Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.
Agglutination Tests/methods/*veterinary ; Animals ; *Animals, Newborn ; Antibodies, Monoclonal/*immunology ; Antigens, Surface/immunology/isolation & purification ; Bacterial Toxins/immunology/isolation & purification ; Cattle ; Cattle Diseases/*immunology/*microbiology ; Chromatography, Gel/veterinary ; Chromatography, Ion Exchange/veterinary ; Chromatography, Liquid/veterinary ; Diarrhea/immunology/*veterinary ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Escherichia coli/*immunology ; Escherichia coli Infections/immunology/*veterinary ; Immunoblotting/veterinary ; Staphylococcus aureus

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.
Animals ; Antigens, Helminth/analysis/*chemistry/immunology ; Clonorchiasis/*diagnosis ; Clonorchis sinensis/*immunology ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel/veterinary ; Humans ; Immunoblotting/veterinary ; Molecular Weight ; Opisthorchiasis/*diagnosis ; Opisthorchis/*immunology

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.
Animals ; Antibodies, Monoclonal/*biosynthesis/immunology ; Chromatography, Affinity/veterinary ; Electrophoresis, Gel, Two-Dimensional/veterinary ; Immunoblotting/veterinary ; Immunoglobulin Heavy Chains/immunology ; Immunoglobulin Light Chains/immunology ; Immunoglobulins/blood/*immunology ; Perciformes/blood/*immunology

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 microg/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.
Animals ; Antigens/immunology/isolation & purification ; Argasidae/immunology ; Chromatography, Affinity/veterinary ; Electrophoresis, Polyacrylamide Gel/veterinary ; Female ; Glycoproteins/*immunology/isolation & purification ; Immunoblotting/veterinary ; Injections, Intramuscular/veterinary ; Ixodidae/growth & development/*immunology ; Life Cycle Stages ; Male ; Rabbits/*immunology/parasitology ; Reproduction ; Species Specificity ; Tick Infestations/immunology/prevention & control/*veterinary

The present study was attempted to compare the Neospora caninum (N. caninum) antigenic bands recognized by different bovine immunoglobulin classes. A total 10, 5, 2, and 6 antigenic bands were exhibited on immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. A 46 kDa band was probed as a common antigenic band except IgA; 69 kDa band was bovine IgM and IgE; 33, 37, 55, and 79 kDa bands were bovine IgM and IgG; 72 kDa band was found IgM and IgA profiles. Based on the analysis, it appeared that different immunoglobulin classes recognizing different antigenic molecules were cooperating to cope with neosporosis.
Animals ; Antigens, Protozoan/immunology ; Cattle ; Cattle Diseases/diagnosis/immunology/*parasitology ; Coccidiosis/diagnosis/immunology/parasitology/*veterinary ; Female ; Immunoblotting ; Immunoglobulin Idiotypes/diagnostic use/*immunology ; Neospora/*immunology

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.
Animals ; Cell Line ; *Chickens ; DNA, Viral/chemistry/genetics ; Herpesvirus 3, Gallid/*genetics/*metabolism/pathogenicity ; Immunoblotting/veterinary ; Marek Disease/*virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Open Reading Frames/*genetics ; Polymerase Chain Reaction/veterinary ; Recombinant Proteins/genetics ; Specific Pathogen-Free Organisms ; Transfection/veterinary ; Viral Proteins/genetics/*metabolism

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.
Amino Acid Sequence ; Animals ; Cattle ; Cattle Diseases/immunology/*parasitology/prevention & control ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Glycoproteins/immunology/*isolation & purification ; Immunoblotting ; Isoelectric Focusing ; Ixodidae/chemistry/*immunology ; Male ; Molecular Weight ; Rabbits ; Sequence Analysis, Protein ; Tick Infestations/immunology/parasitology/prevention & control/*veterinary