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)

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-κB signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated., This work was supported by a MINECO project grant (AGL2013-49137-C3-3-R to BJ and AGL2013-49137-C3-2-R to LL and AGL2013-49137-C3-1 to DdA), a predoctoral fellowship from the UPV/EHU to EV-M (PIF15/361) and a postdoctoral
fellowship from the UPV/EHU to NA (ESPDOC16/43).

The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status. The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping., Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI-fellow of the Spanish MINECO and R. Reina had
a contract of the Public University of Navarra.

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production
and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals
has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between
wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through
modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV
infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells
from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus.
SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from
fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the
potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo
SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling
SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to
control SRLV spread in sheep and goats., Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI fellow of the Spanish MINECO and R. Reina had contracts from the Public University of Navarra and CSIC. The auhors acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis.
Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status., This work was co-funded by the Italian Ministry of Instruction, University and Research PRIN 2008 (no. 20084CSFLT), by Piedmont Region, 'Ricerca Sanitaria Finalizzata' 2008 and 2009, and by University of Turin, 'Fondi ricerca locale (ex-60%)' 2009. R. Reina was supported by Spanish Ministry of Science and Innovation 'Ramón y Cajal' contract (AGL2013-49137-C3-1R).

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).

En esta tesis se han llevado a cabo los siguientes estudios:
1. Identificación y caracterización de la restricción por APOBEC3 ovino frente a los SRLV. Se han estudiado dos tipos celulares que muestran restricción natural frente a los SRLV, monocitos y macrófagos-M1, identificando la isoforma Z1 como la más expresada, entre las proteínas APOBEC3 ovinas. En la caracterización genética de A3Z1 se ha identificado una isoforma adicional resultante de splicing alternativo, también presente en el transcriptoma humano y que carece del dominio citidín desaminasa (A3Z1Tr) característico de este tipo de proteínas. La expresión de estas proteínas se induce tanto tras la incubación con IFN-ɣ como con la infección viral. Ambas son resistentes a la degradación por Vif de SRLV y pueden encapsidarse en los viriones. Mediante ensayos de inmunoprecipitación se ha podido determinar que ambas proteínas pueden interactuar entre sí así como con Vif viral, a pesar de la resistencia que presentan a su degradación. La expresión endógena de A3Z1 en células en cultivo tipo fibroblasto (T-immortalized goat epitelial fibroblasts, TIGEF) tan solo ha sido posible empleando estrategias de expresión transitoria, ya que los intentos de expresión permanente resultaban en cultivos abortivos. Las células TIGEF que expresan A3Z1 muestran una menor producción de SRLV. Ensayos de infectividad de un solo ciclo (single cycle infection) con vectores virales basados en HIV-1, MLV y SIV indican que A3Z1 ovino es capaz de inhibir la infectividad de dichos vectores. El mecanismo de restricción parece implicar la actividad citidín desaminasa en el contexto GA/AA. En ambos casos, la co-expresión de la isoforma truncada mitiga la restricción, probablemente por competición estérica con la proteína nativa en el interior de los viriones.
2. Caracterización de las vías de degradación de APOBEC3. Se ha investigado la capacidad de A3Z1 de restringir la infección con estirpes de SRLV que codifican la proteína Vif. Mediante ensayos de inmunoprecipitación se ha podido comprobar que CYPA y no CBF-β es el cofactor necesario para la degradación de las proteínas APOBEC3 ovinas, requisito esencial en el caso de A3Z1 y A3Z1Tr. Concentraciones altas de CYPA permiten la degradación de A3Z1 por parte de Vif en diferentes tipos celulares, promoviendo así la infección por lentivirus. Sin embargo, no se alcanzan los niveles basales de A3Z1 empleando inhibidores del proteasoma y la degradación se produce también en presencia de Vif mutado, incapaz de reclutar el complejo ubiquitín ligasa y degradar así proteínas por la vía proteasomal, abriendo la posibilidad a la existencia de vías alternativas que permitan degradar A3Z1. Tan solo la combinación de inhibidores del proteasoma junto con inhibidores de la autofagia conseguían restablecer los niveles basales de A3Z1 sugiriendo la implicación de ambas vías. Además, la inhibición de la autofagia parece perjudicar la infección por SRLV, sugiriendo que la inducción podría favorecerla.
3. Estimulación de la respuesta innata ovina a través de la infección con vectores virales basados en el virus murino Sendai. La infección de células ovinas con un vector viral basado en el virus Sendai que codifica la proteína verde fluorescente (SeV-GFP) ha resultado ser del 100%, incluso en macrófagos de distintos orígenes. Sin embargo, la infección con SeV-GFP no se transmite en células ovinas y el vector es no replicativo. La infección con SeV-GFP ha activado una programación diferente dependiendo del tipo celular ovino, siendo la inducción de A3Z1 en células mieloides de especial importancia. La infección con SRLV y la producción de virus se encontraba disminuida en estas células tras el tratamiento con el vector viral. Los sobrenadantes procedentes de estos cultivos eran capaces de transmitir la restricción a células frescas, probablemente debido a la producción de interferón de tipo 1, sugiriendo que podría ser de utilidad en futuras estrategias de inmunización. La restricción también se ha observado frente a vectores basados en lentivirus, como HIV o SIV., Different studies have been carried out in this thesis:
