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.     

Differential activation of mast cells by antigens from Trichinella spiralis muscle larvae,adults, and newborn larvae

Mast cell (MC) hyperplasia and activation are prominent features in Trichinella spiralis infection. Indeed a temporal correlation has been shown between the kinetics of intestinal mastocytosis, release of inflammatory mediators from MC, and adult worm loss, which constitutes a major component of the defense against T. spiralis infection. It is well known that during the intestinal phase of trichinellosis, muscle larvae (ML) and adult worms (AD) enter into contact with the host; however, interaction with MC may also occur during migration of newborn larvae (NBL). Therefore, it is plausible that antigens from these developmental stages could activate MC. We have previously demonstrated by in vitro assays that T. spiralis muscle larval (TSL-1) antigens activate MC through an Ig-independent mechanism leading to the release of histamine, MC protease 5, IL-4 and TNF alpha. In this work we evaluated whether total antigens from AD or NBL could activate unsensitized MC and we compared this activation with the activation seen when MC are stimulated with TSL-1 antigens. MC activation was also tested with affinity chromatography purified antigens from NBL using the monoclonal antibody CE-4 that recognizes NBL surface components. The results obtained in this study showed that AD total extracts and TSL-1 antigens induced the release of histamine but not β-hexosaminidase from unsensitized MC, suggesting a selective secretion of MC mediators. In contrast, NBL total extracts or purified NBL antigens did not induce the release of either histamine or β-hexosaminidase from MC. Interestingly, AD and ML are the stages that interact with the host during the intestinal phase of infection. The mechanisms involved in TSL-1 and AD activation of unsensitized MC may function together with other mechanisms of MC activation in host protection against T. spiralis.     

Th1 response in BALB/c mice immunized with Dirofilaria immitis soluble antigens: a possible role for Wolbachia?

The immune response to filarial infection has been shown to be of both the Th1 and Th2 types. Studies aimed at developing immunization strategies against Dirofilaria immitis infection in dogs have shown that protection against larval challenge is of the Th2 type and that several proteins are recognized by immunized or infected animals. The bacterial endosymbiont Wolbachia, harbored by many filarial species including D. immitis, has recently been shown to interact with the host immune system. Specific antibodies to the Wolbachia recombinant surface protein (WSPr) have been observed in cats infected with D. immitis. In this work the authors have determined cytokine production and antibody response in BALB/c mice inoculated with soluble antigens from third stage larvae or from adult worms of D. immitis. Inoculated mice first produced IFN-gamma followed by a peak in IL-4. Specific antibodies to the Wolbachia protein WSPr were exclusively IgG2a, while antibodies against peptides derived from antigens of D. immitis were in the IgG1 and IgE subclasses. The cytokine response is thus similar to that reported for other filarial infection, where Th1 response shifts towards Th2. Antibody response indicates that Wolbachia may induce preferentially a Th1 response during filarial infection, while nematode antigens may be involved in Th2 response. There is thus an overall agreement with current opinions on the role of bacterial versus nematode molecules in driving the response towards the different directions.     

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.     

Cellular and humoral local immune responses in sheep experimentally infected with Oestrus ovis (Diptera: Oestridae)

Cellular and humoral local responses were investigated following repetitive artificial Oestrus ovis infections in lambs. The presence of larvae induced a huge local recruitment of either leucocytes (T and B lymphocytes, macrophages) or granulocytes (eosinophils, mast cells and globule leucocytes). This cellular response was more pronounced in the ethmoid and sinus (development sites of second and third instar larvae) than in the septum or turbinates where first instar larvae migrate. Infected lambs produced Oestrus ovis specific IgG and IgA antibodies in their mucus. This local humoral response was mainly directed against larval salivary gland antigens and not against larval digestive tract antigens. Compared to the control animals, the sinusal mucosa of infected animals was extremely thickened and the epithelium exhibited hyperplasia, metaplasia and eosinophilic exocytosis. The possible roles of these local immune responses in the regulation of O. ovis larvae populations in sheep are discussed.     

Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues.

Rabbit monoclonal (RM) antibodies appear to have higher affinity for antigens than mouse monoclonal (MM) antibodies. However, RM antibodies have not been used in veterinary diagnostic immunohistochemistry. The authors compared reactivities of RM and MM antibodies on formalin-fixed, paraffin-embedded canine tissues, targeting 11 different antigens: CD3, CD79a, calcitonin, calretinin, chromogranin A, COX-2, estrogen receptor, Ki67, progesterone receptor, synaptophysin, and vimentin. Paraffin-embedded tissue sections were processed by 1 of 2 antigen-retrieval methods: 1) proteinase K digestion or 2) steam heat in citrate buffer. An additional set of slides did not receive antigen retrieval. Immunostaining was performed using an automated stainer, and scores were assigned to the different dilutions and antigen-retrieval methods on the basis of staining intensity and number of positive cells. Steam heat was usually the best antigen-retrieval method. The optimal dilution for each antibody was that which resulted in the highest specific staining and the lowest nonspecific (background) staining. The RM or MM antibodies yielded a specific reaction for all antigens examined except calretinin. The RM and MM antibodies yielded a specific reaction for 4 antigens only: COX-2, Ki67, synaptophysin, and vimentin. Three antigens (CD3, chromogranin A, and progesterone receptor) were detected only with RM antibodies, whereas the other 3 (CD79a, calcitonin, estrogen receptor) were detected only with MM antibodies. The results of this study differed from those reported for human tissues by the manufacturers of the antibodies. These results emphasize that, regardless of manufacturers' recommendations, each antibody must be individually standardized and validated before routine use in canine tissues.     

