Immunization of guinea-pigs and cattle against adult Rhipicephalus appendiculatus ticks using semipurified nymphal homogenates and adult gut homogenate.

Guinea-pigs inoculated with crude homogenate of unfed nymphs of the tick Rhipicephalus appendiculatus and with three semipurified fractions of the homogenate obtained by gel permeation chromatography, acquired a significant degree of immunity to infestation with adults of this tick. Fraction 2 induced the highest reduction (66%) in mean weight of engorged females followed by crude homogenate and fractions 1 and 3. Calves immunized with crude homogenates of unfed nymphs, fraction 2 of nymphal homogenate, and gut homogenate of unfed females also acquired immunity against adults of R. appendiculatus. The mean weight of engorged females fed on calves inoculated with nymphal fraction 2 was the lowest of all five groups of calves on which females fed. The reduction in weight (38%) was not significantly different from that observed for females fed on calves inoculated with crude nymphal homogenate (31%) or females from third infestation of adult ticks. No differences in the weight and hatchability of egg batches produced by engorged females collected from the five groups of calves were observed. Analysis of sera collected from the five groups of calves showed that the concentration of albumin, alpha-1, alpha-2 and beta-globulins fluctuated and no significant differences between the treated groups were observed. The levels of gamma-globulin increased in treated groups including the group inoculated with adjuvant only, but unlike previous reports no increase in gamma-globulin or a correlation between the level of gamma-globulin and the degree of resistance acquired were observed in calves exposed to repeated tick infestations. However, the increase in the concentration of gamma-globulin in calves inoculated with fraction 2 or crude nymphal homogenate was higher than that observed in the other groups.     

Induction of host resistance toRhipicephalus appendiculatus in rabbits: Effects of immunizing with detergent-solubilized tick tissue proteins

Resistance to the hard tick,Rhipicephalus appendiculatus, was induced in rabbits by immunizing them with tick tissue proteins extracted with a detergent, Triton X-100. There was 25% mortality in female ticks fed on immunized rabbits as compared with those fed on controls. Similarly, there was a 40% and 60% reduction in the engorged weight and the weight of egg batches, respectively, of ticks fed on immunized rabbits. Western blot analysis of detergent-solubilized tick tissue Western blot analysis of detergent-solubilized tick tissue proteins, carried out using immune sera, recognized a complex pattern of proteins. A strong reaction was observed with proteins with apparent molecular weights of 94000 and 40000 daltons.This work was carried out at the Institute of Zoology, University of Neuchâtel, Chantemerle 22, CH-2000 Neuchâtel, Switzerland     

Acaricidal effect of herbal extracts against cattle tick Rhipicephalus (Boophilus) microplus using in vitro studies

The crude methanolic extract of Datura stramonium, Azadirachta indica, and Calotropis procera leaves, Allium sativum (AS) cloves, and Carica papaya (CP) seeds collected from Banaskanta, Gujarat (India) was tested for its acaricidal properties against Rhipicephalus (Boophilus) microplus. The percent adult mortality within 15 days, reproductive index, percentage inhibition of oviposition, hatching of laid ova, and percentage larval mortality were studied at concentrations of 12.5, 25, 50, and 100 mg/ml. At the highest concentration (100 mg/ml), the adult tick mortality was 66.67, 73.33, 80.00, and 93.33 % for C. procera, D. stramonium, A. sativum, and C. papaya extracts, respectively, and it was statistically significant (P?<?0.001). However, for A. indica, mortality was low and estimated to be 33.33 %. Inhibition of oviposition at the highest concentration of A. indica, C. procera, D. stramonium, A. sativum, and C. papaya extract-treated ticks was 20.73, 71.34, 77.17, 85.83, and 100.00 %, respectively. Inhibition of fecundity of treated groups differed significantly from the control and was concentration dependent. Larvae treated with all the tested concentrations of A. indica, C. procera, D. stramonium, A. sativum, and C. papaya extracts by larval packet test showed significant mortality (P?<?0.001) than that of control tick larvae, and at the highest concentration, it was 55.2, 63.2, 71.8, 69.0, and 82.2 %, respectively. Garlic cloves and papaya seed extract produced complete failure of eclosion of eggs from the treated ticks even at lower concentrations; however, neem, calotropis, and datura was capable of reducing hatchability by 20, 50, and 70 %, respectively. The results pointed that the crude extracts of A. sativum cloves and C. papaya seeds have very good acaricidal properties and could be a potential component of alternative R. (B.) microplus tick control strategy.     