1. Identification and characterization of the restriction exerted by ovine APOBEC3 against SRLV. Two cellular types that show natural restriction against SRLV have been studied, monocytes and M1-macrophages, identifying the Z1 isoform as the most expressed, among the ovine APOBEC3 proteins. In the genetic characterization of A3Z1, an additional isoform resulting from alternative splicing, also present in the human transcriptome, was described. This new isoform lacks the citidin deaminase domain (A3Z1Tr), characteristic of this type of proteins. Both A3Z1 proteins were induced after incubation with IFN-γ as well as after viral infection, are resistant to Vif SRLV degradation and can be incorporated into virions. Through immunoprecipitation assays we have been able to determine that both A3Z1 proteins can interact, as well as with viral Vif, in spite of their resistance to degradation. A3Z1 endogenous expression in fibroblast type cells (T-immortalized goat epitelial fibroblasts, TIGEF) has only been possible using transient expression strategies, because permanent expression experiments resulted in aborted cultures. TIGEF cells that transiently express A3Z1 show a lower SRLV production. Single cycle infectivity trials with viral vectors based on HIV-1, MLV and SIV showed that ovine A3Z1 is able to inhibit the aforementioned vectors. The restriction mechanism seem to be citidin deaminase dependent in the GA/AA context. In both cases, the co-expression of the truncated isoform mitigates the restriction, probably by steric competition with the native protein inside the virions.
2. Characterization of APOBEC3 degradation pathways. The ability of A3Z1 to restrict SRLVs encoding the Vif protein has been investigated. Through immunoprecipitation assays it has been possible to confirm that CYPA, and not CBF-β is the cofactor for the optimal degradation of ovine APOBEC3, an essential requirement in the case of A3Z1 and A3Z1Tr. High concentrations of CYPA allowed the degradation of A3Z1 by Vif in different cell types, thus promoting lentivirus infection. However, A3Z1 basal levels were not restored when proteasome inhibitors were used. Furthermore, degradation also happened in the presence of a mutated Vif, unable to recruit the ubiquitin ligase complex and degrade proteins by the proteasome pathway, opening alternative pathways to degrade A3Z1. The combination of proteasome and autophagy inhibitors was able to restore A3Z1 basal levels, suggesting the involvement of both pathways. In addition, autophagy inhibition seems to impair SRLV infection, suggesting that induction could be a virus promoting mechanism. 3. Stimulation of innate immune response through murine Sendai virus vectors. The infection of ovine cells with a Sendai virus vector that encodes the green fluorescent protein (SeV-GFP) has been obtained at the 100% level, even in macrophages from different origins. However, SeV-GFP was a non-replicative vector and infection was not transmitted among ovine cells. Infection with SeV-GFP induced a different activation depending on the cell type, being the induction of A3Z1 in myeloid cells of special importance. Infection with SRLV as well as virus production were reduced in these cells after treatment with the viral vector. Supernatants from SeV infected cell cultures were able to transmit the restriction to fresh cells, likely due to type I IFN production, suggesting the potential application of this vector in future immunization strategies. The restriction has also been observed against lentivirus-based vectors from HIV or SIV., Este trabajo ha sido financiado por los siguientes proyectos de investigación: Título del proyecto: 'Inmunidad innata y adquirida frente a lentivirus de pequeños rumiantes: elementos responsables del tropismo viral y de la presentación clínica de la enfermedad'. Entidad financiadora: CICYT (AGL2010-22341-C04-01); Título del proyecto: 'Patogenómica traslacional en el estudio de enfermedades infecciosas con implicaciones económico-sanitarias en especies productivas'. Entidad financiadora: Gobierno de Navarra (referencia IIQ14064.RI1); Título del proyecto: 'Modelos in vitro para evaluación de eficacia inmunológica de β-glucanos de Shiitake'. Entidad financiadora: Agencia de Desarrollo Económico de La Rioja, ADER (en colaboración con UE: FEDER, FEADER) (Ref.: 2013-l-lDD-00074); Título del proyecto: 'Inmunidad implicada en los procesos inmunomediados: síndrome asia ovino y lentivirosis de especies productivas'. Entidad financiadora: MINECO_DGICYT (AGL2013-49137-C3-1-R); Título del proyecto: 'Potenciación del estado inmunológico del ganado cunícula a través de betaglucanos de setas (RABBITGLUCAN)'. Entidad financiadora: CENTRO PARA EL DESARROLLO TECNOLÓGICO INDUSTRIAL (CDTI) (cofinanciable con FEDER) (Ref.: IDI 20140852); Título del proyecto: 'Inmunopotenciación del ganado cunícula mediante suplementos a base de beta glucanos de setas (CUNIGLUCAN)'. Entidad financiadora: MAGRAMA Ayudas IDi (REF.: 20140020001815); Título del proyecto: 'Nuevas estrategias de control frente a lentivirus basadas en terapia génica'. Entidad financiadora: Fundación Caja Navarra Id70558; Título del proyecto: 'Contribución a la epidemiologia molecular y al control innato de las infecciones lentivirales ovi-caprinas'. Entidad financiadora: Gobierno de Navarra. Departamento de Desarrollo Económico PI042 LENTIMOL; Título del proyecto: 'Epidemiología molecular y control innato de las infecciones lentivirales en el ganado Navarro'. Entidad financiadora: Gobierno de Navarra. Plan de Desarrollo Rural; Título del proyecto: 'Herramientas diagnósticas y adyuvantes inmunológicos para el control del Ectima contagioso en Navarra. (CONECTIM)'. Entidad financiadora: Gobierno de Navarra. Centros Tecnológicos., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-κB signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated., This work was supported by a MINECO project grant (AGL2013-49137-C3-3-R to BJ and AGL2013-49137-C3-2-R to LL and AGL2013-49137-C3-1 to DdA), a predoctoral fellowship from the UPV/EHU to EV-M (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to NA (ESPDOC16/43)., Peer reviewed

