Use of a local anaesthetic and antiseptic wound formulation for the treatment of lambs naturally infected with Orf virus.

Contagious ecthyma (CE) is a worldwide highly contagious zoonotic viral skin disease of sheep and goats. Treatment for Orf virus (ORFV) infection usually involves topical and oral antibiotics. An anaesthetic and antiseptic topical gel (Multisolfen® or Tri-Solfen®; MS®, Medical Ethics, Australia) has been documented as an efficacious therapy for lesions from mucosal and epithelial viral infections in ruminants. The present study tested a new treatment protocol of MS® for CE therapy on-farm in 150 lambs naturally infected with ORFV. Lambs were divided into three cohorts of 50 lambs each (C, D and E). Cohort C was treated with MS® 3 times with an interval of 3 days between treatments, cohort D was treated daily with hypochlorous acid, whilst cohort E served as untreated controls. The lambs were examined clinically every two days, weight measured weekly, with whole blood and sterile swabs from ORFV lesions collected for haematological analysis and specific ORFV PCR. Cohort C presented fewer lambs displaying ORFV-associated lesions than other cohorts at different times of the experiment. Further, lesions treated with MS® were milder compared with other cohorts. However, following cessation of therapy, most of the lambs again developed ORFV-associated lesions. No differences between cohorts were observed in weight, haematological and PCR results. These findings suggest that topical treatment with MS® is effective for CE in field conditions, especially in the first stages of the clinical course, although treatment with MS® may need to be extended a minimum of 4 weeks.

Use of a Local Anaesthetic/Antiseptic Formulation for the Treatment of Lambs Experimentally Infected with Orf Virus.

Contagious ecthyma is a highly transmissible eruptive viral disease of the skin and mucosa of sheep and goats distributed worldwide. The treatment of orf lesions is usually based on the use of antiseptics and antibiotics for the management of presumptive secondary infections, increasing risks of antimicrobial resistance. The wound dressing formulation Tri-Solfen® (TS) containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation has been demonstrated to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease virus infection and reduced the orf viral load in lambs. In the present study, experimental infection with the orf virus was conducted in 50 newborn lambs and 25 animals were treated after the presence of the first lesions with TS and repeated three days later. Daily clinical examination, haematological, serological, biomolecular and post-mortem analyses were conducted during 34 days after treatment. Results indicated that treatment had no effect on weight gain and clinical progression of the lesions. It was determined that seroconversion after experimental infection occurs 34 days after infection and suggested that the deep basal epithelial location of the orf lesions may have prevented the therapy from having altered the clinical course.

Gene Expression Profiling Reveals New Pathways and Genes Associated with Visna/Maedi Viral Disease.

Visna/Maedi virus (VMV) is a lentivirus that infects the cells of the monocyte/macrophage lineage in sheep, goats and wild ruminants. Infection with VMV causes a multisystemic inflammatory disorder, which includes pneumonia, encephalitis, mastitis or arthritis. The immune response to VMV infection is complex, and the infection and pathogenesis of this virus are not totally characterized yet. In this work, a gene expression microarray was used to identify the differentially expressed genes in VMV infection and disease development by comparing sheep with different serologic status and with presence of VM-characteristic clinical lesions. The expression profile analysis has revealed many interesting genes that may be associated with the viral infection process. Among them, the OXT gene appeared significantly up-regulated, so the oxytocin-secreting system could play an essential role in VM disease. Moreover, some of the most significantly enriched functions in up-regulated genes appeared the complement pathway, which (in combination with the Toll-like receptor signaling network) could compose a mechanism in the VMV pathogenesis. Identifying the host genetic factors associated with VMV infection can be applied to develop strategies for preventing infection and develop effective vaccines that lead to therapeutic treatments.

Detection of aluminium hydroxide-induced granulomas in sheep by computed tomography: A feasible approach for small ruminant lentiviruses diagnosis and research.

Aluminium (Al) hydroxide use as adjuvant induces local formation of long-lasting subcutaneous granulomas in sheep. Macrophages within these granulomas have been identified as a new small ruminant lentivirus (SRLV) replication site in naturally infected animals. Diagnosis of Al hydroxide-induced granulomas in sheep is mostly based on postmortem observations but little information is available on in vivo detection. Computed tomography (CT) is used for studying these reactions in other animal species. To determine if CT could be a tool for in vivo diagnosis and research of subcutaneous Al hydroxide-induced granulomas in sheep. A retrospective survey on thoracic CT scans was performed on 46 adult sheep. Analysis included absence or presence, number and location of subcutaneous nodules. Thoracic CT scans and pathological studies were prescribed to two further sheep. Single or multiple subcutaneous nodules were detected in 26 (56.52%) sheep. One or two nodules per animal were most often observed (36.95%). Size ranged between 1.5 and 4.5 cm. Pre-contrast two-dimensional (2D) CT images showed focal or multifocal increases in subcutaneous tissue thickness. Post-contrast 2D CT images revealed hypointense areas in the centre. Histopathology indicated the presence of granulomas composed by a large number of activated macrophages, surrounding a central core of necrosis. Large intracytoplasmic Al-positive aggregates were demonstrated by lumogallion staining. CT is a useful tool to detect subcutaneous Al hydroxide-induced granulomas in vivo in sheep. CT provides a diagnostic and research tool that can be very useful in future works in Al hydroxide-induced pathology, SRLV infection, or both.

