Prostaglandin-related immune suppression in cattle.

Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.

Peptidase inhibitors in tick physiology.

Peptidase inhibitors regulate a wide range of physiological processes involved in the interaction between hematophagous parasites and their hosts, including tissue remodeling, the immune response and blood coagulation. In tick physiology, peptidase inhibitors have a crucial role in adaptation to improve parasitism mechanisms, facilitating blood feeding by interfering with defense-related host peptidases. Recently, a larger number of studies on this topic led to the description of several new tick inhibitors displaying interesting novel features, for example a role in pathogen transmission to the host. A comprehensive review discussing these emerging concepts can therefore shed light on peptidase inhibitor functions, their relevance to tick physiology and their potential applications. Here, we summarize and examine the general characteristics, functional diversity and action of tick peptidase inhibitors with known physiological roles in the tick-host-pathogen interaction.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.

Systemic alterations of bovine hemostasis due to Rhipicephalus (Boophilus) microplus infestation.

The tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that causes considerable economic losses to cattle breeding. Although R. microplus saliva contains several molecules that interfere with the blood coagulation process, so far the systemic alterations in the host hemostatic system have not been described. This study aims to determine if R. microplus infestation induces any disturbance to the host's hemostatic system. To address these questions, six calves were experimentally infested with 20,000 R. microplus larvae and their blood was collected before and 7, 14 and 21 days post-infestation. Collagen and ADP-induced platelet aggregation as well as coagulation (activated partial thromboplastin time and prothrombin time) decreased in infested bovines. Platelet blood count and fibrinogen increased during the course of infestation, probably as a compensatory response. These alterations may play a role in host health status, and show that the host cannot fully counteract the tick anti-hemostatic mechanisms.

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines.

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.

Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae).

Glutathione S-transferases (GSTs) are enzymes that act in excretion of physiologic and xenobiotic substances, protecting cells against chemical toxicity and stress. In this work, we characterized the enzymatic activity of GST in eggs and larvae of cattle tick Boophilus microplus, on different days after oviposition and eclosion. The results showed that the GST activity varied depending on the time elapsed after oviposition and eclosion. Molecules involved in mechanism of protection from oxidative stress are correlated with the increase in GST activity. The oxygen consumption kinetics showed a positive correlation with the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In eggs and larvae, GST activity can be correlated to kinetic parameters of oxidative stress such as catalase and glutathione. In addition, GST activity showed strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs.

Cell death during preoviposition period in Boophilus microplus tick.

Programmed cell death (PCD) is present during the development of multicellular organisms and occurs from embryogenesis to death. In females of Boophilus microplus, the mass of several organs is reduced after the detachment from the host. In order to better characterize the cell death process that eliminates unnecessary tissues, the degeneration of salivary glands, ovaries and synganglia was investigated using DNA fragmentation in agarose gel, comet and TUNEL assays, and apoptosis activation pathway by the caspase assay. DNA fragmentation and enzymatic activity of caspase-3 were observed in salivary glands and ovaries at 48 and 72h after tick removal from the host; in synganglia these parameters were maintained at low levels upon 48h. The results obtained suggest that there is a refined control of tissue maintenance through apoptosis.

A Boophilus microplus vitellin-degrading cysteine endopeptidase.

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4.0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS-PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Immunization of bovines with an aspartic proteinase precursor isolated from Boophilus microplus eggs.

The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.