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SUMMARYIn New Zealand, bovine tuberculosis (bTB) is present in domestic cattle and deer herds primarily as the result of on-going disease transmission from the primary wildlife host, the brushtail possum (Trichosurus vulpecula). However, bTB is also present in other introduced free-ranging mammalian species. Between 1996 and 2007, we conducted a series of studies to determine whether poison control of possum populations would have any effect on the prevalence of Mycobacterium bovis infection in sympatric feral pigs (Sus scrofa). We compared trends in the prevalence of bTB infection in feral pigs in six study areas: possum numbers were reduced in three areas, but not in the other three, effectively providing a thrice-replicated before-after-control-intervention design. Before possum control, the overall prevalence of culture-confirmed M. bovis infection in feral pigs was 16·7-94·4%, depending on area. Infection prevalence varied little between genders but did vary with age, increasing during the first 2-3 years of life but then declining in older pigs. In the areas in which possum control was applied, M. bovis prevalence in feral pigs fell to near zero within 2-3 years, provided control was applied successfully at the whole-landscape scale. In contrast, prevalence changed much less or not at all in the areas with no possum control. We conclude that feral pigs in New Zealand acquire M. bovis infection mainly by inter-species transmission from possums, but then rarely pass the disease on to other pigs and are end hosts. This is in contrast to the purported role of pigs as bTB maintenance hosts in other countries, and we suggest the difference in host status may reflect differences in the relative importance of the oral route of infection in different environments. Despite harbouring M. bovis infection for a number of years, pigs in New Zealand do not sustain bTB independently, but are good sentinels for disease prevalence in possum populations. [ABSTRACT FROM AUTHOR]

Vaccination of cattle with Mycobacterium bovis BCG has been shown to protect against infection with virulent strains of M. bovis, and against resultant bovine tuberculosis (TB). Here we report on a large-scale trial in New Zealand where free-ranging cattle were vaccinated with 3 x 10 5 BCG via injection, a lower dose than any previously trialed in cattle against exposure to a natural force of M. bovis infection. In a multi-year enrolment study involving >800 animals, three cohorts of 1-2 year old cattle were randomised to receive vaccine or to serve as non-vaccinated controls. Cattle were slaughtered and subject to standard abattoir post mortem examination for M. bovis culture-positive TB lesions after up to 3.7 years of in-field exposure; additionally, lymph node samples from approximately half of the cattle were examined further to identify infection in the absence of lesions. Overall TB prevalence, as identified by gross lesions detected at slaughter, was low among farmed cattle at the study site (<4% annually). There were two lesioned cases among 520 vaccinated trial cattle (0.38%) compared to eight among 297 non-vaccinated trial cattle (2.69%). Trial vaccine efficacy was 85.7% against abattoir-detectable TB (statistically significant protection), and 86.7% when adjusted for duration of exposure. BCG vaccination did not significantly affect the response rates of cattle to ante mortem skin- or blood-tests in diagnostic tests conducted >7 months post-vaccination. Use of a reduced, yet effective, dose of BCG would increase the cost effectiveness of using this vaccine in a bovine TB control programme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Oral-delivery Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine in a lipid matrix has been shown to confer protection against M. bovis infection and reduce the severity of tuberculosis (TB) when fed to brushtail possums (Trichosurus vulpecula), the major wildlife vector of bovine TB in New Zealand. Here we demonstrate the feasibility of aerial delivery of this live vaccine in bait form to an M. bovis-infected wild possum population, and subsequently assess vaccine uptake and field efficacy. Pre-trial studies indicated a resident possum population at very low density (<0.6 possums/ha) at the field site, with a 5.1% prevalence of macroscopic TB lesions. Pilot studies indicated that flavoured lipid matrix baits in weather-proof sachets could be successfully sown aerially via helicopter and were palatable to, and likely to be consumed by, a majority of wild possums under free-choice conditions. Subsequently, sachet-held lipid baits containing live BCG vaccine were sown at 3 baits/ha over a 1360 ha area, equating to >5 baits available per possum. Blood sampling conducted two months later provided some evidence of vaccine uptake. A necropsy survey conducted one year later identified a lower prevalence of culture-confirmed M. bovis infection and/or gross TB lesions among adult possums in vaccinated areas (1.1% prevalence; 95% CI, 0-3.3%, n = 92) than in unvaccinated areas (5.6%; 0.7-10.5%, n = 89); P = 0.098. Although not statistically different, the 81% efficacy in protecting possums against natural infection calculated from these data is within the range of previous estimates of vaccine efficacy in trials where BCG vaccine was delivered manually. We conclude that, with further straightforward refinement to improve free-choice uptake, aerial delivery of oral BCG vaccine is likely to be effective in controlling TB in wild possums. We briefly discuss contexts in which this could potentially become an important complementary tool in achieving national eradication of TB from New Zealand wildlife., Competing Interests: Two of the authors (FA & BB) are named co-inventors of the vaccine that was being trialed in this study. This does not alter these authors’ or any of their co-authors’, adherence to PLOS ONE policies on sharing data and materials.

