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Abstract

Objective—To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis.

Animals—Two 3-year-old Argentine boa constrictors.

Procedure—Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 µg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity.

Results—A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas.

Conclusions and Clinical Relevance—Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens. (Am J Vet Res 2003;64:388–395)

Full access
in American Journal of Veterinary Research

Abstract

Objectives

To produce monoclonal antibodies (MAB) with specificity for the heavy chain of macaw IgG; to incorporate these MAB into an ELISA to measure IgG responses of macaws inoculated with bovine serum albumin (BSA); and to evaluate the antigenicity of BSA in Blue and Gold Macaws.

Animals

Four 1-year-old Blue and Gold Macaws, 2 males and 2 females.

Procedure

1 male and 1 female 1 were randomly assigned to each of 2 study groups. Group-1 birds were inoculated with 200 μg of BSA on days 0, 14, 28, and 42. Group-2 birds were inoculated with 200 μg of BSA on days 0 and 28. Blood was collected weekly for measurement of anti-BSA titer. Hybridomas were prepared from mice immunized with Scarlet Macaw (Ara macao) IgG purified by salt precipitation and gel chromatography. Specificity for IgG of Scarlet Macaw and other macaw species was confirmed by ELISA and western blot analysis. Hybridoma HL544 was cloned and the antibody purified. Following biotinylation, MAB HL544 was incorporated into an ELISA that measured IgG responses of macaws inoculated with BSA.

Results

Adult Blue and Gold Macaws developed strong primary and secondary anti-BSA antibody titers 14 days after inoculation with 200 μg of BSA. An inoculation interval of 28 days resulted in stronger secondary responses than an interval of only 14 days.

Conclusions

MAB specific for macaw immunoglobulins can be used in ELISA to evaluate the humoral immune responses of macaws. 1-year-old Blue and Gold Macaws developed strong anti-BSA titer when inoculated with 200 μg of BSA. An inoculation interval of 28 days resulted in stronger secondary responses than did an interval of only 14 days.

Clinical Relevance

These MAB, the first reported to have specificity for a psittacine antibody class, will be useful in the evaluation of psittacine antibody responses and in the development of psittacine diagnostic assays. (Am J Vet Res 1996; 57:1157-1161)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate Mycoplasma agassizii-specific maternal antibodies in desert tortoise (Gopherus agassizii) hatchlings.

Sample Population

Plasma from 43 captive-reared desert tortoise hatchlings.

Procedure

ELISA for M agassizii-specific antibodies was performed. Four hatchlings from 4 clutches of 3 M agassizii-seropositive females with chronic upper respiratory tract disease (URTD) were tested on the day of hatching (set 1), and 20 hatchlings from 4 clutches of 4 M agassizii-seropositive females with URTD and 19 hatchlings from 4 M agassizii-seronegative healthy females were tested at 4, 8, 12, and 29 months old (set 2). Immunoblot analysis was performed to determine immunoglobulin classes in yolk and plasma of hatchlings. To determine infection status of hatchlings, yolk, egg shell membranes (set 1), and nasal lavage fluid (sets 1 and 2) were examined for M agassizii by use of polymerase chain reaction.

Results

Yolk and hatchling plasma had significantly lower amounts of specific antibodies than did plasma from adult females. The IgG and IgM antibodies were transferred, but M agassizii-specific antibodies were of the IgG class. Hatchlings were not infected with mycoplasmas. Offspring of sick females had significantly higher specific antibody titers than did offspring of healthy females. Titers were still significantly different in 1-year-old hatchlings.

Conclusions

Desert tortoise females transfer specific IgG and IgM antibodies to their offspring that are still detectable after 1 year.

Clinical Relevance

Infection with M agassizii may be misdiagnosed in hatchlings with persistent maternal antibodies. Passively acquired antibodies may have a role in pathogenesis of mycoplasma-induced respiratory tract disease and other diseases. (Am J Vet Res 1999;60:826–831)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To document the maternal transfer of IgG antibodies from Blue and Gold Macaw hens to chicks via the egg; to measure serum IgG half-life in macaw chicks; and to measure the ability of 2- to 10-week-old macaw chicks to generate primary and secondary IgG responses.

Procedure

4 adult Blue and Gold Macaw hens were inoculated with 200 μg of bovine serum albumin (BSA) every 21 days throughout the breeding season. Eggs laid by these hens were incubator hatched to eliminate the possibility of antibody transfer through crop secretions during feeding. Anti-BSA titer was measured just prior to each inoculation in hens and in chicks from 14 to 42 days of age. 1 chick from each of 5 macaw clutches hatched to nonimmunized hens was assigned to 1 of 2 experimental groups. Group-1 chicks were inoculated with 200 μg of BSA at 2 and 6 weeks of age. Group-2 chicks were inoculated with 200 μg of BSA at 6 and 10 weeks of age. Anti-BSA titer was measured weekly for 8 weeks after primary inoculation.

Blood samples were centrifuged, and serum was harvested and frozen at −85 C until analyzed. Anti-BSA IgG titers were measured by ELISA. In the maternal transfer experiment, an exponential decay model was used to calculate the half-life of BSA antibodies in chicks. In the BSA antibody response experiment, comparison of primary and secondary anti-BSA responses of 2- and 6- week-old chicks was performed, using a two-way repeated measures ANOVA, with significance set at P < 0.05.

Results

Hens maintained high anti-BSA titer throughout the breeding season. Maternal transfer of anti-BSA IgG antibodies was documented in all 7 chicks. Anti-BSA titer in chicks decreased in exponential fashion with an average serum IgG half-life of 3.85 days. By 42 days of age, antibodies to BSA were virtually undetectable in all chicks. The primary antibody response of 6-week-old chicks was significantly higher than that of 2-week-old chicks (P = 0.016). No significant difference was observed in the magnitude of the secondary antibody responses between these age groups. Peak anti-BSA IgG antibody responses were reached by 14 days after primary and secondary immunization. Chicks of both age groups generated lower anti-BSA IgG titer than did adult Blue and Gold Macaws.

Conclusions

Blue and Gold Macaw hens transfer IgG antibodies to their chicks through the egg. The half-life of IgG in newly hatched chicks is approximately 3.85 days. 6-week-old chicks develop higher anti-BSA titers than do 2-week-old chicks, but significantly lower titers than do adult macaws.

Clinical Relevance

Information on the nondomestic avian immune system will be useful in the development of vaccination and other preventive health programs for psittacine birds. (Am J Vet Res 1996;57:1162-1167)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs.

Animals—111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved.

Procedures—58 dogs received an initial series of 4 injections of huTyr vaccine (102 μg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death.

Results—Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising.

Conclusions and Clinical Relevance—Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM.

Impact for Human Medicine—Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Full access
in American Journal of Veterinary Research