Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Effect of reducing or eliminating BCG load on cell-mediated immunity.

Oral delivery of lipid-encapsulated BCG represents an effective method for vaccination against tuberculosis (Tb). This method establishes live, replicating BCG in the lymphatic tissues of the alimentary tract, and promotes systemic-level cell-mediated immunity (CMI) and consequent protection against virulent mycobacterial challenge. Here, we investigated the effects of reducing or eliminating the BCG load on CMI responses in mice. Mice receiving a standard immunising dose of approximately 10(7) BCG (range, 1-5 x 10(7)) developed mycobacterial antigen-specific lymphocyte transformation (LT) responses, as well as interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) secretion, at 8 and 18 weeks post-oral vaccination. These responses were concurrent with establishment of viable, replicating BCG in the alimentary tract lymphatics in over 90% of cases. Reducing the immunising dose by 10-fold reduced the magnitude of CMI, concurrent with abridged establishment of BCG in the lymphatics; reducing the dose 100-fold ablated BCG establishment, and diminished the production of IFN-gamma by antigen-stimulated lymphocytes of these mice. In mice immunised using the standard dose, replicating BCG were eliminated from the alimentary tract lymphatics using selective antibiotics. Interestingly, while lymphocyte transformation and interleukin-2 responses remained largely unaltered in these mice, levels of IFN-gamma produced by antigen-stimulated lymphocytes were shown to be reduced significantly. This study identifies a dosage threshold for effective oral vaccination using lipid-encapsulated BCG, and furthermore highlights the requirement of on-going intra-lymphatic BCG replication for the maintenance of strong IFN-gamma production, above other indicator CMI responses.

Oral delivery of lipid-encapsulated Mycobacterium bovis BCG extends survival of the bacillus in vivo and induces a long-term protective immune response against tuberculosis.

The success of oral-route vaccination using Mycobacterium bovis bacille Calmette-Guérin (BCG) relies on delivery of live, actively metabolising bacilli to confer protection. Here, we describe that lipid-microencapsulation can extend the in vivo survival of bacilli when fed to mice, and can induce a long-lasting protective immune response. Feeding mice with lipid-encapsulated BCG (L-BCG) resulted in greater recovery of viable BCG bacilli from the mesenteric lymph nodes (MLN) compared to mice fed non-encapsulated BCG. A time-course study indicated persistence of viable BCG bacilli in MLN up to 30 weeks post-vaccination, similar to the duration of viable BCG recovery from the spleen following subcutaneous vaccination. The persistence of viable bacilli in the MLN of L-BCG mice invoked long-lasting systemic cell-mediated immune reactivity, with responses similar to those observed in subcutaneously-vaccinated mice. Further, L-BCG-vaccinated mice showed a high degree of protection against aerogenic challenge with virulent M. bovis at 30 weeks post-vaccination, with significant reductions in lung and spleen pathogen burdens. This study identifies that lipid-encapsulation of live BCG bacilli can facilitate increased in vivo survival and immunogenicity of the vaccine in orally-vaccinated mice, and highlights protection via this route for up to 7 months post-immunisation.

Transmission of Mycobacterium bovis from experimentally infected ferrets to non-infected ferrets (Mustela furo).

AIMS: To demonstrate the transmission of Mycobacterium bovis infection from experimentally infected ferrets (Mustela furo) to non-infected ferrets in a laboratory setting, using three different isotypes of M. bovis, and to observe ferret behaviour that might be implicated in disease transmission. METHODS: Three female ferrets, each experimentally infected with a unique strain of M. bovis, were housed together with six female and two male non-infected ferrets in an isolation facility. Transmission of infection was monitored clinically, serologically (using an ELISA test), bacteriologically, histologically, and by isotype analysis of M. bovis isolates using spoligotyping to determine whether or not transmission of each strain occurred. Ferret behaviour was observed using a time-lapse video recorder. RESULTS: Transmission of M. bovis infection was confirmed in two male and four female ferrets. Isotype analysis showed that of the experimentally infected females, one did not infect any other ferret, another transmitted M. bovis to one ferret before it died prematurely 49 days post-infection, and the third, which was cannibalised, appears to have transmitted M. bovis to both males and three females. However, two of these latter three females had died before the event of cannibalism took place. One female was infected with two strains. Several behavioural interactions were observed that could have resulted in M. bovis transmission, including den sharing, sniffing of orifices and faeces, cannibalism and aggressive breeding behaviour. CONCLUSIONS: Horizontal transmission of M. bovis infection was demonstrated in ferrets under experimental housing conditions. Routes of transmission may involve cannibalism and factors such as den sharing, playing, fighting, mating, and sniffing of faeces.

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel.

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.

Localization and genomic organization of sheep antimicrobial peptide genes.

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.

Physical mapping confirms that sheep chromosome 10 has extensive conserved synteny with cattle chromosome 12 and human chromosome 13.

The following loci, on human chromosome 13, have been newly assigned to sheep chromosome 10 using chromosomally characterized sheep-hamster cell hybrids: gap junction protein, beta 2, 26 kDa (connexin 26) (GJB2); gap junction protein, alpha 3, 46 kDa (connexin 46) (GJA3), and esterase D/formylglutathione hydrolase (ESD). This assignment of ESD is consistent with comparative mapping evidence, but not with an earlier report of it on sheep chromosome 3p26-p24. Cell hybrid analysis confirmed the location of another chromosome 13 locus, retinoblastoma 1 (including osteosarcoma) (RB1), and the anonymous ovine genomic sequence RP11 on sheep chromosome 10. Isotopic in situ hybridization was used to regionally localize RP11 on to sheep 10q15-q22. The location of microsatellites AGLA226, OarDB3, OarHH41, OarVH58, and TGLA441, previously assigned to sheep chromosome 10 by linkage analysis, was confirmed by polymerase chain reaction using the cell hybrid panel. These mapping data provide further evidence that sheep chromosome 10 is the equivalent of cattle chromosome 12, and that these chromosomes show extensive conserved synteny with human chromosome 13.

Vasodilation and reduction in forearm vascular resistance after acute administration of dilevalol.

Dilevalol is a long-acting antihypertensive drug that has been demonstrated in animals to combine specific beta 2-agonist-mediated vasodilation with nonspecific beta blockade. To document vasodilation in humans, single oral doses of dilevalol, 200 mg, and placebo were randomly administered to 12 untreated hypertensive patients. Dilevalol produced significant reductions (p less than or equal to 0.01) in diastolic blood pressure throughout a 24-hour period relative to placebo, without changing heart rate. Forearm blood flow, measured hourly over the initial 4 hours after dosing, demonstrated a shift to a more vasodilated state after dilevalol administration, with significant increases in minimal forearm blood flow (4.0 vs 2.9 ml/dl tissue/min, dilevalol vs placebo, respectively; p = 0.05) and in mean average forearm blood flow (5.3 vs 4.0 ml/dl tissue/min, dilevalol vs placebo; p = 0.04). Similarly, dilevalol produced a decrease in mean forearm vascular resistance (26.5 vs 34.6 mm Hg/ml/dl tissue/min, dilevalol vs placebo; p = 0.02). In the absence of a change in heart rate, the acute hypotensive response to dilevalol in these patients appears to have resulted primarily from vasodilation and reduced vascular resistance.