Immunization of guinea-pigs against Rhipicephalus appendiculatus adult ticks using homogenates from unfed immature ticks.

Guinea-pigs immunized with homogenates of unfed larvae and nymphs of the tick Rhipicephalus appendiculatus developed significant levels of protective immunity to infestation with adults of this species. The mean engorged weight of female ticks feeding on immunized animals (181.96 +/- 05.63 mg and 170.11 +/- 11.54 mg) was reduced by an average of 46% and 51%, respectively, compared to that of female ticks feeding on control guinea-pigs, although in some individual animals the reduction was as high as 86%; the mean egg mass weight was also significantly reduced. Electrophoretic separation of the homogenates followed by immunostaining with post-infestation sera revealed several antigen bands common to all stages. Two bands of 36,500 and 23,000 molecular weight (MW) were recognized in all homogenates by post-adult infestation serum, but not by post-larval or post-nymphal infestation sera, suggesting that these may be antigens specifically involved in feeding by adult ticks, and are either not presented to the host's immune system or presented only in minimal amounts during feeding by immature stages. Sera from animals immunized with the homogenates did not recognize either of these antigens. Post-immunization sera did, however, stain two bands of 84,000 and 60,000 MW in the homogenates which were not recognized by post-infestation sera.     

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and interleukin-2 for protection against bovine tuberculosis

In this study vaccines prepared from culture filtrate proteins (CFP) of Mycobacterium bovis and interleukin-2 (IL-2) were tested in cattle for their capacity to stimulate immune responses and to protect against an intratracheal challenge with virulent M. bovis. Nine groups of cattle were vaccinated with combinations of different doses of CFP and bovine IL-2 mixed with a monophosphoryl lipid A (MPL) adjuvant. An additional group was vaccinated with M. bovis BCG. Immune responses in b1P-IL-2-vaccinated animals differed from those seen in BCG-vaccinated animals by inducing high antigen-specific antibody responses and low levels of gamma interferon and IL-2 released from purified protein derivative-stimulated whole-blood cultures. In a concurrent experiment, additional animals were added to the high-dose CFP-IL-2, MPL control, and BCG groups and these expanded groups of animals were challenged intratracheally with virulent M. bovis. Although the lung lesion scores were significantly lower for both the CFP-IL-2-and BCG-vaccinated groups compared to the MPL control group, the overall level of protection was greatest for the BCG-vaccinated animals. There were more animals with extrathoracic spread of disease in the CFP-IL-2 group than in the other groups. While vaccination of cattle with M. bovis CFP gave an encouraging reduction in tuberculous lesions and did not induce a delayed-type hypersensitivity response to PPD, future CFP vaccines must prevent any extrathoracic spread of disease.     

Vaccination of goats with 31 kDa and 32 kDa Schistosoma japonicum antigens by DNA priming and protein boosting

Two Schistosomajaponicum vaccine candidate antigens Sj 31 and Sj 32, which have shown particular promise to induce protective immunity in mice, were used to immunize goats by using a DNA priming-protein boosting strategy in present work. DNA vaccine formulations of the two antigens （VRSj31 and VRSj32） were produced and injected intramuscularly twice at a 2-week interval and then recombinant proteins （rSj31 and rSj32） together with Freund Complete Adjuvant （FCA） were used to boost the goats. The experiment was repeated in different batche cercariae. A strong anamnestic antibody response was induced after boost. A significant reduction of liver egg counts and miracidial hatching was showed in both experiments. Significant protections against challenge infection were elicited with 31.6% of percentage reduction for worm recovery in the second experiment and 20.9% in the first experiment, respectively.     