In New Zealand, managing the threat of bovine tuberculosis (TB) to livestock includes population reduction of potentially infectious wildlife, primarily the brushtail possum (Trichosurus vulpecula). Population control is often targeted on forested buffer zones adjacent to farmland, in order to limit movements of possums across the buffer and reduce the risk of disease transmission to livestock. To assess the effectiveness of buffers in protecting livestock we analysed GPS telemetry data from possums located in untreated forest adjacent to buffers, and used these data to characterise patterns of movement that could lead to possums reaching farmland during the season when most dispersal occurs. Analyses of movement data showed that the direction of dispersal by sub-adult and adult possums and the extent of long exploratory movements were not biased toward forest buffers, even though these provided vacant habitat as suitable for possums as untreated forest. Instead, dispersal and exploratory movements were uncommon even for sub-adult possums and such events typically lasted <10 days. Dispersing possums settled predominantly in river valleys. A simulation model was developed for the 3-6-month dispersal season; it demonstrated a probability of <0.001 that an infected possum, originating from a low-density population with low disease prevalence in untreated forest, would move across 3 km of recently controlled forest buffer to reach farmland. Our results indicate short-term reduction in the risk of TB transmission from possums to livestock in New Zealand by the use of depopulated buffer zones, while acknowledging that the threat of disease spread from untreated forest is likely to increase over time as possum population density and, potentially, TB prevalence among those possums, increase in the buffer zone.

In New Zealand, the introduced marsupial brushtail possum (Trichosurus vulpecula) is a pest species subject to control measures, primarily to limit its ability to transmit bovine tuberculosis (TB) to livestock and for conservation protection. To better define parameters for targeted possum control and TB surveillance, we here applied a novel approach to analyzing GPS data obtained from 44 possums fitted with radio-tracking collars, producing estimates of the animals' short-term nocturnal foraging patterns based on 1-, 3- or 5-nights' contiguous data. Studies were conducted within two semi-arid montane regions of New Zealand's South Island High Country: these regions support low-density possum populations (<2 possums/ha) in which the animals' home ranges are on average larger than in high-density populations in forested habitat. Possum foraging range width (FRW) estimates increased with increasing monitoring periods, from 150-200 m based on a single night's movement data to 300-400 m based on 5 nights' data. The largest average FRW estimates were recorded in winter and spring, and the smallest in summer. The results suggest that traps or poison-bait stations (for lethal control) or monitoring devices (for TB surveillance), set for > 3 consecutive nights at 150 m interval spacings, would likely place >95% of the possums in this type of habitat at risk of encountering these devices, year-round. Modelling control efficacy against operational expenditure, based on these estimations, identified the relative cost-effectiveness of various strategies that could be applied to a typical aerial poisoning operation, to reduce the ongoing TB vectorial risk that possums pose in the High Country regions. These habitat-specific findings are likely to be more relevant than the conventional pest control and monitoring methodologies developed for possums in their more typical forested habitat.

In New Zealand, wild pigs acquire Mycobacterium bovis infection by scavenging tuberculous carrion, primarily carcasses of the main disease maintenance host, the brushtail possum (Trichosurus vulpecula). We investigated the utility of captive-reared, purpose-released pigs as sentinels for tuberculosis (TB) following lethal possum control and subsequent population recovery. Within 2-3 years of possum control by intensive poisoning, TB prevalence and the incidence rate of M. bovis infection in released sentinel pigs were lower than in an adjacent area where possums had not been poisoned. Unexpectedly, TB did not decline to near zero levels among pigs in the poisoned area, a fact which reflected an unanticipated rapid increase in the apparent abundance of possums. Monitoring infection levels among resident wild pigs confirmed that TB prevalence, while reduced due to possum control, persisted in the poisoned area at >20% among pigs born 2-3 years after poisoning, while remaining >60% among resident wild pigs in the nonpoisoned area. When fitted with radio-tracking devices, purpose-released pigs provided precise spatial TB surveillance information and facilitated effective killing of wild pigs when employed as "Judas" animals to help locate residents. Sentinel pigs offer value for monitoring disease trends in New Zealand, as TB levels in possums decline nationally due to large-scale possum control.

Sentinel species are increasingly used by disease managers to detect and monitor the prevalence of zoonotic diseases in wildlife populations. Characterizing home-range movements of sentinel hosts is thus important for developing improved disease surveillance methods, especially in systems where multiple host species co-exist. We studied ranging activity of major hosts of bovine tuberculosis (TB) in an upland habitat of New Zealand: we compared home-range coverage by ferrets (Mustela furo), wild deer (Cervus elaphus), feral pigs (Sus scrofa), brushtail possums (Trichosurus vulpecula) and free-ranging farmed cattle (Bos taurus). We also report in detail the proportional utilization of a seasonal (4-monthly) range area for the latter four species. Possums covered the smallest home range (<30 ha), ferrets covered ~100 ha, pigs ~4 km(2), deer and cattle both >30 km2. For any given weekly period, cattle, deer and pigs were shown to utilize 37–45% of their estimated 4-month range, while possums utilized 62% during any weekly period and 85% during any monthly period of their estimated 4-month range. We suggest that present means for estimating TB detection kernels, based on long-term range size estimates for possums and sentinel species, probably overstate the true local surveillance coverage per individual.

Introduced brushtail possums (Trichosurus vulpecula) are wildlife maintenance hosts for Mycobacterium bovis in New Zealand, often living sympatrically with other potential hosts, including wild red deer (Cervus elaphus scoticus). Population control of possums has been predicted to eradicate tuberculosis (TB) from New Zealand wildlife ; however, there is concern that long-lived M. bovis-infected deer could represent a ‘ spillback’ risk for TB re-establishment (particularly when possum populations recover after cessation of intensive control). We constructed a time-, age- and sex-structured, deer/TB population generic model and simulated the outcomes of deer control on this potential spillback risk. Maintaining intensive possum control on a 5-year cycle, the predicted spillback risk period after TB eradication from possums is ~7 years, while the probability of TB re-establishing in possums over that period is ~6%. Additional targeted control of deer would reduce the risk period and probability of spillback; however, even with high population reductions (up to 80%) only modest decreases in risk and risk period would be achieved. We conclude that possum control alone remains the best strategy for achieving TB eradication from New Zealand habitats in which possums and wild deer are the main M. bovis hosts.

