title: Oral vaccination of brushtail possums (Tichosurus vulpecula) with BCG: immune responses, persistence of BCG in lymphoid organs and excretion in faeces.
abstract: OBJECTIVE
  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 degrees 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.
authors.affiliation: AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand. neil.wedlock@agresearch.co.nz
authors.forename: D N
authors.initials: DN
authors.lastname: Wedlock
authors.forename: F E
authors.initials: FE
authors.lastname: Aldwell
authors.forename: D
authors.initials: D
authors.lastname: Keen
authors.forename: M A
authors.initials: MA
authors.lastname: Skinner
authors.forename: B M
authors.initials: BM
authors.lastname: Buddle
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000284:Administration, Oral; D000818:Animals; D001500:BCG Vaccine / Q000008:administration & dosage* / Q000276:immunology; D015169:Colony Count, Microbial / Q000662:veterinary; D004197:Disease Reservoirs / Q000662:veterinary; D005243:Feces / Q000382:microbiology*; D005260:Female; D008198:Lymph Nodes / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D009163:Mycobacterium bovis / Q000276:immunology*; D013997:Time Factors; D048330:Trichosurus / Q000097:blood / Q000276:immunology* / Q000382:microbiology; D014376:Tuberculosis / Q000276:immunology / Q000517:prevention & control / Q000635:transmission / Q000662:veterinary*
chemical_list: D000276:Adjuvants, Immunologic; D001500:BCG Vaccine