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis.

Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status., This work was co-funded by the Italian Ministry of Instruction, University and Research PRIN 2008 (no. 20084CSFLT), by Piedmont Region, “Ricerca Sanitaria Finalizzata” 2008 and 2009, and by University of Turin, “Fondi ricerca locale (ex-60%)” 2009. The Authors acknowledge Mr. R. Maritano, CISRA for his valuable contribution in animal management, and Mr. D. Arnulfo and R. Vanni for their competent work and assistance during animal autopsies.
R. Reina was supported by Spanish Ministry of Science and Innovation ‘Ramón y Cajal’ contract (AGL2013-49137-C3-1R)., Peer reviewed

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).

Resumen del trabajo presentado en el XXX Keele Meeting on Aluminium, celebrado en Yucatán (México), del 23 al 27 de marzo de 2019, Aluminium (Al)-containing adjuvants promote an effective immune response but in sheep, they also induce local granulomatous reactions and accumulate in regional lymph nodes1. 21 lambs, divided into 3 groups (n=7 each) were inoculated during 15 months with 19 doses of different substances: A) Vaccines containing Al hydroxide; B) Al hydroxide only; C) PBS. Lumbar spinal cord and frontal lobe of the brain were studied by fluorescence microscopy with lumogallion, Graphite Furnace Atomic Absorption Spectroscopy and immunohistochemistry against glial fibrillary acidic protein (GFAP). Groups A and B showed higher Al concentration in spinal cord than group C (p=0.001). Yellowish fluorescence emission (590 nm), typical of Al, was detected as small aggregates associated to myelin sheets. The highest individual measurements were obtained in group B. GFAP was overexpressed in group B compared to group C (p=0.047). In sheep, Al from adjuvants may translocate from subcutaneous tissue to the CNS, inducing astrocyte activation., This work was funded by grants from the Spanish Ministry of Economy and Industry
(AGL2013-49137-C3-1-R and AGL2013-49137-C3-2-R). Asín J is a PhD student
funded by the Spanish Ministry of Education, Culture and Sports.

Resumen del trabajo presentado en el XXX Keele Meeting on Aluminium, celebrado en Yucatán (México), del 23 al 27 de marzo de 2019, In sheep, aluminium-containing vaccines induce the recruitment of macrophages and
formation of granulomas1. Small ruminant lentiviruses (SRLV) are retroviruses that
replicate in activated macrophages2. Eleven adult, naturally SRLV-infected sheep were
inoculated with eight doses of aluminium-containing vaccines. In vitro cultures of
granuloma, spleen, peripheral blood and bronchoalveolar lavage macrophages were
established and monitored for retrotranscriptase activity, PCR and virus identification
by gag gene sequencing. Immunohistochemistry was also performed with CAEP5A1
monoclonal antibody. SRLV could be detected in post-vaccination granulomas in 5
sheep (45.5%). In three animals, viral sequences in the granuloma were highly
divergent compared to sequences isolated elsewhere in the same sheep
(compartmentalization). Location of SRLV was mostly associated to Al-containing
phagolysosomes. Persistent granulomatous inflammatory reactions induced by
vaccines may allow in situ SRLV replication, thus favouring mutation and
recombination. The role of Al-induced, post-vaccination granulomas in the spread and
pathogenesis of SRLV need to be addressed and considered., This work was funded by grants from the Spanish Ministry of Economy and Industry
(AGL2013-49137-C3-1-R and AGL2013-49137-C3-2-R). De Miguel R is a PhD
student funded by the Department of Innovation, Research and University of Aragon.

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).

