- Email: [email protected]
University of California, Davis
UNIVERSITY OF CALIFORNIA, DAVIS
MEASUREMENT OF IMMUNE REGULATURY MOLECULES (CYTOKINES) IN EXPERIMENTALLY INDUCED EQUINE SARCOIDS Johanna L. Watson, Elizabeth A. Carr, and David W. Horohov In a previous Center for Equine Health study, tissue samples were collected from experimentally induced tumors, immediately frozen, and stored at ⫺70°C. Researchers harvested genetic material from each sample and determined the quantity of messenger RNA for specific equine hormones by using quantitative reverse transciptase polymerase chain reaction (RT- PCR). A mathematical model was used to quantify the messenger RNA expressed. Researchers evaluated the types of cytokines expressed as well as the relative expression over time.
TREATMENT OF EQUINE MELANOMAS WITH A TUMORCELL VACCINE USING MODIFIED TUMOR CELLS FROM THE AFFECTED HORSE Alain P. Theon Researchers surgically removed melanoma cells from affected horses and then processed the cells. The tumor cells were infected with certain viruses, which adds to the immunity production of tumor antigens. This process converts nonimmunogenic or weakly immunogenic tumor antigens to an immunogenic tuCopyright 2002, Elsevier Science (USA). All rights reserved. 0737-0806/02/2211-0009$35.00/0 doi:10.1053/jevs.2002.37430
510
mor by placing weak tumor antigens next to strong viral antigens capable of inducing a strong immune response. Researchers further augmented tumor cell immunogenicity through the use of a nonspecific immunostimulant, cyclophosphamide, a potent immunosuppressive agent that can, in some instances, augment the immune response. The cyclophosphamide was given before antigen presentation (ie, vaccine administration) to augment the immune response through reduction in T-suppressor cell activity. Researchers have shown that a reduction in T-suppressor cell activity may be an essential component of successful immunotherapy.
STRATEGIES TO PROMOTE MORE EFFECTIVE PRIMARY IMMUNIZATION OF FOALS AGAINST TETANUS, VIRAL ENCEPHALOMYELITIS, INFLUENZA, EHV-1, AND EHV-4 INFECTION W. David Wilson and Judy E. Mihalyi Researchers conducted this study over a 2-year period and included 31 foals. Their dams received booster doses of inactivated vaccines containing tetanus, WEE, EEE, influenza EHV-l, and EHV-4 antigens 4 to 6 weeks before foaling. Researchers collected blood samples from foals at 2 to 7 days of age and then at monthly intervals thereafter while foals remained in the research herd (usually until they were 10 to 15 months of age). Researchers confirmed the adequacy of passive transfer using a CITE® test for immunoglobin G on samples collected during the first week of life. All serum samples were stored frozen in
quadruplicate until assayed for titers of specific antibody against the antigens of interest using the following tests: (1) tetanus—isotype-specific ELISA, (2) influenza— hemagglutination inhibition and isotype-specific enzyme-linked immunosorbent assay, (3) WEE, EEE, EHV-1, and EHV-4 —serum neutralization. Researchers clinically monitored the foals throughout the study. The foals received thorough examinations by veterinarians and appropriate laboratory diagnostic tests to identify the etiology of any illness that might confound results of serologic testing. Foals were assigned to 1 of 2 groups. Group 1 foals were first vaccinated at 12 weeks of age and received additional doses of vaccine at 16 and 26 weeks of age. Group 2 foals were vaccinated at 26, 30, and 40 weeks of age. Antibody titers were determined for each antigen on each sampling day. For each group, the total number and percentage of foals showing seroconversion (an increase in antibody titer) was determined for each sampling time to further define responses to individual antigens.