Effectiveness of antigens from various developmental stages of Ascaris suum in the deomonstration of the migratory phase of experimental ascariasis in pigs]

Tests of the efficiency of antigens prepared from different developmental stages of Ascaris suum in indirect haemagglutination test in the course of proving the migration phase of experimental ascariasis in pigs show that the antigens prepared by ultrasound from the invasive larval stage of A. suum in comparison with antigens of sexually mature stages have higher serological activity. By using this antigen it is possible to prove specific antibodies in experimentally invaded pigs from 6 to 120 days after invasion as opposed to the other tested antigens (the detectability of antibodies from the 7th-8th day to the 64th day after invasion).     

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.     

Carbohydrate profiling and protein identification of tegumental and excreted/secreted glycoproteins of adult Schistosoma bovis worms

Schistosoma bovis is a parasite of wild and domestic ruminants that is broadly distributed throughout many tropical and temperate regions of the old world. S. bovis causes severe health problems and significant economic losses in livestock, but in contrast to human schistosomes, S. bovis has been little investigated at a molecular level. Since schistosome glycans and glycoproteins can play important roles in the host-parasite interplay, the aims of the present work were: (i) to characterize the glycans expressed by adult S. bovis worms on their excreted/secreted (ES) and tegumental (TG) glycoproteins and (ii) to identify their carrier protein backbones by mass spectrometry. Using a panel of lectins and monoclonal and polyclonal anti-glycan antibodies, we observed: (i) the absence of sialic acid in S. bovis; (ii) the presence of complex-type N-glycans and LDN antennae on ES glycoproteins; (iii) the presence of glycans containing the Fucalpha1-2Galbeta motif in many TG glycoproteins, and (iv) the presence of glycans containing the Fucalpha1-3GlcNAc motif on many ES and TG glycoproteins but, simultaneously, the absence of the F-LDN(-F) glycans from both the ES and TG glycoproteins. Interestingly, we also found the Lewis(X) and Lewis(Y) antigens co-expressed on several TG isoforms of ATP:guanidino kinase and glyceraldehyde-3-phosphate dehydrogenase. Finally, by ELISA we observed the presence of antibodies against Lewis(X), Lewis(Y) and F-LDN(-F) in the sera of sheep experimentally infected with S. bovis.     

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins.In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.     

Induction of antibodies to canine herpesvirus in mice by immunization with anti-idiotypic antibodies.

Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers.     

Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses.     

Monoclonal antibodies to human antigens recognise feline myeloid cells

Immunological techniques have been used to study the expression of a series of cell surface antigens in cat haemopoietic tissues. Forty-two monoclonal antibodies raised against well-defined antigens of human origin were tested by indirect immunofluorescence on feline blood, bone marrow, spleen and thymus. Myeloid cells from all tissues reacted with antibodies to CD9, CD10 and CD18 antigens. No antibodies specific for T or B lymphocytes were found to react with cat lymphoid cells. Osteoclasts, isolated from juvenile bone marrow, were found to express the 23C6 human osteoclast-specific antigen. The potential use of such antibodies in experimental and diagnostic veterinary haematology are discussed.     

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens.     

Detection of antibodies against membrane-glycoproteins of swine erythrocytes after experimental infection with Mycoplasma hyopneumoniae

During enzootic pneumonia of pig, antibodies against membrane glycoproteins of erythrocytes were detected with an enzyme-linked immunosorbent assay (ELISA). These antibodies arise simultaneously with the specific antibodies. They are not identical to cold agglutinins.     

Antibody response of chickens to serotype 1, 2, or 3 Marek's disease vaccines based on ELISA with infected cells as antigen

An enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the antibody response of commercial White Leghorn chickens to vaccination against Marek's disease (MD) at hatch (day 0) with serotype-1 (Rispens), -2 (SB-1), or -3 (turkey herpesvirus, HVT) vaccine virus and to challenge on day 21 with MD virus. Antigens for the test were whole chicken embryo fibroblast cells infected with Rispens, SB-1, or HVT. The chickens were progeny of stock that had been vaccinated with HVT, and on day 21 the nonvaccinated group had higher levels of maternal antibodies to HVT than to other antigens (P < 0.05). Only SB-1 vaccine had induced antibodies by day 21, and this was detected only against homologous antigens. On day 49, all three vaccines had induced higher levels of antibodies to homologous than to heterologous antigens. Marek's Disease virus (MDV) induced antibodies to all three antigens, but challenging vaccinated chicks did not significantly increase levels of antibodies on day 81 to any of the three antigens. It was concluded that an ELISA using whole cells as antigens would have potential value for monitoring the antibody response induced by MD vaccines and virulent MDV.     

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.     

Characterization of monoclonal antibodies against fowl poxvirus

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.     

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).     

Persistence of passively transferred antibodies in porcine Taenia solium cysticercosis. Cysticercosis Working Group in Peru.

We evaluated the presence and persistence of anticysticercal antibodies in piglets born to Taenia solium infected sows. Infected sows from a disease-endemic area of Peru were transported to a nondisease-endemic area and impregnated. Serum samples were collected from sows and piglets on Day 2 through Week 35 after birth. Using an immunoblot specific for cysticercosis, Ig isotypes to 7 cyst antigens were measured and quantified. Serum samples from the piglets contained detectable antibodies from Week 1 through Week 35 (27 weeks after weaning). The primary Ig isotype present in both sows and piglets was IgG. Antibodies did not appear in piglet serum samples until after suckling, demonstrating that anti-cysticercal antibodies are transferred solely via colostrum. Our data have shown that maternally transferred antibodies to cyst antigens may persist through much of a pig's life. Therefore, the presence of passively transferred antibodies must be considered in studies that examine the prevalence of cysticercosis in pigs. Furthermore, when designing control strategies for cysticercosis, careful evaluation and selection of sentinel pigs becomes a crucial component of sentinel selection.