Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Nanoarchitectonic E-Tongue of Electrospun Zein/Curcumin Carbon Dots for Detecting Staphylococcus aureusin Milk.

We report a nanoarchitectonic electronic tongue made with flexible electrodes coated with curcumin carbon dots and zein electrospun nanofibers, which could detect Staphylococcus aureus(S. aureus) in milk using electrical impedance spectroscopy. Electronic tongues are based on the global selectivity concept in which the electrical responses of distinct sensing units are combined to provide a unique pattern, which in this case allowed the detection of S. aureus through non-specific interactions. The electronic tongue used here comprised 3 sensors with electrodes coated with zein nanofibers, carbon dots, and carbon dots with zein nanofibers. The capacitance data obtained with the three sensors were processed with a multidimensional projection technique referred to as interactive document mapping (IDMAP) and analyzed using the machine learning-based concept of multidimensional calibration space (MCS). The concentration of S. aureus could be determined with the sensing units, especially with the one containing zein as the limit of detection was 0.83 CFU/mL (CFU stands for colony-forming unit). This high sensitivity is attributed to molecular-level interactions between the protein zein and C-H groups in S. aureus according to polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) data. Using machine learning and IDMAP, we demonstrated the selectivity of the electronic tongue in distinguishing milk samples from mastitis-infected cows from milk collected from healthy cows, and from milk spiked with possible interferents. Calibration of the electronic tongue can also be reached with the MCS concept employing decision tree algorithms, with an 80.1% accuracy in the diagnosis of mastitis. The low-cost electronic tongue presented here may be exploited in diagnosing mastitis at early stages, with tests performed in the farms without requiring specialized laboratories or personnel.

Lipopolysaccharide triggers different transcriptional signatures in taurine and indicine cattle macrophages: Reactive oxygen species and potential outcomes to the development of immune response to infections.

Macrophages are classified upon activation as classical activated M1 and M2 anti-inflammatory regulatory populations. This macrophage polarization is well characterized in humans and mice, but M1/M2 profile in cattle has been far less explored. Bos primigenius taurus (taurine) and Bos primigenius indicus (indicine) cattle display contrasting levels of resistance to infection and parasitic diseases such as C57BL/6J and Balb/c murine experimental models of parasite infection outcomes based on genetic background. Thus, we investigated the differential gene expression profile of unstimulated and LPS stimulated monocyte-derived macrophages (MDMs) from Holstein (taurine) and Gir (indicine) breeds using RNA sequencing methodology. For unstimulated MDMs, the contrast between Holstein and Gir breeds identified 163 Differentially Expressed Genes (DEGs) highlighting the higher expression of C-C chemokine receptor type five (CCR5) and BOLA-DQ genes in Gir animals. LPS-stimulated MDMs from Gir and Holstein animals displayed 1,257 DEGs enriched for cell adhesion and inflammatory responses. Gir MDMs cells displayed a higher expression of M1 related genes like Nitric Oxide Synthase 2 (NOS2), Toll like receptor 4 (TLR4), Nuclear factor NF-kappa-B 2 (NFKB2) in addition to higher levels of transcripts for proinflammatory cytokines, chemokines, complement factors and the acute phase protein Serum Amyloid A (SAA). We also showed that gene expression of inflammatory M1 population markers, complement and SAA genes was higher in Gir in buffy coat peripheral cells in addition to nitric oxide concentration in MDMs supernatant and animal serum. Co-expression analyses revealed that Holstein and Gir animals showed different transcriptional signatures in the MDMs response to LPS that impact on cell cycle regulation, leukocyte migration and extracellular matrix organization biological processes. Overall, the results suggest that Gir animals show a natural propensity to generate a more pronounced M1 inflammatory response than Holstein, which might account for a faster immune response favouring resistance to many infection diseases.

Microarray analysis of tick-infested skin in resistant and susceptible cattle confirms the role of inflammatory pathways in immune activation and larval rejection.