EFFECT OF AGE AND MATERNAL ANTIBODY STATUS ON THE SEROLOLOGICAL RESPONSES OF THOROUGHBRED FOALS TO VACCINATION WITH AN INACTIVATED EHV-1/4 VACCINE W. David Wilson, Peter D. Rossdale, Jules Minke, and Judy E. Mihalyi Fifty-three healthy foals on 2 branches of a commercial thoroughbred farm were randomly assigned to 2 groups, G3 and G5, and were vaccinated with a commercially available inacti-
JOURNAL OF EQUINE VETERINARY SCIENCE
vated carbomer adjuvanted EHV-1/4 vaccine. Foals were pastured in distinct subgroups of 6 to 10 foals. Each subgroup included both G3 and G5 foals, both before and after weaning. The foals in G3 were first vaccinated at 12 (⫾ 1) weeks of age and the foals in G5 were first vaccinated at 20 (⫾ 1) weeks of age. Vaccination was repeated 4 and 8 weeks later. Researchers collected blood samples each time the vaccine was given and 3 weeks after the fourth dose. Researchers determined serum virus neutralization and complement fixation titers against both EHV-1 and EHV-4, and they computed geometric mean titers for each group at each sampling time. Researchers statistically compared mean titers at different sampling times within each group and between the groups.
IMPLEMENTATION OF ALLERGY TESTING IN HORSES USING BLOOD SAMPLES TO DETERMINE THE CAUSE OF THE ALLERGY Laurel I. Gershwin, Johanna L. Watson, H. David Pettigrew, and Warren V. Kalina In a previous Center for Equine Health study, researchers used molecular biology techniques to develop special antibody reagents to detect immunoglobin E. An assay has been developed by using these antibody reagents to detect immunoglobin E in horse blood that binds to the substance causing the allergy. Researchers have developed a blood bank that includes medical history about the allergic signs. Blood samples were evaluated for reactivity with many different allergens. Assay results were correlated with clinical signs to determine the allergen responsible for the reaction. To address allergic reactivity to vaccines, researchers are developing assays using equine vaccine components to test blood samples from horses with doc-
Volume 22, Number 11, 2002
umented histories of allergic reactivity after vaccination.
EQUINE INTERLEUKIN-4 RECEPTOR ALPHA CHAIN Johanna L. Watson Researchers have identified the gene for the interleukin-4 receptor in the mouse, rat, and human. Using these gene sequences, researchers designed primer sequences to detect a part of the equine sequence in the polymerase chain reaction. Researchers used this piece of the equine sequence to select the full-length gene from a library of genes produced in a previous study. Researchers completely sequenced this gene and target areas for likely mutation were identified by comparing them with the human gene sequence.
FIELD INVESTIGATION OF A CLUSTER OF TYZZER’S DISEASE ON A SOUTHERN CALIFORNIA THOROUGHBRED FARM David W. Hird, Deryck H. Read, Richard L.Walker, and Geoffrey T. Fosgate Researchers analyzed data from 1998-2000 farm records on mares and foaling records from a southern California farm where 9 cases of Tyzzer’s disease occurred in 3 years. Researchers also analyzed data from a detailed investigation of the farm and associated management practices. They identified 3 risk factors.
EVALUATION OF ANTIPROTOZOAL THERAPIES USED TO TREAT EQUINE PROTOZOAL MYELOENCEPHALITIS Patricia A. Conrad and Antoinette E. Marsh Researchers evaluated the ability of laboratory parasites to grow in cells, with or without drugs added to the nutrient
media. Parasite growth was monitored using several methods with a special emphasis on quantitative analysis of parasite numbers. Researchers tested drug treatments shown to be efficacious under these conditions in experimentally infected Sarcocystis neurona mice to determine if the same effects were seen in an animal model.
DEVELOPMENT OF A REVERSETRANSCRIPTION POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF NORTH AMERICAN AND EUROPEAN STRAINS OF EQUINE ARTERITIS VIRUS IN SEMEN AND OTHER CLINICAL SAMPLES N. James MacLachlan, Udeni B.R. Balasuriya, Jodi F. Hedges, Peter J. Timoney, William H. McCollum, W. David Wilson, and Irwin K.M. Liu Researchers evaluated 49 samples that contained Equine Arteritis Virus (EAV). These included 23 tissue culture fluids containing North American and European strains of EAV, 18 different samples of seminal plasma from 12 stallions, and 6 tissue samples collected from foals affected during a recent outbreak of EVA. Researchers also evaluated tissue culture fluids containing modified live virus vaccine and the recombinant virus derived from an infectious complementary DNA clone of EAV. Viral nucleic acids from each sample were extracted according to a standard protocol. Twenty different primer pairs specific for regions of the leader sequence and ORF’s 1b, 3, 4, 5, 6, and 7 of EAV were used for reversetranscription polymerase chain reaction amplification according to a standard protocol.
