Report on the international workshop on alternative methods for Leptospira vaccine potency testing: state of the science and the way forward.

Routine potency testing of Leptospira vaccines is mostly conducted using a vaccination-challenge test that involves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their international partners organized a workshop to review the state of the science of alternative methods that might replace, reduce, and refine the use of animals for veterinary Leptospira vaccine potency testing and to identify ways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate or continue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacements for potency testing of four common Leptospira serogroups. Participants discussed the potential for eliminating the back-titration procedure in the hamster challenge assay, which could reduce animal use by 50% for each individual potency test. Further animal reduction may also be possible by using cryopreserved Leptospira stock to replace continual passaging through hamsters. Serology assays were identified as a way to further reduce and refine animal use but should be considered only after attempting in vitro assays. Workshop participants encouraged consideration of analgesics and use of earlier humane endpoints when the hamster vaccination-challenge potency assay is used. International harmonization of alternative potency methods was recommended to avoid duplicative potency testing to meet regionally different requirements.

Histamine production by Haemophilus somnus.

Ten Haemophilus somnus isolates were grown on blood agar plates under a 5% CO2 atmosphere for 48 h. Harvested whole cells were washed and evaluated for the presence of histamine by ELISA. All H. somnus isolates had cell-associated histamine concentrations of between 18.5 and 200 ng/ml. In a separate study, the ability of H. somnus to secrete histamine into BHI growth medium was evaluated using H. somnus strains 8025 and 156A as well as a recent 156A respiratory isolate. Each strain or isolate was grown under various concentrations of CO2 to approximate the CO2 concentration in the bronchi. The histamine content of washed whole cells and medium supernatant were determined at various stages of incubation. Highest histamine concentrations were detected in the recent respiratory isolate; whole cells (225 ng/ml) after 120 h incubation in 15% CO2 and supernatant (1721 ng/ml) after incubation for 41 h in 25% CO2. This study indicates that different H. somnus isolates can produce and secrete histamine which may be enhanced by CO2 concentrations which approximate those in the bronchial tree. Results of this study may partially explain some of the post-vaccination reactions occasionally observed with H. somnus bacterins. Additional studies are needed to determine the actual role of H. somnus-derived histamine in the pathogenesis of bovine respiratory disease and airway hyperresponsiveness.