Tick bites promote activation of an inflammatory process that is influenced by bovine genetic composition and its history of previous exposure. Taurine and indicine breeds are known to differ on its immune response development against Rhipicephalus microplus. Nevertheless, further investigation about the complex molecular pathways involved in the development of immune response to tick infestation in cattle presenting the same genetic background is mandatory. The aim of this work was to access the early immune response triggered by R. microplus larvae attachment in previously selected resistant and susceptible animals in a bovine F2 population derived from Gyr (Bos indicus)×Holstein (Bos taurus) crosses. Microarray data analysis of RNA samples from tick infested skin was used to evaluate the gene expression at 0, 24 and 48h after R. microplus larvae attachment. Our experimental design allowed us to deeply explore the immune response related to R. microplus infestation avoiding the innate differences between these breeds. The differentially expressed genes found reveal networks and pathways that suggest a key role of lipid metabolism in inflammation control and impairment of tick infestation in resistant animals. Acute phase response also seems to be impaired in susceptible animals. These results provide new insights about early immune response against ticks and raise the possibility of using immunomodulation processes to improve and develop novel tools for tick control.

Haplotypes of the bovine IgG2 heavy gamma chain in tick-resistant and tick-susceptible breeds of cattle.

Bovines present contrasting, heritable phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus. Tick salivary glands produce IgG-binding proteins (IGBPs) as a mechanism for escaping from host antibodies that these ectoparasites ingest during blood meals. Allotypes that occur in the constant region of IgG may differ in their capacity to bind with tick IGBPs; this may be reflected by the distribution of distinct allotypes according to phenotypes of tick infestations. In order to test this hypothesis, we investigated the frequency of haplotypes of bovine IgG2 among tick-resistant and tick-susceptible breeds of bovines. Sequencing of the gene coding for the heavy chain of IgG2 from 114 tick-resistant (Bos taurus indicus, Nelore breed) and tick-susceptible (B. t. taurus, Holstein breed) bovines revealed SNPs that generated 13 different haplotypes, of which 11 were novel and 5 were exclusive of Holstein and 3 of Nelore breeds. Alignment and modeling of coded haplotypes for hinge regions of the bovine IgG2 showed that they differ in the distribution of polar and hydrophobic amino acids and in shape according to the distribution of these amino acids. We also found that there was an association between genotypes of the constant region of the IgG2 heavy chain with phenotypes of tick infestations. These findings open the possibility of investigating if certain IgG allotypes hinder the function of tick IGBPs. If so, they may be markers for breeding for resistance against tick infestations.

Rhipicephalus (Boophilus) microplus: clotting time in tick-infested skin varies according to local inflammation and gene expression patterns in tick salivary glands.

Ticks deposit saliva at the site of their attachment to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses. The anti-haemostatic properties of tick saliva have been described by many studies, but few show that tick infestations or its anti-haemostatic components exert systemic effects in vivo. In the present study, we extended these observations and show that, compared with normal skin, bovine hosts that are genetically susceptible to tick infestations present an increase in the clotting time of blood collected from the immediate vicinity of haemorrhagic feeding pools in skin infested with different developmental stages of Rhipicepahlus microplus; conversely, we determined that clotting time of tick-infested skin from genetically resistant bovines was shorter than that of normal skin. Coagulation and inflammation have many components in common and we determined that in resistant bovines, eosinophils and basophils, which are known to contain tissue factor, are recruited in greater numbers to the inflammatory site of tick bites than in susceptible hosts. Finally, we correlated the observed differences in clotting times with the expression profiles of transcripts for putative anti-haemostatic proteins in different developmental stages of R. microplus fed on genetically susceptible and resistant hosts: we determined that transcripts coding for proteins similar to these molecules are overrepresented in salivary glands from nymphs and males fed on susceptible bovines. Our data indicate that ticks are able to modulate their host's local haemostatic reactions. In the resistant phenotype, larger amounts of inflammatory cells are recruited and expression of anti-coagulant molecules is decreased tick salivary glands, features that can hamper the tick's blood meal.

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines.

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly more resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick's blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules - intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin - was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host's ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks.

Rhipicephalus (Boophilus) microplus: distinct acute phase proteins vary during infestations according to the genetic composition of the bovine hosts, Bos taurus and Bos indicus.

Tick bites may trigger acute phase responses. Positive and negative acute phase proteins were measured in infested cattle genetically resistant and susceptible to ticks. During heavier infestations levels of haptoglobin increased significantly in susceptible bovines; levels of serum amyloid A increased in resistant bovines; levels of alpha-1-acid glycoprotein decreased significantly in resistant bovines; levels of transferrin decreased significantly in susceptible bovines. In conclusion, tick infestations trigger acute phase responses and enhancement of specific acute phase proteins differs according to the genetic composition of hosts. Acute phase proteins may constitute useful biological signatures for monitoring the stress induced by tick infestations.