Gross pathology due to tuberculosis can be established experimentally in brushtail possums (Trichosurus vulpecula) within 7 weeks of injection of virulent Mycobacterium bovis into subcutaneous connective tissues of the peripheral limbs. This pathology involves lymphadenomegaly and development of gross lesions in peripheral lymph nodes, with subsequent gross lesions in the lungs and reticuloendothelial organs. Using this artificial infection model, we here assessed the mortality rate for possums in the wild, to provide new information on the likely survival period for New Zealand's major wildlife host. Possums were trapped and inoculated with <50 CFU of M. bovis, then fitted with mortality signal emitting radio tracking collars, released and re-tracked for 6 months. Possum survival probability was 89% up to 12 weeks post-injection (p.i.), but cumulative mortality was rapid from then on. The median survival period, based on study of 38 possums, was 18 weeks p.i.; this corresponds with a predicted time interval of 11 weeks between first presentation of TB as palpable lymphadenomegaly and death for an average possum, shorter than period values currently used in possum TB epidemiological modelling. We also examined gross pathology in 11 possums by post mortem necropsy, and confirmed lymphadenomegaly and tuberculous lesions at 7 and 12 weeks p.i. Extra-peripheral gross lesions were more frequent among possums at 12 weeks p.i. than at 7 weeks, while the occurrence of lung lesions (the most likely cause of disease-induced mortality) was apparent in animals at 12 weeks but not at 7 weeks p.i. Our results suggest that the time course of TB from development of gross lesions to mortality may be shorter than previously estimated from field studies of naturally tuberculous possums., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation., Methods: Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis., Results: Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens., Discussion: The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Brushtail possums (Trichosurus vulpecula) are the major wildlife reservoir of Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), in New Zealand. Primary diagnosis of BTB in wild possums is by palpation to detect peripheral lymphadenomegaly followed by necropsy examination, which frequently identifies gross tuberculous lesions in the peripheral lymph nodes and lungs. Experimental infection studies were conducted with wild possums in an attempt to emulate field BTB, focussing on percutaneous administration of virulent M. bovis in the paws. In a preliminary study, viable M. bovis bacilli were recovered from lymph nodes draining fore- or hindlimbs 12 days after percutaneous injection. Subsequently, 21 wild possums were injected interdigitally with 500 colony forming units (cfu) of M. bovis, radio-collared and released; 17/18 possums recaptured 8 weeks later had an established M. bovis lymphatic infection, with 16 having culture-positive gross lesions in the superficial and/or deep axillary lymph nodes. A dual-site infection model was established, involving simultaneous interdigital injection of 100 cfu of M. bovis into front and rear paws of 19 wild possums; this identified that the average degree of lymphadenitis involved 30-fold enlargement of the draining lymph node by 7-8 weeks post injection (wpi). A time-course study demonstrated establishment of M. bovis infection in peripheral lymph nodes of 9/11 possums at 3-5 wpi of doses ranging from 60 to 190 cfu, but with no development of gross lesions; by 7 weeks, 8/8 animals injected similarly had both an established infection and gross lesions of peripheral lymph nodes. The incidence and progression of peripheral lesion development, together with indications of sequential infection of the lungs, liver and mesenteric lymph nodes(MLNs), indicates that a low-dose percutaneous M. bovis infection model is likely to emulate natural disease in possums., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The metabolic toxin sodium fluoroacetate ('compound 1080') is widely used for controlling introduced mammalian pests in New Zealand. For large-scale operations, 1080 is distributed aerially in bait to kill brushtail possums (Trichosurus vulpecula Kerr) and ship rats (Rattus rattus L.). While usually successful in reducing pest populations by > 80%, widespread distribution of toxic bait is relatively expensive and raises concerns from some members of the public. Here, trials with spatial aggregation of baits in forested habitats were conducted to determine whether this can reduce toxin usage while maintaining operational efficacy., Results: When 1080 baits were aggregated into clusters (by hand sowing) or into strips (by precision aerial deployment), indices of possum relative abundance were reduced by 92-100%, compared with 73-100% reductions using conventional aerial broadcasting, while all methods reduced relative abundance indices of rats by 88% or greater. Radio tracking indicated a kill rate of > 90% against possums, regardless of bait distribution method., Conclusions: Simply by modifying bait distribution patterns, spatial aggregation can be used to maintain the high encounter rate of pests with 1080 bait that is necessary for operational efficacy, while reducing current toxin usage by up to 80%. Aggregated bait delivery could have relevance for other mammalian pest control scenarios internationally., (Copyright © 2012 Society of Chemical Industry.)

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Aim: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock., Methods: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map., Results: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples., Conclusions: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits., Clinical Relevance: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in New Zealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.

Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: This study investigated the effect of bovine colostrum (BC) on mucosal defense in the respiratory tracts of athletes and a nonexercising control group., Methods: An athlete cohort (ATH) of 25 (12 male, 13 female) elite swimmers (age 14-23 yr) and a control cohort (CON) of 28 (9M, 19F) students (age 18-27 yr) were randomly allocated in a double-blind manner to receive either 25 g BC (low-protein colostrum powder) or isocaloric placebo (PL) per day for 10 wk. Postprandial saliva samples were analyzed for immunoglobulins (A, G, and M) and osmolality at baseline, after 4 and 10 wk, and 2 wk postsupplementation. Blood samples were analyzed for immunoglobulins and C-reactive protein at baseline, after 5 and 10 wk, and 2 wk postsupplementation. Dietary intake was assessed by self-recorded dietary records. Upper respiratory tract symptoms (URS) and exercise were also self-recorded daily., Results: There was no significant time-related effect of the BC supplement on either saliva or plasma immunoglobulin levels for either cohort. After 4 wk supplementation fewer ATH/BC (25%) than ATH/PL participants (61%) reported URS incidents (p = .062). No significant difference occurred in URS reports in the control group., Conclusion: There was no measurable effect on immunoglobulin levels of consuming BC, which is in contrast to effects that have been reported previously in marathon runners, indicating that the effect of BC supplementation is not universal in all groups of athletes. Fewer athletes reported URS (although cause unknown) when consuming BC, which may be advantageous for training.

Alpha integrins play an important role in cell to cell and cell to extra-cellular matrix interactions required for an effective T-lymphocyte-mediated immune response, however little is known about age related differences in expression of alpha integrins on T-cells in humans. We here measured alpha-4 (alpha4) integrin (CD49d) expression on T-lymphocytes via peripheral blood sampling, comparing parameters between cohorts of young and old adults. No age-related differences were found for the absolute numbers of T-cells, although the percentage of CD4+ T-cells in older adults was significantly greater and the percentage of CD8+ T-cells lower than in younger cohorts. Percentage and absolute numbers of CD3+ T-cells co-expressing CD49d were significantly lower in older adults compared to younger cohorts, and the percentage of gated CD4+ and CD8+ cells that co-labelled positively for CD49d was also reduced in this group. There were no age-related differences in circulating levels of cytokines (Type I interferons) that are known to regulate cell surface integrin expression. Reduced expression of alpha integrins on T-cells may be an early indicator of the loss of homeostatic control that occurs with ageing, contributing to diminished effector T-cell responses during senescence.

In in vitro co-culture experiments, the ovine-derived cathelicidin OaBac5mini showed antimicrobial activity against Escherichia coli cells and modulated production of a cytokine by a mammalian inflammatory cell type (macrophage). Using atomic force microscopy, the morphology of peptide-treated E. coli bacteria showed no cell lysis, indicating an intracellular mode of action of the peptide leading to bacterial cell inhibition. At a concentration of 50microg/mL OaBac5mini, the peptide suppressed production of the inflammatory cytokine interleukin-12 by murine J774A cells that had been stimulated with Staphylococcus aureus strain Cowan; levels of other cytokines were unaffected. Thus, certain cationic peptides can enter and disrupt invading Gram-negative pathogens and may be able to modulate inflammatory responses induced by Gram-positive bacterial products., (Copyright 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Oral delivery of live Mycobacterium bovis BCG in a lipid matrix invokes cell-mediated immune (CMI) responses in mice and consequent protection against pulmonary challenge with virulent mycobacteria. To investigate the influence of prior BCG sensitization on oral vaccine efficacy, we assessed CMI responses and BCG colonization of the alimentary tract lymphatics 5 months after oral vaccination, in both previously naive mice and in mice that had been sensitized to BCG by injection 6 months previously. CMI responses did not differ significantly between mice that received subcutaneous BCG followed by oral BCG and those that received either injected or oral BCG alone. In vivo BCG colonization was predominant in the mesenteric lymph nodes after oral vaccination; this colonizing ability was not influenced by prior BCG sensitization. From this murine model study, we conclude that although prior parenteral-route BCG sensitization does not detrimentally affect BCG colonization after oral vaccination, there is no significant immune-boosting effect of the oral vaccine either.

Cultures of Mycobacterium bovis BCG, comprising predominantly single-cell bacilli, were prepared in broth without animal-derived reagents. When formulated into a vegetable-derived lipid matrix, the vaccine was stable in vitro and was immunogenic in vivo upon feeding it to mice. This formulation could be useful for oral vaccination of wildlife against tuberculosis, where concern over transmissible prions may preclude the field use of vaccines containing animal products.

Bovine tuberculosis (Tb) caused by Mycobacterium bovis has proved refractory to eradication from domestic livestock in countries with wildlife disease reservoirs. Vaccination of wild hosts offers a way of controlling Tb in livestock without wildlife culling. This study was conducted in a Tb-endemic region of New Zealand, where the introduced Australian brushtail possum (Trichosurus vulpecula) is the main wildlife reservoir of Tb. Possums were trapped and vaccinated using a prototype oral-delivery system to deliver the Tb vaccine bacille Calmette-Guerin. Vaccinated and control possums were matched according to age, sex and location, re-trapped bimonthly and assessed for Tb status by palpation and lesion aspiration; the site was depopulated after 2 years and post-mortem examinations were conducted to further identify clinical Tb cases and subclinical infection. Significantly fewer culture-confirmed Tb cases were recorded in vaccinated possums (1/51) compared with control animals (12/71); the transition probability from susceptible to infected was significantly reduced in both males and females by vaccination. Vaccine efficacy was estimated at 95 per cent (87-100%) for females and 96 per cent (82-99%) for males. Hence, this trial demonstrates that orally delivered live bacterial vaccines can significantly protect wildlife against natural disease exposure, indicating that wildlife vaccination, along with existing control methods, could be used to eradicate Tb from domestic animals.

