFs) were stimulated through infection with SeV encoding green fluorescent protein (GFP). SeV efficiently infected ovine cells, inducing an antiviral state in AM from SRLV naturally-infected animals, as well as in in vitro SRLV-infected BDM and OSF from non-infected animals. Supernatants from SeV-infected AM induced an antiviral state when transferred to fresh cells challenged with SRLV. Similar to SRLV, infectivity of an HIV-1-GFP lentiviral vector was also restricted in ovine cells infected with SeV. In myeloid cells, an M1-like proinflammatory polarization was observed together with an APOBEC3Z1 induction, among other lentiviral restriction factors. Our observations may boost new approximations in ameliorating the SRLV burden by stimulation of the innate immune response using SeV-based vaccine vectors.

Small ruminant lentiviruses (SRLVs) are endemic in most areas of Europe, causing a chronic infection and a multisystemic disease affecting the udder, carpal joints, lungs, and central nervous system. Due to the lack of treatments and protective vaccination strategies, infection control is focused on the identification of infected animals through serological or molecular techniques. However, antigenic and genetic heterogeneity of SRLVs represent a clear drawback for diagnosis. Infected animals may present lower animal production parameters such as birth weight or milk production and quality, depending on productive systems considered and, likely, to the diagnostic method applied. In this study, four sheep flocks dedicated to dairy or meat production were evaluated using three different ELISA and two PCR strategies to classify animal population according to SRLV infection status. Productive parameters were recorded along one whole lactation or reproductive period and compared between positive and negative animals. SRLV was present in 19% of the total population, being unequally distributed in the different flocks. Less than half of the infected animals were detected by a single diagnostic method, highlighting the importance of combining different diagnostic techniques. Statistical analysis employing animal classification using all the diagnostic methods associated lambing size, lamb weight at birth, and daily weight gain with SRLV infection status in meat flocks. Milk production, somatic cell count, fat, and protein content in the milk were associated with SRLV infection in dairy flocks, to a greater extent in the flock showing higher seroprevalence. A multi-platform SRLV diagnostic strategy was useful for ensuring correct animal classification, thus validating downstream studies investigating production traits.

Sheep health management strategies often include the use of aluminum (Al)-containing vaccines. These products were associated with the appearance of the ovine autoimmune/inflammatory syndrome induced by adjuvants (ASIA syndrome), which included an array of ethological changes in the affected animals. The aim of this pilot study was to investigate cognitive and behavioral changes in sheep subjected to a protocol of repetitive inoculation with Al-containing products. Twenty-one lambs were assigned to three groups (n = 7 each): Control, Adjuvant-only, and Vaccine. Vaccine group was inoculated with commercial Al- hydroxide containing vaccines; Adjuvant-only group received the equivalent dose of Al only (Alhydrogel®), and Control group received Phosphate-buffered saline. Sixteen inoculations were administered within a 349-day period. Ethological changes were studied in late summer (7 inoculations) and mid-winter (16 inoculations). Animals in Vaccine and Adjuvant-only groups exhibited individual and social behavioral changes. Affiliative interactions were significantly reduced, and aggressive interactions and stereotypies increased significantly. They also exhibited a significant increase in excitatory behavior and compulsive eating. There were increased levels of stress biomarkers in these two groups. In general, changes were more pronounced in the Vaccine group than they were in the Adjuvant-only group. Some changes were already significant in summer, after seven inoculations only. This study is the first to describe behavioral changes in sheep after having received repetitive injections of Al-containing products, and may explain some of the clinical signs observed in ovine ASIA syndrome.

Aluminum (Al) hydroxide is an effective adjuvant used in sheep vaccines. However, Al-adjuvants have been implicated as potential contributors to a severe wasting syndrome in sheep— the so-called ovine autoimmune-inflammatory syndrome induced by adjuvants (ASIA syndrome). This work aimed to characterize the effects of the repetitive injection of Al-hydroxide containing products in lambs. Four flocks (Flocks 1–4; n = 21 each) kept under different conditions were studied. Three groups of seven lambs (Vaccine, Adjuvant-only, and Control) were established in each flock. Mild differences in average daily gain and fattening index were observed, indicating a reduced growth performance in Vaccine groups, likely related to short-term episodes of pyrexia and decreased daily intake. Clinical and hematological parameters remained within normal limits. Histology showed no significant differences between groups, although there was a tendency to present a higher frequency of hyperchromatic, shrunken neurons in the lumbar spinal cord in the Adjuvant-only group. Although Al-hydroxide was linked to granulomas at the injection site and behavioral changes in sheep, the results of the present experimental work indicate that injected Al-hydroxide is not enough to fully reproduce the wasting presentation of the ASIA syndrome. Other factors such as sex, breed, age, production system, diet or climate conditions could play a role.

Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation.

Small Ruminant Lentiviruses (SRLV) are highly prevalent retroviruses with significant genetic diversity and antigenic heterogeneity that cause a progressive wasting disease of sheep called Maedi-visna. This work provides a systematic review and meta-analysis of the last 40 years (1981–2020) of scientific publications on SRLV individual and flock prevalence. Fifty-eight publications and 314 studies were included. Most articles used a single diagnostic test to estimate prevalence (77.6%), whereas articles using three or more tests were scarce (6.9%). Serological tests are more frequently used than direct methods and ELISA has progressively replaced AGID over the last decades. SRLV infection in sheep is widespread across the world, with Europe showing the highest individual prevalence (40.9%) and being the geographical area in which most studies have been performed. Africa, Asia, and North America show values between 16.7% to 21.8% at the individual level. South and Central America show the lowest individual SRLV prevalence (1.7%). There was a strong positive correlation between individual and flock prevalence (¿ = 0.728; p = 0.001). Despite the global importance of small ruminants, the coverage of knowledge on SRLV prevalence is patchy and inconsistent. There is a lack of a gold standard method and a defined sampling strategy among countries and continents.