Immunization against ticks: use of salivary gland antigens and infestations with Rhipicephalus appendiculatus (Acari: Ixodidae) in rabbits

Comparative immunogenicity of salivary gland antigens (SGA) derived from adult Rhipicephalus appendiculatus (Neumann) and experimental infestations with the three stages of the tick was investigated. The best immunization schedule judged by reduction of engorgement weights and hatchability resulted from nymphal and adult tick infestations. Inoculation of rabbits with SGA in Freund's incomplete adjuvant (FIA) induced immune responses comparable to those associated with larval infestations, but less than those produced by nymphal or adult tick infestations. High antibody titers directed against SGA were detected only in the vaccinated and adult tick-infested rabbits. The results of this experiment suggest that SGA prepared from partially fed ticks can be used in the experimental induction of immunity to ticks and that the antigen has potential as a vaccine against R. appendiculatus ticks.     

Chemical composition and larvicidal properties of the essential oils from Drimys brasiliensis Miers (Winteraceae) on the cattle tick Rhipicephalus (Boophilus) microplus and the brown dog tick Rhipicephalus sanguineus

The essential oil obtained from leaves and stem barks of the Southern Brazilian native Drimys brasiliensis Miers, a tree with medicinal properties, was analyzed by gas chromatography (GC) and GC/mass spectrometry (MS). The oil was characterized by sesquiterpenoids (66%), cyclocolorenone being the most abundant (30.4%), followed by bicyclogermacrene (11.8%) and alpha-gurjunene (6.0%). Laboratory tests were carried out to determine the toxicity of the essential oil on larvae of the cattle tick Rhipicephalus (Boophilus) microplus and the brown dog tick Rhipicephalus sanguineus by the larval immersion test. It was observed that the oil was lethal, killing 100% of the larvae of both ticks at the doses of 25, 12.5, and 6.25 μl/ml. The lowest dose tested, 3.125 μl/ml, was also toxic, killing 95–98% of the larvae.     

Some structural properties of thymus leukemia antigen (TL) solubilized with detergent.

Plasma membranes were isolated from the leukemia cell line ASL1w and extracted with detergent (DOC). DOC solubilized more TL activity than could be detected on isolated membranes. However, extraction of membranes with LDS or EDTA solubilized only 17% and 4%, respectively, of the activity. This indicated that TL was not loosely associated with the membrane but rather was integrated into the lipid bilayer. At low concentrations of DOC (0.05%), TL was found to be largely aggregated and was also prone to autolysis. Neither aggregation nor autolysis was observed at a higher DOC concentration (0.5%). The apparent molecular weight of TL in 0.5% DOC was determined by Sephadex G-200 chromatography to be about 65,000-70,000. Digestion of a 0.5% DOC extract of TL with either papain or trypsin produced a fragment of TL of about 35,000 molecular weight. These fragments were similar in size to a fragment produced by autolysis. These data suggested that a region of the TL molecule was very prone to proteolytic attack. The 35,000 molecular weight proteolytic fragments bound specifically to lentil lectin affinity columns, which indicated that they retained at least part of the carbohydrate present on the native molecule.     

Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations

The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, we describe the analysis of R. microplus glutathione-S transferase, ubiquitin (UBQ), selenoprotein W, elongation factor-1 alpha, and subolesin (SUB) complementary DNAs (cDNAs) by RNA interference (RNAi) in R. microplus and Rhipicephalus annulatus. Candidate protective antigens were selected for vaccination experiments based on the effect of gene knockdown on tick mortality, feeding, and fertility. Two cDNA clones encoding for UBQ and SUB were used for cattle vaccination and infestation with R. microplus and R. annulatus. Control groups were immunized with recombinant Bm86 or adjuvant/saline. The highest vaccine efficacy for the control of tick infestations was obtained for Bm86. Although with low immunogenic response, the results with the SUB vaccine encourage further investigations on the use of recombinant subolesin alone or in combination with other antigens for the control of cattle tick infestations. The UBQ peptide showed low immunogenicity, and the results of the vaccination trial were inconclusive to assess the protective efficacy of this antigen. These experiments showed that RNAi could be used for the selection of candidate tick-protective antigens. However, vaccination trials are necessary to evaluate the effect of recombinant antigens in the control of tick infestations, a process that requires efficient recombinant protein production and formulation systems.     