[ES] En esta tesis se han llevado a cabo los siguientes estudios: 1. Identificación y caracterización de la restricción por APOBEC3 ovino frente a los SRLV. Se han estudiado dos tipos celulares que muestran restricción natural frente a los SRLV, monocitos y macrófagos-M1, identificando la isoforma Z1 como la más expresada, entre las proteínas APOBEC3 ovinas. En la caracterización genética de A3Z1 se ha identificado una isoforma adicional resultante de splicing alternativo, también presente en el transcriptoma humano y que carece del dominio citidín desaminasa (A3Z1Tr) característico de este tipo de proteínas. La expresión de estas proteínas se induce tanto tras la incubación con IFN-ɣ como con la infección viral. Ambas son resistentes a la degradación por Vif de SRLV y pueden encapsidarse en los viriones. Mediante ensayos de inmunoprecipitación se ha podido determinar que ambas proteínas pueden interactuar entre sí así como con Vif viral, a pesar de la resistencia que presentan a su degradación. La expresión endógena de A3Z1 en células en cultivo tipo fibroblasto (T-immortalized goat epitelial fibroblasts, TIGEF) tan solo ha sido posible empleando estrategias de expresión transitoria, ya que los intentos de expresión permanente resultaban en cultivos abortivos. Las células TIGEF que expresan A3Z1 muestran una menor producción de SRLV. Ensayos de infectividad de un solo ciclo (single cycle infection) con vectores virales basados en HIV-1, MLV y SIV indican que A3Z1 ovino es capaz de inhibir la infectividad de dichos vectores. El mecanismo de restricción parece implicar la actividad citidín desaminasa en el contexto GA/AA. En ambos casos, la co-expresión de la isoforma truncada mitiga la restricción, probablemente por competición estérica con la proteína nativa en el interior de los viriones. 2. Caracterización de las vías de degradación de APOBEC3. Se ha investigado la capacidad de A3Z1 de restringir la infección con estirpes de SRLV que codifican la proteína Vif. Mediante ensayos de inmunoprecipitación se ha podido comprobar que CYPA y no CBF-β es el cofactor necesario para la degradación de las proteínas APOBEC3 ovinas, requisito esencial en el caso de A3Z1 y A3Z1Tr. Concentraciones altas de CYPA permiten la degradación de A3Z1 por parte de Vif en diferentes tipos celulares, promoviendo así la infección por lentivirus. Sin embargo, no se alcanzan los niveles basales de A3Z1 empleando inhibidores del proteasoma y la degradación se produce también en presencia de Vif mutado, incapaz de reclutar el complejo ubiquitín ligasa y degradar así proteínas por la vía proteasomal, abriendo la posibilidad a la existencia de vías alternativas que permitan degradar A3Z1. Tan solo la combinación de inhibidores del proteasoma junto con inhibidores de la autofagia conseguían restablecer los niveles basales de A3Z1 sugiriendo la implicación de ambas vías. Además, la inhibición de la autofagia parece perjudicar la infección por SRLV, sugiriendo que la inducción podría favorecerla., [EN] Different studies have been carried out in this thesis: 1. Identification and characterization of the restriction exerted by ovine APOBEC3 against SRLV. Two cellular types that show natural restriction against SRLV have been studied, monocytes and M1-macrophages, identifying the Z1 isoform as the most expressed, among the ovine APOBEC3 proteins. In the genetic characterization of A3Z1, an additional isoform resulting from alternative splicing, also present in the human transcriptome, was described. This new isoform lacks the citidin deaminase domain (A3Z1Tr), characteristic of this type of proteins. Both A3Z1 proteins were induced after incubation with IFN-γ as well as after viral infection, are resistant to Vif SRLV degradation and can be incorporated into virions. Through immunoprecipitation assays we have been able to determine that both A3Z1 proteins can interact, as well as with viral Vif, in spite of their resistance to