BUSCADOR RECOLECTA

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).

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.

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.

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., 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). RdM was a PhD student funded by the Department of Innovation, Research and University of Aragón. JA and ARL were PhD students funded by the Spanish Ministry of Science, Innovation and Universities (formerly Spanish Ministry of Education). IE was a PhD student funded by the Universidad Pública de Navarra.