Hereford cattle protected against Boophilus microplus with antigens purified by immunoaffinity chromatography from larval and adult ticks.

The subpopulations of lymphocytes in the pregnant and non-pregnant mammary glands of the sheep were delineated by a panel of monoclonal antibodies. The most striking feature observed was that in the mammary gland of both pregnant and non-pregnant sheep the great majority of the lymphocytes in the ductal and alveolar epithelium were agranulated CD8+ CD5- cells. A small subpopulation of granulated lymphocytes in the epithelium expressed the CD45R antigen but not the major histocompatibility complex (MHC) class II molecules. Other subpopulations, especially B lymphocytes, were present in much lower concentrations and were located mainly in the periductal and intralobular connective tissues. Patches of lymphocytes clustering around venules were observed and the majority of them were shown to be CD5+ CD4+, while some were CD5+ CD8+ but none were CD45R+ (B cell). It is suggested that selective traffic of T cells occurs at these sites.     

Antibodies against polypeptides of purified Epstein-Barr virus in sera from patients with connective tissue diseases.

Antibodies to Epstein-Barr virus (EBV) were investigated in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) by immunoblotting using purified virus. Compared with sera from Epstein-Barr virus seropositive healthy individuals who served as control, sera from patients less frequently recognized several polypeptides. In particular, a 100 kDa envelope polypeptide was recognized by 92% of healthy subjects and only 11% of patients (P less than 0.001). On the other hand, 62% of the patient sera had antibodies to the 42 kDa envelope polypeptide, whereas these antibodies were only present in 4% of the sera from the control subjects (P less than 0.001). These results could reflect either perturbations of the immune response associated with connective tissue disorders or a possible pathogenic role of EBV.     

Vaccination of cattle against bovine ephemeral fever with live attenuated virus followed by killed virus

Summary Serial passage of bovine ephemeral fever virus in hamster lung cells, HmLu-1, deprived rapidly the virus of virulence for cattle with simultaneous loss of the ability to produce neutralizing antibody. The attenuated virus thus obtained, however, could prime cattle to produce neutralizing antibody rapidly to high titers following a subsequent inoculation of formalin-inactivated, AIPO₄ gel-adsorbed vaccine (K). Although one subcutaneous dose of live vaccine (L) prepared from the attenuated virus produces no detectable amounts of neutralizing antibody unless as heavy a dose as 10⁷ TCID₅₀ was used, the priming effect of L vaccine was shown even with 10 TCLD₅₀. Similar priming effect was also shown in mice by intracerebral inoculation of L vaccine. Maximal antibody response of cattle was obtained by one subcutaneous L vaccine dose of lO⁵]TCID₅₀ followed by 3 ml of K vaccine administered intramuscularly at intervals of 2 to 4 weeks. This LK method induced seroconversion in nearly all of initially seronegative cattle, and the neutralizing antibody thus produced persisted much longer than that following two doses of K vaccine given at one-month intervals. The LK method exerted little side effects in cattle. Cows vaccinated during pregnancy delivered healthy calves in term. No adverse effect on milk production was shown. The vaccine virus could not be passaged serially in calves by intravenous inoculation of the blood. Lyophilized L vaccine was stored at 4 C with little loss of infectivity for at least one year. Reconstituted vaccine could be used safely at least for 6 hours when kept at 4 C or even at room temperature.All these findings indicate the efficacy and safety of the LK vaccination method, although the final evaluation rests on large-scale field trials.     

Association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens

The association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens was analyzed by a direct binding assay using radiolabeled beta 2-microglobulin and an immunoadsorbent containing a monoclonal antibody to the HLA-A,B heavy chains. Binding of beta 2-microglobulin to HLA-A,B heavy chains could be saturated with respect to the amount of membrane glucoprotein in the system and reached steady state after 6 h at 37 degrees C. Inhibition of [125I]beta 2-microglobulin binding to HLA-A,B heavy chains by beta 2-microglobulin purified from human urine or bovine colostrum resulted in identical inhibition curves and apparent dissociation constants of 1 X 10(-8) M. This evidence suggests that beta 2-microglobulins from different species have similar binding sites for HLA-A,B heavy chains.     