degradation. A3Z1 endogenous expression in fibroblast type cells (T-immortalized goat epitelial fibroblasts, TIGEF) has only been possible using transient expression strategies, because permanent expression experiments resulted in aborted cultures. TIGEF cells that transiently express A3Z1 show a lower SRLV production. Single cycle infectivity trials with viral vectors based on HIV-1, MLV and SIV showed that ovine A3Z1 is able to inhibit the aforementioned vectors. The restriction mechanism seem to be citidin deaminase dependent in the GA/AA context. In both cases, the co-expression of the truncated isoform mitigates the restriction, probably by steric competition with the native protein inside the virions. 2. Characterization of APOBEC3 degradation pathways. The ability of A3Z1 to restrict SRLVs encoding the Vif protein has been investigated. Through immunoprecipitation assays it has been possible to confirm that CYPA, and not CBF-β is the cofactor for the optimal degradation of ovine APOBEC3, an essential requirement in the case of A3Z1 and A3Z1Tr. High concentrations of CYPA allowed the degradation of A3Z1 by Vif in different cell types, thus promoting lentivirus infection. However, A3Z1 basal levels were not restored when proteasome inhibitors were used. Furthermore, degradation also happened in the presence of a mutated Vif, unable to recruit the ubiquitin ligase complex and degrade proteins by the proteasome pathway, opening alternative pathways to degrade A3Z1. The combination of proteasome and autophagy inhibitors was able to restore A3Z1 basal levels, suggesting the involvement of both pathways. In addition, autophagy inhibition seems to impair SRLV infection, suggesting that induction could be a virus promoting mechanism., Este trabajo ha sido financiado por los siguientes proyectos de investigación: Título del proyecto: 'Inmunidad innata y adquirida frente a lentivirus de pequeños rumiantes: elementos responsables del tropismo viral y de la presentación clínica de la enfermedad'. Entidad financiadora: CICYT (AGL2010-22341-C04-01); Título del proyecto: 'Patogenómica traslacional en el estudio de enfermedades infecciosas con implicaciones económico-sanitarias en especies productivas'. Entidad financiadora: Gobierno de Navarra (referencia IIQ14064.RI1); Título del proyecto: 'Modelos in vitro para evaluación de eficacia inmunológica de β-glucanos de Shiitake'. Entidad financiadora: Agencia de Desarrollo Económico de La Rioja, ADER (en colaboración con UE: FEDER, FEADER) (Ref.: 2013-l-lDD-00074); Título del proyecto: 'Inmunidad implicada en los procesos inmunomediados: síndrome asia ovino y lentivirosis de especies productivas'. Entidad financiadora: MINECO_DGICYT (AGL2013-49137-C3-1-R); Título del proyecto: 'Potenciación del estado inmunológico del ganado cunícula a través de betaglucanos de setas (RABBITGLUCAN)'. Entidad financiadora: CENTRO PARA EL DESARROLLO TECNOLÓGICO INDUSTRIAL (CDTI) (cofinanciable con FEDER) (Ref.: IDI 20140852); Título del proyecto: 'Inmunopotenciación del ganado cunícula mediante suplementos a base de beta glucanos de setas (CUNIGLUCAN)'. Entidad financiadora: MAGRAMA Ayudas IDi (REF.: 20140020001815); Título del proyecto: 'Nuevas estrategias de control frente a lentivirus basadas en terapia génica'. Entidad financiadora: Fundación Caja Navarra Id70558; Título del proyecto: 'Contribución a la epidemiologia molecular y al control innato de las infecciones lentivirales ovi-caprinas'. Entidad financiadora: Gobierno de Navarra. Departamento de Desarrollo Económico PI042 LENTIMOL; Título del proyecto: 'Epidemiología molecular y control innato de las infecciones lentivirales en el ganado Navarro'. Entidad financiadora: Gobierno de Navarra. Plan de Desarrollo Rural; Título del proyecto: 'Herramientas diagnósticas y adyuvantes inmunológicos para el control del Ectima contagioso en Navarra. (CONECTIM)'. Entidad financiadora: Gobierno de Navarra. Centros Tecnológicos.