511
MEASUREMENT OF IMMUNE REGULATURY MOLECULES (CYTOKINES) IN EXPERIMENTALLY INDUCED EQUINE SARCOIDS Johanna L. Watson, Elizabeth A. Carr, and David W. Horohov In a previous Center for Equine Health study, tissue samples were collected from experimentally induced tumors, immediately frozen, and stored at ⫺70°C. Researchers harvested genetic material from each sample and determined the quantity of messenger RNA for specific equine hormones by using quantitative reverse transciptase polymerase chain reaction (RT- PCR). A mathematical model was used to quantify the messenger RNA expressed. Researchers evaluated the types of cytokines expressed as well as the relative expression over time.
TREATMENT OF EQUINE MELANOMAS WITH A TUMORCELL VACCINE USING MODIFIED TUMOR CELLS FROM THE AFFECTED HORSE Alain P. Theon Researchers surgically removed melanoma cells from affected horses and then processed the cells. The tumor cells were infected with certain viruses, which adds to the immunity production of tumor antigens. This process converts nonimmunogenic or weakly immunogenic tumor antigens to an immunogenic tuCopyright 2002, Elsevier Science (USA). All rights reserved. 0737-0806/02/2211-0009$35.00/0 doi:10.1053/jevs.2002.37430
510
mor by placing weak tumor antigens next to strong viral antigens capable of inducing a strong immune response. Researchers further augmented tumor cell immunogenicity through the use of a nonspecific immunostimulant, cyclophosphamide, a potent immunosuppressive agent that can, in some instances, augment the immune response. The cyclophosphamide was given before antigen presentation (ie, vaccine administration) to augment the immune response through reduction in T-suppressor cell activity. Researchers have shown that a reduction in T-suppressor cell activity may be an essential component of successful immunotherapy.
STRATEGIES TO PROMOTE MORE EFFECTIVE PRIMARY IMMUNIZATION OF FOALS AGAINST TETANUS, VIRAL ENCEPHALOMYELITIS, INFLUENZA, EHV-1, AND EHV-4 INFECTION W. David Wilson and Judy E. Mihalyi Researchers conducted this study over a 2-year period and included 31 foals. Their dams received booster doses of inactivated vaccines containing tetanus, WEE, EEE, influenza EHV-l, and EHV-4 antigens 4 to 6 weeks before foaling. Researchers collected blood samples from foals at 2 to 7 days of age and then at monthly intervals thereafter while foals remained in the research herd (usually until they were 10 to 15 months of age). Researchers confirmed the adequacy of passive transfer using a CITE® test for immunoglobin G on samples collected during the first week of life. All serum samples were stored frozen in
quadruplicate until assayed for titers of specific antibody against the antigens of interest using the following tests: (1) tetanus—isotype-specific ELISA, (2) influenza— hemagglutination inhibition and isotype-specific enzyme-linked immunosorbent assay, (3) WEE, EEE, EHV-1, and EHV-4 —serum neutralization. Researchers clinically monitored the foals throughout the study. The foals received thorough examinations by veterinarians and appropriate laboratory diagnostic tests to identify the etiology of any illness that might confound results of serologic testing. Foals were assigned to 1 of 2 groups. Group 1 foals were first vaccinated at 12 weeks of age and received additional doses of vaccine at 16 and 26 weeks of age. Group 2 foals were vaccinated at 26, 30, and 40 weeks of age. Antibody titers were determined for each antigen on each sampling day. For each group, the total number and percentage of foals showing seroconversion (an increase in antibody titer) was determined for each sampling time to further define responses to individual antigens.
