Oral vaccination of brushtail possums (Trichosurus vulpecula) with BCG: immune responses, persistence of BCG in lymphoid organs and excretion in faeces

Abstract

AIMS: To determine immune responses, and the localisation and persistence of Mycobacterium bovis bacille Calmette-Guérin (BCG) in gut-associated lymphoid tissues (GALT) and other organs in possums vaccinated orally with lipid-formulated BCG vaccine. To determine the duration of excretion and longevity of survival of BCG in the faeces of vaccinated animals.

METHODS: Possums (n=28) were vaccinated with lipid-formulated BCG (1 x 10 ⁸ colony forming units (cfu) of formulated BCG) by the oral route. Control possums (n=17) were fed oral bait pellets containing formulation medium only. Possums were sacrificed at 3 days and at 1, 3, 6 and 8 weeks after vaccination or ingestion of bait. Proliferation responses to bovine purified protein derivative (PPD) were measured in lymphocytes from blood and mesenteric lymph nodes (MLN) and samples of lung, spleen, liver, MLN and Peyer's patches (PP) were cultured for the presence of BCG. The number of BCG organisms excreted in faeces and the duration of excretion were determined in eight vaccinated possums and eight control possums over a 3-week period. In a separate experiment, a further six possums were vaccinated with oral BCG vaccine (5–10 x 10 ⁸ cfu BCG/possum) and their faeces collected over 48–72 h, for culture of BCG. The longevity of survival of BCG in these faeces was determined by storing faecal samples (n=12) under three different conditions: in an incubator (22.5°C), and conditions which simulated the forest floor and open pasture. A proportion (1–2 g) of these faecal samples was collected after storage for 1, 3, 5, 8 or 20 weeks, and cultured for BCG.

RESULTS: Possums vaccinated orally with BCG vaccine showed strong proliferation responses to bovine PPD in peripheral blood lymphocytes at 6–8 weeks post-vaccination (p.v.). Positive lymphocyte proliferation assay (LPA) responses to bovine PPD were first evident in MLN at 3 weeks p.v. BCG was cultured from MLN and PP in a proportion of animals at 3–8 weeks p.v. BCG was not cultured from sections of spleen, lung or liver at any time p.v. BCG was recovered in low to moderate numbers from the faeces of vaccinated possums for up to 7 days, and maximal numbers were cultured in faeces collected 48–72 h p.v. After storage for 1 week, BCG was cultured from all faecal samples placed in the incubator and from a proportion of faeces exposed to conditions similar to those on the forest floor and pasture. With the exception of one faecal sample stored under forest floor conditions which was culture-positive for BCG at 3 and 5 weeks, BCG was not cultured from any other faecal sample stored for more than 1 week.

CONCLUSIONS: Ingestion of oral BCG vaccine by possums was associated with the development of strong cell-mediated immunity in both blood and MLN. Following oral vaccination with BCG, the organisms were localised and persisted in GALT but did not spread to the spleen, liver or lungs. BCG was shed in low to moderate numbers in the faeces for up to 7 days p.v. The viability of BCG excreted in faeces decreased rapidly, particularly when faeces were exposed to an open pasture environment. Oral vaccination of possums with formulated BCG is unlikely to result in undue contamination of the environment with BCG.

KEY WORDS:

• Possum
• Trichosurus vulpecula
• BCG
• vaccine
• immune responses
• oral vaccination

Acknowledgements

We thank Keith Hamel, Natalie Parlane, Jessica Koach and Gary Yates for expert technical assistance; Lilian Morrison for statistical analyses; and Michel Denis for helpful advice. This work was supported financially by the Animal Health Board (New Zealand) and the New Zealand Ministry of Agriculture and Forestry (Policy Management).