Effect of a Topical Formulation on Infective Viral Load in Lambs Naturally Infected with Orf Virus.

INTRODUCTION: Orf is a highly contagious eruptive viral disease of the skin and mucosa of sheep and goats. Although vaccination with live or attenuated orf virus is the preferred option for disease control, the vaccine is unavailable in many countries. Treatment of orf lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was developed for pain relief in sheep undergoing surgical husbandry procedures in Australia. Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control. METHODS: One-month-old lambs (n=14), naturally infected with orf, were recruited from a farm during a natural outbreak of the disease. The animals were selected at the early stages of the infection and randomly divided into two cohorts: Group A (n=11) treated with the topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat embryonic fibroblasts (TIGEF). RESULTS: Although no significant differences were found in the clinical progression of the lesions and PCR quantification (p=0.722) between these small cohorts, there was a significant difference (p<0.05) in reduction in infective viral load between the groups when assessed in OSF cell cultures between T0 and T3. CONCLUSION: These preliminary findings suggest that treatment of early stage lesions with this TS may reduce the infective viral load present in orf lesions.

Worldwide Prevalence of Small Ruminant Lentiviruses in Sheep: A Systematic Review and Meta-Analysis.

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 (ρ = 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.

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping.

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.

Growth Performance and Clinicopathological Analyses in Lambs Repetitively Inoculated with Aluminum-Hydroxide Containing Vaccines or Aluminum-Hydroxide Only.

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.

Replication of Small Ruminant Lentiviruses in Aluminum Hydroxide-Induced Granulomas in Sheep: a Potential New Factor for Viral Dissemination.

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.

Multi-Platform Detection of Small Ruminant Lentivirus Antibodies and Provirus as Biomarkers of Production Losses.

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.

Sendai Virus, a Strong Inducer of Anti-Lentiviral State in Ovine Cells.

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.

Detection of aluminum in lumbar spinal cord of sheep subcutaneously inoculated with aluminum-hydroxide containing products.

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.

Cognition and behavior in sheep repetitively inoculated with aluminum adjuvant-containing vaccines or aluminum adjuvant only.

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.

A new approach for Small Ruminant Lentivirus full genome characterization revealed the circulation of divergent strains.

Small Ruminant Lentiviruses (SRLV) include at least 4 viral highly divergent genotypes. Genotypes A and B are widely distributed and genotypes C and E have been recognized in restricted geographic areas. New phylogroups have been identified targeting conserved regions. However, this approach suffers from the potential risk to misamplify highly divergent strains. Pathogenic strains are easily adapted to fibroblastic cells, but non-pathogenic strains isolation may require a different approach. We developed a fast and effective method for SRLV full genome characterization after cell culture isolation. Spleen samples were collected during regular slaughter from sheep and goats in northwestern Italy. Spleen-derived macrophage cultures were monitored for reverse transcriptase activity and RNA was extracted from the supernatant of positive cultures. Using Illumina MiSeq platform 22 new full genome sequences were obtained. The success of this approach is based on the following features: spleen is one of the main target for SRLV persistence; red pulp is a reserve of resident macrophages, the main target for SRLV replication in vivo; RTA is a sensitive assay for any replicating retrovirus; de novo sequencing do not require genetic knowledge in advance.

Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq.

BACKGROUND: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. RESULTS: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. CONCLUSIONS: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.

Granulomas Following Subcutaneous Injection With Aluminum Adjuvant-Containing Products in Sheep.

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.

Molecular Signature of Aluminum Hydroxide Adjuvant in Ovine PBMCs by Integrated mRNA and microRNA Transcriptome Sequencing.

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.

Prospects in Innate Immune Responses as Potential Control Strategies against Non-Primate Lentiviruses.

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.

Lentinula edodes ß-glucan enriched diet induces pro- and anti-inflammatory macrophages in rabbit.

ß-glucans exhibited in cell walls of several pathogens as bacteria or fungi are sensed by pathogen recognition receptors such as scavenger receptors present in antigen presenting cells, i.e., macrophages. ß-glucans obtained from Shiitake mushrooms were chemically characterized. A ß-glucan supplemented diet was assayed for 30 days in rabbits aiming to characterize the immune response elicited in blood-derived macrophages. M1 and M2 profiles of macrophage differentiation were confirmed in rabbits by in vitro stimulation with IFN-γ and IL-4 and marker quantification of each differentiation pathway. Blood derived macrophages from rabbits administered in vivo with the ß-glucan supplemented diet showed higher IL-4, IFN-γ and RAGE together with lower IL-10 relative expression, indicative of an ongoing immune response. Differences in IL-1ß, IL-13 and IL-4 expression were also found in rabbit sera by ELISA suggesting further stimulation of the adaptive response. Recent challenges in the rabbit industry include the search of diet supplements able to elicit an immune stimulation with particular interest in facing pathogens such as viruses or bacteria. ß-glucans from fungi may contribute to maintain an immune steady state favouring protection and thus reducing antibiotic treatment.