EFFECT OF AGE AND MATERNAL ANTIBODY STATUS ON THE SEROLOLOGICAL RESPONSES OF THOROUGHBRED FOALS TO VACCINATION WITH AN INACTIVATED EHV-1/4 VACCINE W. David Wilson, Peter D. Rossdale, Jules Minke, and Judy E. Mihalyi Fifty-three healthy foals on 2 branches of a commercial thoroughbred farm were randomly assigned to 2 groups, G3 and G5, and were vaccinated with a commercially available inacti-
JOURNAL OF EQUINE VETERINARY SCIENCE
vated carbomer adjuvanted EHV-1/4 vaccine. Foals were pastured in distinct subgroups of 6 to 10 foals. Each subgroup included both G3 and G5 foals, both before and after weaning. The foals in G3 were first vaccinated at 12 (⫾ 1) weeks of age and the foals in G5 were first vaccinated at 20 (⫾ 1) weeks of age. Vaccination was repeated 4 and 8 weeks later. Researchers collected blood samples each time the vaccine was given and 3 weeks after the fourth dose. Researchers determined serum virus neutralization and complement fixation titers against both EHV-1 and EHV-4, and they computed geometric mean titers for each group at each sampling time. Researchers statistically compared mean titers at different sampling times within each group and between the groups.
IMPLEMENTATION OF ALLERGY TESTING IN HORSES USING BLOOD SAMPLES TO DETERMINE THE CAUSE OF THE ALLERGY Laurel I. Gershwin, Johanna L. Watson, H. David Pettigrew, and Warren V. Kalina In a previous Center for Equine Health study, researchers used molecular biology techniques to develop special antibody reagents to detect immunoglobin E. An assay has been developed by using these antibody reagents to detect immunoglobin E in horse blood that binds to the substance causing the allergy. Researchers have developed a blood bank that includes medical history about the allergic signs. Blood samples were evaluated for reactivity with many different allergens. Assay results were correlated with clinical signs to determine the allergen responsible for the reaction. To address allergic reactivity to vaccines, researchers are developing assays using equine vaccine components to test blood samples from horses with doc-
Volume 22, Number 11, 2002
umented histories of allergic reactivity after vaccination.
EQUINE INTERLEUKIN-4 RECEPTOR ALPHA CHAIN Johanna L. Watson Researchers have identified the gene for the interleukin-4 receptor in the mouse, rat, and human. Using these gene sequences, researchers designed primer sequences to detect a part of the equine sequence in the polymerase chain reaction. Researchers used this piece of the equine sequence to select the full-length gene from a library of genes produced in a previous study. Researchers completely sequenced this gene and target areas for likely mutation were identified by comparing them with the human gene sequence.
FIELD INVESTIGATION OF A CLUSTER OF TYZZER’S DISEASE ON A SOUTHERN CALIFORNIA THOROUGHBRED FARM David W. Hird, Deryck H. Read, Richard L.Walker, and Geoffrey T. Fosgate Researchers analyzed data from 1998-2000 farm records on mares and foaling records from a southern California farm where 9 cases of Tyzzer’s disease occurred in 3 years. Researchers also analyzed data from a detailed investigation of the farm and associated management practices. They identified 3 risk factors.
EVALUATION OF ANTIPROTOZOAL THERAPIES USED TO TREAT EQUINE PROTOZOAL MYELOENCEPHALITIS Patricia A. Conrad and Antoinette E. Marsh Researchers evaluated the ability of laboratory parasites to grow in cells, with or without drugs added to the nutrient
media. Parasite growth was monitored using several methods with a special emphasis on quantitative analysis of parasite numbers. Researchers tested drug treatments shown to be efficacious under these conditions in experimentally infected Sarcocystis neurona mice to determine if the same effects were seen in an animal model.
DEVELOPMENT OF A REVERSETRANSCRIPTION POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF NORTH AMERICAN AND EUROPEAN STRAINS OF EQUINE ARTERITIS VIRUS IN SEMEN AND OTHER CLINICAL SAMPLES N. James MacLachlan, Udeni B.R. Balasuriya, Jodi F. Hedges, Peter J. Timoney, William H. McCollum, W. David Wilson, and Irwin K.M. Liu Researchers evaluated 49 samples that contained Equine Arteritis Virus (EAV). These included 23 tissue culture fluids containing North American and European strains of EAV, 18 different samples of seminal plasma from 12 stallions, and 6 tissue samples collected from foals affected during a recent outbreak of EVA. Researchers also evaluated tissue culture fluids containing modified live virus vaccine and the recombinant virus derived from an infectious complementary DNA clone of EAV. Viral nucleic acids from each sample were extracted according to a standard protocol. Twenty different primer pairs specific for regions of the leader sequence and ORF’s 1b, 3, 4, 5, 6, and 7 of EAV were used for reversetranscription polymerase chain reaction amplification according to a standard protocol.
511