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

Background: Long non-coding RNAs (lncRNAs) are involved in several immune processes, including the immune response to vaccination, but most of them remain uncharacterised in livestock species. The mechanism of action of aluminium adjuvants as vaccine components is neither not fully understood. Results: We built a transcriptome from sheep PBMCs RNA-seq data in order to identify unannotated lncRNAs and analysed their expression patterns along protein coding genes. We found 2284 novel lncRNAs and assessed their conservation in terms of sequence and synteny. Differential expression analysis performed between animals inoculated with commercial vaccines or aluminium adjuvant alone and the co-expression analysis revealed lncRNAs related to the immune response to vaccines and adjuvants. A group of co-expressed genes enriched in cytokine signalling and production highlighted the differences between different treatments. A number of differentially expressed lncRNAs were correlated with a divergently located protein-coding gene, such as the OSM cytokine. Other lncRNAs were predicted to act as sponges of miRNAs involved in immune response regulation. Conclusions: This work enlarges the lncRNA catalogue in sheep and puts an accent on their involvement in the immune response to repetitive vaccination, providing a basis for further characterisation of the non-coding sheep transcriptome within different immune cells., This research was supported by the Spanish Ministry of Economy and Industry project number AGL2013-49137-C3 to BMJ and University of the Basque Country (UPV/EHU) predoctoral fellowships to MB-A (PIF17/ 306) and EV-M ( PIF15/361) and a postdoctoral fellowship to NA (ESP-COC16/43).