Bovine tuberculosis (Tb), due to infection with virulent Mycobacterium bovis, represents a threat to New Zealand agriculture due to vectorial transmission from wildlife reservoir species, principally the introduced Australian brushtail possum (Trichosurus vulpecula). An oral-delivery wildlife vaccine has been developed to immunize possums against Tb, based on formulation of the human Tb vaccine (M. bovis BCG) in edible lipid matrices. Here BCG bacilli were shown to be stable in lipid matrix formulation for over 8 mo in freezer storage, for 7 wk under room temperature conditions, and for 3-5 wk under field conditions in a forest/pasture margin habitat (when maintained in weatherproof bait-delivery sachets). Samples of the lipid matrix were flavored and offered to captive possums in a bait-preference study: a combination of 10% chocolate powder with anise oil was identified as the most effective attractant/palatability combination. In a replicated field study, 85-100% of wild possums were shown to access chocolate-flavored lipid pellets, when baits were applied to areas holding approximately 600-800 possums/km(2). Finally, in a controlled vaccination/challenge study, chocolate-flavored lipid vaccine samples containing 10(8) BCG bacilli were fed to captive possums, which were subsequently challenged via aerosol exposure to virulent M. bovis: vaccine immunogenicity was confirmed, and protection was identified by significantly reduced postchallenge weight loss in vaccinated animals compared to nonvaccinated controls. These studies indicate that, appropriately flavored, lipid delivery matrices may form effective bait vaccines for the control of Tb in wildlife.

In New Zealand, possums (Trichosurus vulpecula) are the main wildlife reservoir for bovine tuberculosis (Tb), which they transmit to livestock. This study investigated oral vaccination with lipid-formulated Mycobacterium bovis BCG and subsequent protection against virulent M. bovis challenge in wild-caught possums. Possums were trapped from the field and either hand-vaccinated and released back into the wild, or acclimatised to captive conditions prior to voluntary uptake of flavoured vaccine. Possums were subsequently exposed to pulmonary challenge with virulent M. bovis, administered either by instillation of a liquid suspension as an intra-tracheal challenge (field animals) or in micro-droplets as an aerosol (captive animals). Field studies indicated that the relative risk of death in wild possums due to Tb was 2.4 times greater in control compared with orally-vaccinated possums, with the vaccine conferring protection to possums in both good and poor body condition. Laboratory studies indicated that oral vaccination conferred protection in cage-acclimatised possums, with >3log(10) reduction in lung bacterial burdens among vaccinated animals. This study provides evidence that lipid-formulated BCG oral vaccine can provide significant protection to possums in field as well as laboratory conditions, which may favour the use of this formulation as a delivery method for controlling wildlife Tb.

Lipid formulations containing BCG strains Danish 1331 or Moreau (Rio de Janeiro) were trialled as oral vaccines in rodent models. In mice, oral-delivery of either strain resulted in BCG colonisation of the alimentary tract lymphatics and induction of gamma-interferon responses. In guinea pigs, both strains provided pulmonary protection against Mycobacterium tuberculosis aerosol challenge, as shown by significantly reduced bacterial loads and lung:body weight ratios. Lipid-formulated BCG provided superior protection against M. tuberculosis over unformulated orally-delivered BCG (Moreau), and equivalent protection to sub-cutaneous BCG (Danish) immunisation. Oral-delivery of lipid-formulated BCG may offer a practical alternative to parenteral-route BCG vaccination.

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Meles meles) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guérin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for an oral BCG vaccine in badgers.

Increased incidence of bovine tuberculosis (TB) in the United Kingdom caused by infection with Mycobacterium bovis is a cause of considerable economic loss to farmers and the government. The Eurasian badger (Meles meles) represents a wildlife source of recurrent M. bovis infections of cattle in the United Kingdom, and its vaccination against TB with M. bovis bacillus Calmette-Guérin (BCG) is an attractive disease control option. Delivery of BCG in oral bait holds the best prospect for vaccinating badgers over a wide geographical area. Using a guinea pig pulmonary challenge model, we evaluated the protective efficacy of candidate badger oral vaccines, based on broth-grown or ball-milled BCG, delivered either as aqueous suspensions or formulated in two lipids with differing fatty acid profiles (one being animal derived and the other being vegetable derived). Protection was determined in terms of increasing body weight after aerosol challenge with virulent M. bovis, reduced dissemination of M. bovis to the spleen, and, in the case of one oral formulation, restricted growth of M. bovis in the lungs. Only oral BCG formulated in lipid gave significant protection. These data point to the potential of the BCG-lipid formulation for further development as a tool for controlling tuberculosis in badgers.

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid-formulation induces a gamma-interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non-replicating mycobacterial antigens. Single oral immunization with lipid-formulated live BCG invoked secreted and cellular IFN-gamma responses in mice 8 weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8-week period. Single oral immunization with live BCG also invoked an interleukin-2 response (but not TNF-alpha or IL-4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid-formulated soluble or particulate non-replicating mycobacterial antigens failed to invoke cytokine responses, except for a low-level IFN-gamma response in mice multiple immunized with lipid-formulated heat-killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral-route immunization with live or non-replicating mycobacterial antigens and with practical reference to the development of oral-delivery vaccines against tuberculosis.