Virulence of Isaria sp. and Purpureocillium lilacinum to Rhipicephalus microplus tick under laboratory conditions

Rhipicephalus microplus (Canestrini) is an ectoparasite accountable for great economic losses. The use of entomopathogenic fungi to control arthropods has shown promising responses. The present study evaluated the virulence of Isaria farinosa (Holmsk.) Fr., Isaria fumosorosea (Wize) Brown and Smith, and Purpureocillium lilacinum (=Paecilomyces lilacinus) (Thom.) Samson to engorged females, eggs, and larvae of R. microplus. There were four treatment groups (10(5), 10(6), 10(7), and 10(8) conidia?ml(-1)) and the control group (water and Tween 80, 0.1?%?v/v). The treatment was based on immersion of the specimen in 1?ml of the suspension or control solution. The study observed changes in egg viability and larval mortality after treatment. The results showed that I. farinosa, P. lilacinum, and I. fumosorosea caused alterations in the biological parameters of R. microplus ticks. I. fumosorosea presented the greatest potential to control R. microplus engorged females in vitro, causing a 49?% decrease in nutritional index. All fungal isolates presented significant reduction in the egg production index. I. farinosa reduced the hatching percentage if the eggs were treated with the two highest conidial concentrations. All conidial concentrations of I. fumosorosea were able to reduce the hatching percentage significantly. All tested isolates showed pathogenicity toward unfed R. microplus larvae. As far as we know, this is the first study reporting the effect in vitro of I. farinosa, I. fumosorosea, and P. lilacinum to different developmental stages of R. microplus ticks.     

Immune response region-associated antigens of the mouse. Biochemical properties of detergent and papain solubilized molecules

Immune response region-associated (Ia) antigens and classical H-2 antigens are glycoproteins which differ in their molecular weight and tissue distribution. Ia and H-2 molecules also show differences in overall charge, apparent isoelectric point and susceptibility to proteolytic degradation, acid treatment and exposure to 56°C. In the present paper, Ia.11^d antigens were shown to have a molecular weight of about 35 000 after solubilization with non- ionic detergents. Purified Ia^d antigens, solubilized by controlled papain digestion, display a molecular weight of about 26 000 after final purification by indirect immune coprecipitation. This figure corresponds to the molecular weight of a highly purified H-2 polypeptide fragment obtained after papain solubilization. The remarkable size similarity of proteolytic fragments from Ia and H-2 antigens could suggest that these genetically and functionally related molecules possess some structural features in common.     

Babesia bovis: Vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes

The distilled water lysate of erythrocytes infected withBabesia bovis was separated by gel filtration on Sephadex G200. The void volume fraction or a pool of retained fractions which had immunodiffusion activity were injected into two groups of five cattle. These were challenged 2 weeks after the final vaccination with a heterologous strain ofB. bovis. A control group of five cattle was similarly challenged. Two of the five control animals died from the challenge, whereas none of the vaccinated animals died. There were significant differences in parasitaemia and pathophysiological parameters between the vaccinated groups and the control group.     

Immunization with purified protein antigens from Streptococcus mutans against dental caries in rhesus monkeys.

Protein antigens I, I/II, II, and III were prepared from Streptococcus mutans (serotype c). Their immunogenicities and protective effects against dental caries were investigated in 40 rhesus monkeys kept entirely on a human-type diet, containing about 15% sucrose. Antigens I, I/II and, to a lesser extent, antigen II induced significant reductions in dental caries, as compared with sham-immunized monkeys. This was achieved with 1 or 2 doses of antigen, the first of which was administered with adjuvant (Freund incomplete adjuvant or aluminum hydroxide). There was no reduction in caries in monkeys immunized with antigen III. The reduction in caries in the animals immunized with antigens I or I/II was comparable to that in monkeys immunized with whole cells. Protection against caries was associated predominantly with serum and gingival crevicular fluid immunoglobulin G antibodies, which appeared to be directed against the antigen I determinant, but antibodies to antigen II, though not to antigen III, were also protective.