Identification and partial purification of serologically defined Boophilus microplus larval antigens by natural ectoparasite exposure

In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. microplus. Six putative B. microplus-specific antigens were identified by antibodies in the sera of calves repeatedly exposed to B. microplus larvae. One of the antigens, a 19.1 kDa protein, was used in the development of a diagnostic kELISA for previous exposure to B. microplus. The 19.1 kDa protein did not have tryptic protease activity or inhibit bovine trypsin activity, but appeared to be allergenic in that a partially pure fraction elicited immediate-type hypersensitivity responses in calves previously exposed to B. microplus.     

rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization

Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.     

Vaccine potential of a tick vitellin-degrading enzyme (VTDCE)

VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.     

Characterization of tick antigens inducing host immune resistance. II. Description of rabbit-acquired immunity to Amblyomma americanum ticks and identification of potential tick antigens by Western blot analysis

Feeding by adult Amblyomma americanum ticks induced a level of immunity in rabbits to subsequent tick feeding that resulted in a significant decrease in tick feeding success and fecundity. Histological analysis of tick feeding sites in hosts expressing resistance revealed a predominant eosinophil response, with weak basophil and neutrophil infiltrates. While the basophil was never the dominant granulocyte at the tick feeding sites in resistant hosts, this cell exhibited the greatest increase in density (tenfold) over levels observed in hosts experiencing their first infestation; eosinophils and neutrophils exhibited increases of five- and twofold, respectively. Serum from animals that expressed resistance was tested for the presence of anti-tick antibodies to tick-derived salivary gland substances (SGA) by Western blotting. Western blot analysis of female-derived SGA compared to male-derived SGA, using the Avidin/Biotin technique, resulted in the identification of approximately 25 proteins from the female preparation, but only seven from the male. The use of 125I labeled protein-A as the probe for anti-tick antibody in Western blot analysis resulted in fewer recognized proteins. Serum from rabbits immunized with A. americanum-derived SGA emulsified with complete (CFA) Freund's adjuvant recognized most of the proteins identified by active serum, whereas serum from animals immunized with SGA in incomplete (IFA) Freund's adjuvant did not. Furthermore, both sera recognized a multiplicity of proteins from extracts of larval A. americanum Dermacentor variabilis and Boophilus microplus ticks, suggesting the presence of common antigens between these distantly related ticks. The results from this study demonstrate that rabbits acquire a strong immunity to A. americanum ticks characterized by the production of antibody. Furthermore, ticks secrete a number of substances into rabbits during feeding, as seen by Western blot analysis but only three may be crucial to the induction of host immunity; proteins at 41, 40 and 39 kDa. The purified anti-tick antibody will be used for subsequent isolation and characterization of crucial antigens.     

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.     

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.     

Cross-reactions between crude antigens of larval Taenia solium (Cysticercus cellulosae) and other helminths of pigs

A crude antigen extract of larval Taenia solium was shown by immunodiffusion (ID) and immunoelectrophoresis (IEP) to cross-react with rabbit antisera against pig serum proteins and larval T. hydatigena, and by enzyme-linked immunosorbent assay (ELISA) with antisera against pig serum proteins, Fasciolopsis buski, larval T. hydatigena, hydatid cyst, Hymenolepis diminuta and Dipylidium caninum. Immunoblotting demonstrated that the crude antigens extract contained epitopes of pig serum proteins of 48 and 66 kDa. The crude extract also contained a subunit of antigen B (95 kDa) which was also found in T. hydatigena and H. diminuta. Immunoperoxidase and indirect immunofluorescence studies showed that cross-reacting antigens were distributed mainly on the tegument of T. solium.     

Purification and characterization of two larval glycoproteins from the cattle tick, Boophilus annulatus

The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.     

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.     

DNA restriction endonuclease cleavage pattern and protein antigen profile of Ehrlichia risticii

Molecular characterization of Ehrlichia risticii, the etiological agent of Potomac horse fever, was performed. Restriction endonuclease cleavage of E. risticii DNA generated distinct patterns by different enzymes. The DNA cleavage patterns of E. risticii isolates obtained from different geographic regions were similar. Protein analysis identified thirty-five distinct proteins with molecular weights ranging from 160 to 16 kilodalton (kDa). Antigenic analysis by radioimmunoprecipitation using 125I surface labeled E. risticii and by Western blotting determined the presence of eighteen antigens (160, 110, 86, 84, 81, 70, 55, 51, 49, 44, 41, 36, 33, 31, 28, 24, 22 and 16 kDa) of which nine (110, 86, 70, 55, 51, 49, 44, 33, and 28 kDa) were major antigens. Fourteen of these antigens, which included the major antigens, were apparent surface components. There were no heat-modifiable proteins but lipopolysaccharide components of 245 and 14 kDa, resistant to proteinase K and of non-antigenic character, were detected in the organism.     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

Relationship between the resistance of cattle to Haemaphysalis longicornis and to Boophilus microplus

Cattle that had been exposed to Haemaphysalis longicornis were as susceptible to Boophilus microplus as cattle that had never been exposed to either species of tick. Cattle with acquired resistance to both species ranked consistently for levels of resistance to each when infested separately. Concurrent infestation with H. longicornis had no effect on ranking for resistance to B. microplus. The coefficient of concordance between the rankings of individuals on their levels of resistance to both species of tick was positive, but was not statistically significant. We conclude that the tick antigens that stimulate host resistance are species-specific and do not cross protect. The apparent correlation in rankings for resistance to the 2 species may be a consequence of either an individual's immunological responsiveness to tick antigens or to non-specific host factors which determine levels of resistance. The apparent correlation suggests that co-selection for resistance to different tick species is practicable.     