Numerous infectious diseases caused by bacteria or viruses persist in developed and developing countries due to ongoing transmission among wildlife reservoir species. Such diseases become the target of control and management programmes in cases where they represent a threat to public health (for example rabies, sylvatic plague, Lyme disease), or livestock production (for example bovine tuberculosis, brucellosis, pseudorabies), or where they threaten the survival of endangered animal populations. In the majority of cases, lethal control operations are neither economically feasible nor publicly supported as a practical means for disease management. Prophylactic vaccination has emerged over the last 15 years as an alternative control strategy for wildlife diseases, mainly driven by the success of widescale oral rabies vaccination programmes for meso-carnivores in North America and Northern Europe. Different methods have been trialled for the effective delivery of wildlife vaccines in the field, however oral vaccination remains the most widely used approach. Successful implementation of an oral wildlife vaccine is dependent on a combination of three components: an efficacious immunogen, a suitable delivery vehicle, and a species-specific bait. This review outlines the major wildlife disease problems for which oral vaccination is currently under consideration as a disease management tool, and also focuses on the technological challenges that face wildlife vaccine development. The major conclusion is that attenuated or recombinant live microbes represent the most widely-used vaccines that can be delivered by the oral route; this in turn places major emphasis on effective delivery systems (to maintain vaccine viability), and on selective baiting systems, as the keys to wildlife vaccine success. Oral vaccination is a valuable adjunct or alternative strategy to culling for the control of diseases which persist in wildlife reservoirs.

The use of a bacillus Calmette-Guerin (BCG)-based vaccine could represent a viable strategy for controlling bovine tuberculosis (TB), principally in those cases where a wildlife disease vector exists. This article focuses on recent progress in animal TB vaccinology, outlining that oral-route vaccination represents the most feasible means of distributing a vaccine to control disease in wildlife. Drawing on historical successes of previous wildlife vaccination programs, the article suggests how, and in what form, an oral-delivery BCG-based vaccine might become operational, considering the wide diversity of TB reservoir species and the inherent problems associated with field delivery of a live-attenuated microbial vaccine.

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.

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.

Secretory IgA in saliva (s-IgA) is a potential mucosal immune correlate of upper respiratory tract infection (URTI) status. Nutritional supplements may improve mucosal immunity, and could be beneficial to athletes who are at increased risk of URTI. In this study, 35 distance runners (15 female, 20 male, age 35 to 58 y) consumed a supplement of either bovine colostrum or placebo for 12 wk. Saliva samples were taken prior to training at baseline, monthly during supplementation, and 2 wk post supplementation. Median levels of s-IgA increased by 79% in the colostrum group after 12 wk intervention, and the time-dependent change from baseline value was significant (P = 0.0291). This significance was still apparent after adjusting for training volume and self-reporting of upper respiratory symptoms. This study has demonstrated increased s-IgA levels among a cohort of athletes following colostrum supplementation. While this result is statistically significant, its physiological interpretation must be viewed with caution due to the small numbers in this study and the large variability in s-IgA levels.

Lipid microencapsulation of Mycobacterium bovis bacille Calmette-Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell-mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium-specific IFN-gamma-secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid-encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyer's patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium-specific IFN-gamma production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid-encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.

Rotavirus-induced diarrhea is a common infection that results in the death of nearly 500,000 children annually. Currently, no large-scale preventative treatments or vaccines exist. Because some whey protein concentrates (WPC) were shown to contain bioactive ingredients that may activate immune cells and/or prevent infection, the current study was conducted to assess whether the proprietary WPC IMUCARE (WPC-IC) could protect against rotavirus. Suckling BALB/c mice were treated by gavage once daily with WPC-IC or with the control protein bovine serum albumin from the age of 9 to 17 d, and were infected with murine rotavirus at the age of 11 d. Disease symptoms were graded as mild, moderate, or severe, and viral shedding was measured in fecal samples during the postinfection period. Severe diarrhea occurred in 63% of control mice; this was significantly reduced to 36% in WPC-IC-fed mice. Severe diarrhea occurred for a 4-d period in the control group but only for a 2-d period in the WPC-IC group. Although the mean viral load per mouse did not differ between the groups, the proportion of mice shedding high levels of the virus in the feces postinfection was significantly lower in the WPC-IC group on d 13, 16, and 17, and significantly higher on d 14. Rotavirus-specific antibody levels in serum and gut fluid did not differ between groups. Thus, prophylactic treatment with WPC-IC may reduce rotaviral disease by decreasing the prevalence of severe diarrhea and by decreasing the time period during which severe symptoms and high viral shedding occur.

Bacteria used in commercial probiotic preparations are most commonly gram-positive lactic acid-producing species, although there are also some probiotic products which utilise gram-negative coliform bacteria. Characterising how the innate immune system responds to these bacteria in vitro may give an indication as to the likely immunomodulatory events that can be triggered following probiotic administration in vivo. Here, an established gram-positive probiotic (Lactobacillus casei Shirota) was compared against a novel gram-negative probiotic strain (Escherichia coli Nissle 1917) for its ability to induce cytokine production in a cell type representative of the innate immune system; in addition, responses were contrasted against those induced by an enteropathogenic coliform, E. coli 2282. We investigated the ability of these three bacterial strains to modulate production of interleukins-10, -12 and -18; tumour necrosis factor-alpha; interferon-alpha; and transforming growth factor-beta, via a series of in vitro culture experiments involving the murine monocyte/macrophage cell line J774A.1. All bacteria induced marked secretion of IL-12 and TNFalpha by cells, while only coliforms induced production of IL-10; there was minimal or no induction of IL-18 or TGFbeta. Activation of cells with recombinant gamma-interferon promoted increased production of IL-12, but decreased production of IL-10, in response to the co-culture of coliform bacteria, indicating differential cytokine induction depending on the activation status of the target cell. In general, live bacteria stimulated higher levels of IL-10, IL-12 and TNFalpha secretion than heat-killed preparations, while only live coliforms induced IFNalpha. These findings are discussed in relation to the likely immunomodulatory effects of gram-positive and gram-negative bacteria on the innate immune system in vivo, with particular emphasis on the marked similarity in cytokine response patterns observed between probiotic versus pathogenic coliform bacteria.