Small ruminant lentiviruses (SRLV) globally affect welfare and production of sheep and goats and are mainly controlled through elimination of infected animals, independently of the viral kinetics within the single animal. Control programs are based on highly sensitive serological tests, however the existence of low antibody responders leads to the permanent presence of seronegative infected animals in the flock, thus perpetuating the infection. On the other hand, long-term non-progressors show a detectable antibody response not indicative of a shedding animal, suggesting immune contention of infection. In this study, we analyse two goat populations within the same herd, harbouring low or high proviral SRLV loads respectively, both showing a robust antibody response. In vivo findings were confirmed in vitro since fibroblastic cell lines obtained from one high and one low proviral load representative goats, showed respectively a high and a faint production of virus upon infection with reference and field circulating SRLV strains. Differences in virus production were relieved when strain CAEV-Co was used for experimental infection. We analysed LTR promoter activity, proviral load, entry step and production of virus and viral proteins. Intriguingly, proteasomal activity was higher in fibroblasts from low proviral load animals and proteasome inhibition increased viral production in both cell lines, suggesting the implication of active proteasome-dependent restriction factors. Among them, we analysed relative expression and sequences of TRIM5α, APOBEC3 (Z1, Z2, Z3 and Z2-Z3) and BST-2 (Tetherin) and found a global antiviral status in low proviral carriers that may confer protection against viral shedding and disease onset., Funded by Spanish CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1) and Italian “Fondo di Ricerca Locale 2014, Università degli Studi di Torino - Indagini virologiche, biochimiche e molecolari sui meccanismi di immunità innata nella capra durante l’infezione da lentivirus” and partly funded by: RC IZSPLV06/09 “Studio di geni candidati per ‘selezione assistita da marcatori’, ai fini dell’incremento della resistenza a malattie in specie animali di interesse zootecnico”, finanziata dal Ministero della Salute. R. Reina was supported by Spanish Ministry of Economy and Competitiveness “Ramón y Cajal” contract., We acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Peer Reviewed

Lentiviruses are infectious agents of a number of animal species, including sheep, goats, horses, monkeys, cows, and cats, in addition to humans. As in the human case, the host immune response fails to control the establishment of chronic persistent infection that finally leads to a specific disease development. Despite intensive research on the development of lentivirus vaccines, it is still not clear which immune responses can protect against infection. Viral mutations resulting in escape from T-cell or antibody-mediated responses are the basis of the immune failure to control the infection. The innate immune response provides the first line of defense against viral infections in an antigen-independent manner. Antiviral innate responses are conducted by dendritic cells, macrophages, and natural killer cells, often targeted by lentiviruses, and intrinsic antiviral mechanisms exerted by all cells. Intrinsic responses depend on the recognition of the viral pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), and the signaling cascades leading to an antiviral state by inducing the expression of antiviral proteins, including restriction factors. This review describes the latest advances on innate immunity related to the infection by animal lentiviruses, centered on small ruminant lentiviruses (SRLV), equine infectious anemia virus (EIAV), and feline (FIV) and bovine immunodeficiency viruses (BIV), specifically focusing on the antiviral role of the major restriction factors described thus far., This research was funded by MINECO grant number [AGL2013-49137-C3-1-R], Peer reviewed

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats., Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI fellow of the Spanish MINECO and R. Reina had contracts from the Public University of Navarra and CSIC. We acknowledge Marta Gil Antona for sampling in hunting expeditions and Hunting Associations (FEDEMCA, Tragsa, INTIA, etc.) for their collaboration in the obtention of samples. We acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Peer reviewed

The use of vaccines including aluminum (Al)–based adjuvants is widespread among small ruminants and other animals. They are associated with the appearance of transient injection site nodules corresponding to granulomas. This study aims to characterize the morphology of these granulomas, to understand the role of the Al adjuvant in their genesis, and to establish the presence of the metal in regional lymph nodes. A total of 84 male neutered lambs were selected and divided into 3 treatment groups of 28 animals each: (1) vaccine (containing Al-based adjuvant), (2) adjuvant-only, and (3) control. A total of 19 subcutaneous injections were performed in a time frame of 15 months. Granulomas and regional lymph nodes were evaluated by clinicopathological means. All of the vaccine and 92.3% of the adjuvant-only lambs presented injection-site granulomas; the granulomas were more numerous in the group administered the vaccine. Bacterial culture in granulomas was always negative. Histologically, granulomas in the vaccine group presented a higher degree of severity. Al was specifically identified by lumogallion staining in granulomas and lymph nodes. Al median content was significantly higher (P <.001) in the lymph nodes of the vaccine group (82.65 µg/g) compared with both adjuvant-only (2.53 µg/g) and control groups (0.96 µg/g). Scanning transmission electron microscopy demonstrated aggregates of Al within macrophages in vaccine and adjuvant-only groups. In these two groups, Al-based adjuvants induce persistent, sterile, subcutaneous granulomas with macrophage-driven translocation of Al to regional lymph nodes. Local translocation of Al may induce further accumulation in distant tissues and be related to the appearance of systemic signs.

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.