Temporal pattern of isotype-specific antibody responses in primary and challenge infestations of sheep with Psoroptes ovis--the sheep scab mite

In sheep Psoroptes ovis provokes an allergic dermatitis with significant P. ovis antigen-specific IgE responses. The kinetics of the IgE response to primary and challenge infestations of P. ovis were reported earlier [Parasite Immunol. 22 (2000) 407]. The present study examines IgG, IgM and IgA responses to primary and challenge infestations of P. ovis and the profile of antigens/allergens reacting with IgG and IgE antibodies. Antigen-specific enzyme-linked immunosorbent assays (ELISAs) demonstrate that primary infestations elicited significant increases in levels of IgG and IgM but not IgA antibodies. IgG and IgM responses to primary and challenge infestations were not significantly different. Western blots of reduced P. ovis proteins indicate that IgG antibodies reacted with five major antigens following primary infestation and only three of these after challenge infestation. IgE antibodies bound to three major and five minor allergens after primary infestation and two additional minor allergens after challenge infestation. Immunodominant antigens >100 and <15 kDa and allergens >100 kDa were most consistent in stimulating substantial IgG and IgE antibody responses, respectively. These antigens/allergens may be exploited in immunodiagnosis and modulation of the host immune response.     

Immune responses of infected and vaccinated Hereford cattle to antigens of the cattle tick, Boophilus microplus

Responses of infested and vaccinated Hereford cattle to Boophilus microplus antigens were measured by enzyme-linked immunosorbent assay (ELISA), lymphocyte blastogenesis assay (LBA) and intradermal skin tests. Responses against soluble salivary gland extracts (SGS), salivary gland membrane (SGM), soluble gut extracts (GS), gut membrane (GM), soluble larval extracts (LS) and larval membrane (LM) antigens were tested. In one experiment, cattle infested with up to 160,000 ticks had positive cellular responses to SGS and significant antibodies against LM, GM, SGM, and SGS. Cellular responses to Concanavalin A were not depressed following infestation. Cattle vaccinated with GM, using Quil A as adjuvant, had positive cellular responses to gut and salivary gland antigens and significant antibody responses to all antigens tested. The antibody levels of vaccinated cattle were significantly higher than the antibody levels of infested cattle (P less than 0.05). In a second experiment, immune responses of cattle infested with 40,000 ticks were studied during 38 days. Cellular responses in LBA to several tick antigens were transiently elevated and significant levels of antibody were measured against LM, GM, SGM and SGS, from day 25 (P less than 0.05). Infested cattle had positive skin reactions following intradermal injection of larval and adult tick antigens (P less than 0.05).     

Western blot analysis of Amblyomma americanum-derived stage-specific and shared antigens using serum from guinea pigs expressing resistance

Serum from guinea pigs expressing resistance to larval, nymphal and adult Amblyomma americanum ticks was used in Western blot analyses to identify potential antigens from egg, larval and nymphal, and female salivary gland extract preparations. The results demonstrate multiple antigens unique to each life stage, as well as several shared proteins between the three life stages. However, it appears as if two particular proteins of 25 and 38 kDa may be more important than others, based upon their prevalence and intensity of recognition in this assay relative to other polypeptides.     

Immunogenicity of Major Cell Surface Protein(s) of Brucella melitensis Rev 1

Brucella melitensis Rev 1 organisms were salt-extracted and the cell surface proteins (BCSPs) were found to be mainly 39-42 kDa (group 2 porin proteins) in addition to 31.6, 32.5, 58.5 and 14.7 kDa proteins. DEAE-Sephadex anion-exchange column chromatography of BCSPs yielded fraction 1, which contained one major protein (39.8-42.0 kDa) and a minor protein (31.6 kDa). All these proteins were found to be immunogenic by Western blotting. Fraction 1 along with monophosphoryl lipid A and trehalose dicorynomycolate adjuvants as well as BCSPs alone induced significant (p < or = 0.05) protection in BALB/c mice. Both these immunizing agents produced almost equivalent protection to live B. melitensis Rev 1 vaccine at 15 and 30 days post challenge. Lymphocyte stimulation test as well as delayed-type hypersensitivity reaction revealed that both these preparations induced cell-mediated immune response. These preparations also induced humoral immune response as indicated by indirect ELISA. Neither of the immune responses was significantly less (p < or = 0.05) than that with live B. melitensis Rev 1 vaccine, except that their duration was short.     

Species composition and geographic distribution of ticks infesting cattle, goats and dogs in a temperate and in a subtropical region of south-east Africa

The species and distribution of ticks infesting cattle, goats and dogs in the eastern region of the Eastern Cape Province, South Africa and Maputo Province, Mozambique were determined from collections made from these animals at 72 localities in the former region and 30 in the latter. Eleven ixodid and one argasid species were recovered in the Eastern Cape Province and 15 ixodid species in Maputo Province. The most common ticks infesting cattle and goats in both provinces were Amblyomma hebraeum, Rhipicephalus (Boophilus) microplus, Rhipicephalus appendiculatus and Rhipicephalus evertsi evertsi. The dominant species on dogs were Haemaphysalis elliptica and Rhipicephalus simus. The geographic distributions of the major species and some of the minor species in both regions were plotted. The partial or complete displacement of the indigenous tick Rhipicephalus (Boophilus) decoloratus by the introduced species R. (B.) microplus was a major feature of both surveys.     

Demonstration of the Specificity of the Salmonid Hurnoral Response to Renibacterium salmoninarum with a Monoclonal Antibody against Salmonid Immunoglobulin

Abstract

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytscha was compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarum preparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarum antibody.