Proteins derived from the whey fraction of bovine milk are known to modulate immune responses. We have previously described a rennet whey protein concentrate (WPC) that can boost intestinal tract antibody responses to orally administered T-dependent antigens. In the present study, we investigated the effects of feeding WPC to mice on specific antibody responses to several orally or parenterally administered antigens, including influenza vaccine, diphtheria and tetanus toxoids, poliomyelitis vaccine, ovalbumin and cholera toxin sub-unit. WPC-fed mice produced elevated levels of antigen-specific intestinal tract and serum antibodies against all tested antigens, compared to mice that were fed a standard chow diet. Both primary and secondary intestinal tract antibody responses were elevated by WPC feeding, while only secondary serum responses were increased in WPC-fed mice. Significant up-regulation of intestinal tract antibody was observed within 2 weeks of primary oral immunizations. A period of pre-feeding with WPC, prior to commencement of immunization, did not alter the kinetics or magnitude of immune enhancement. These results identify bovine WPC as a potentially important dietary protein supplement, capable of enhancing humoral immune responses to a range of heterologous antigens.

Probiotic lactic acid bacteria can signal the immune system through innate cell surface pattern recognition receptors or via direct lymphoid cell activation. In some cases, this action has been shown to be sufficient to modulate local- and systemic-level in vivo immune responses. Practical applications of probiotics include their use in anti-tumour and anti-allergy immunotherapy, but there is also increasing evidence that some probiotics can stimulate a protective immune response sufficiently to enhance resistance to microbial pathogens. This review outlines the experimental and clinical evidence for enhanced anti-microbial immune protection by probiotic lactic acid bacteria, focussing on those studies where a correlative or suggestive link has been shown between immune modulation and enhanced protection.

Probiotic lactobacilli have been proposed as a potential oral bacteriotherapeutic means of modulating immune phenotype expression in vivo, via their ability to promote cytokine production. This study investigated the ability of a known interferon (IFN)gamma-promoting probiotic (Lactobacillus rhamnosus HNOOI) to modulate cytokine production in mice expressing an on-going Th2-type immune response. BALB/c mice were primed to ovalbumin in alum adjuvant to invoke antigen-specific Th2 cytokine-secreting cell populations. Mice that were fed Lb. rhamnosus HN001 during antigen sensitization produced higher levels of lymphocyte-derived IFNgamma, but also interleukin (IL)-4 and IL-5, in comparison to control animals. Although HN001 was additionally shown to induce pro-IFNgamma monokine (IL-12, IL-18) secretion in macrophages in vitro, its ability to invoke mixed lymphocyte cytokine production during an on-going Th2-type immune response in vivo suggests that this probiotic is a general immunostimulatory agent, in contrast to the pro-Th1/anti-Th2 immunoregulation reported for some strains of IFNgamma-promoting lactobacilli.

Background: The aging process can lead to a decline in cellular immunity. Therefore, the elderly could benefit from safe and effective interventions that restore cellular immune functions., Objective: We determined whether dietary supplementation with the known immunostimulating probiotic Bifidobacterium lactis HN019 could enhance aspects of cellular immunity in elderly subjects., Design: Thirty healthy elderly volunteers (age range: 63-84 y; median: 69 y) participated in a 3-stage dietary supplementation trial lasting 9 wk. During stage 1 (run-in), subjects consumed low-fat milk (200 mL twice daily for 3 wk) as a base-diet control. During stage 2 (intervention), they consumed milk supplemented with B. lactis HN019 in a typical dose (5 x 10(10) organisms/d) or a low dose (5 x 10(9) organisms/d) for 3 wk. During stage 3 (washout), they consumed low-fat milk for 3 wk. Changes in the relative proportions of leukocyte subsets and ex vivo leukocyte phagocytic and tumor-cell-killing activity were determined longitudinally by assaying peripheral blood samples., Results: Increases in the proportions of total, helper (CD4(+)), and activated (CD25(+)) T lymphocytes and natural killer cells were measured in the subjects' blood after consumption of B. lactis HN019. The ex vivo phagocytic capacity of mononuclear and polymorphonuclear phagocytes and the tumoricidal activity of natural killer cells were also elevated after B. lactis HN019 consumption. The greatest changes in immunity were found in subjects who had poor pretreatment immune responses. In general, the 2 doses of B. lactis HN019 had similar effectiveness., Conclusion: B. lactis HN019 could be an effective probiotic dietary supplement for enhancing some aspects of cellular immunity in the elderly.

The probiotic lactic acid bacterium Lactobacillus rhamnosus (strain HN001) is known to stimulate enhanced innate and acquired immune responses in mice. following oral delivery. Here, the ability of HN001 to confer immune enhancement and protection against an oral challenge of Salmonella tYphimurium was investigated. HN001-fed and non-probiotic-fed control BALB/c mice were challenged with either a single dose of S. typhimurium (ATCC strain 1772), or with five repeated daily doses of the pathogen; post-challenge clinical, behavioural, bacteriological and immunological parameters were assessed. Mice began to show ostensible signs of infection 3-4 days following the initiation of Salmonella challenge, and the first mortalities were observed after 6 days. Following single-dose Salmonella challenge, HN001-fed mice maintained a higher mean pre-mortality general health score than control mice; retained significantly greater food and water intake and weight gain, produced higher titres of serum and intestinal tract anti-Salmonella antibodies, and showed greater overall survival of infection (27/30 mice surviving at 21 days post-challenge, compared to 2/29 in the control group). Following repeated-dose Salmonella challenge, HN001-fed mice had significantly lower mean pathogen burdens in visceral organs (spleen, liver) compared to controls, and additionally, blood and peritoneal leucocytes obtained from HN001-fed mice exhibited significantly higher ex vivo phagocytic capacity compared to control-mice. This study affirms that Lb. rhamnosus strain HN001 displays immuno-enhancing properties in S. typhimurium-infected mice, and demonstrates that oral delivery of this probiotic can promote increased protection against a highly virulent enteric bacterial pathogen.

Many elderly subjects are at increased risk of infectious and noninfectious diseases due to an age-related decline in lymphoid cell activity (immunosenescence). Noninvasive means of enhancing cellular immunity are therefore desirable in the elderly. Previous reports have suggested that dietary supplementation could represent an effective means of enhancing the activity of circulating natural killer (NK) cells in the elderly. In the present study, we have conducted a pre-post intervention trial to determine the impact of dietary supplementation with probiotic lactic acid bacteria (LAB) on peripheral blood NK cell activity in healthy elderly subjects. Twenty-seven volunteers consumed low-fat/low-lactose milk supplemented with known immunostimulatory LAB strains (Lactobacillus rhamnosus HN001 or Bifidobacterium lactis HN019) for a period of 3 weeks. A dietary run-in of milk alone was shown to have no significant effect on NK cells. In contrast, the proportion of CD56-positive lymphocytes in peripheral circulation was higher following consumption of either LAB strain, and ex vivo PBMC tumoricidal activity against K562 cells was also increased. Supplementation with HN001 or HN019 increased tumoricidal activity by an average of 101 and 62%, respectively; these increases were significantly correlated with age, with subjects older than 70 years experiencing significantly greater improvements than those under 70 years. These results demonstrate that dietary consumption of probiotic LAB in a milk-based diet may offer benefit to elderly consumers to combat some of the deleterious effects of immunosenescence on cellular immunity.

Studies in gnotobiotic animals have suggested that the intestinal bacterial flora may play an important role in priming the immune system during ontogeny to limit dysfunctional responses, including allergy. Prospective clinical studies have identified a higher incidence of allergy expression in early childhood among children who have low enteric populations of lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, further supporting a role for gut-colonizing bacteria in regulating immunological atopy. There is some evidence to suggest that supplementing the human diet with probiotic LAB might combat both allergy development and expression of atopy in allergy sufferers; however, definitive information, in the form of controlled intervention trials, remains scant. Recent immunological evidence has indicated that certain strains of LAB can stimulate the production of type I and II interferons and pro-interferon monokines (IL-12 and IL-18), following contact with the immune system; therefore, probiotic forms of immunoregulatory LAB could be used as dietary supplements to modify the gut microflora and provide pro-T helper cell 1 (Th1) STAT-activating signals sufficient to deviate the immune phenotype and correct the Th2-type bias which promotes allergy. This review outlines the clinical and laboratory evidence of a role for LAB in combating allergies, and attempts to explain this phenomenon in terms of our current understanding of immunoregulatory signals produced by gut-colonizing microbes., (Copyright 2001 S. Karger AG, Basel)

Clinical reports have suggested that dietary consumption of fermented foods, such as yogurt, can alleviate some of the symptoms of atopy and might also reduce the development of allergies, possibly via a mechanism of immune regulation. Controlled studies have indicated that consumption of fermented milk cultures containing lactic acid bacteria (LAB) can enhance production of Type I and Type II interferons at the systemic level. In animal models, LAB have been shown to promote interferon expression, and to reduce allergen-stimulated production of IL-4 and IL-5 in some cases. Recent results have shown that LAB are potent inducers of pro-interferon monokines (IL-12 and IL-18), and that cytokine secretion is stimulated by the interaction of Gram-positive cell wall components with surface receptors of mononuclear phagocytes, via NF-kappa B and STAT signalling pathways. However, it is clear that the extent and quality of LAB-induced immunoregulation is strain-dependent. This review discusses the clinical and laboratory evidence for anti-allergy properties of fermented foods, and proposes a model for the mechanism by which some well-defined strains of immunoregulatory LAB might down-regulate a Th2 allergic phenotype.

The elderly represent a group for whom health issues are of paramount concern. Physiological processes, including the functioning of the immune and digestive systems, are known to decline with age, and consequently aging can increase susceptibility to infectious and non-infectious disease. Dietary supplementation may offer a safe and effective means of countering physiological senescence, by optimizing important immune and gut functions. This review outlines our current understanding of the ways in which aging affects immune and gut function, and discusses clinical evidence for a role of dietary supplementation in countering age-related deficiencies in these processes.