The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

AIM: To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease. METHODS: Thirty-three adult possums were inoculated via the endo-bronchial route with 20-100 colony forming units of Mycobacterium bovis. The possums were killed at 1,2,3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopathologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally. RESULTS: Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised. CONCLUSIONS: Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.     

Effects of rapamycin-treated HSP60-pulsed dendritic cells on th e progression of the atherosclerotic plaque in mice

AIM:To evaluate whether tolerogenic dendri tic cells (DC) loaded with heat shock protein 60 (HSP60) inhibit the progression of aortic atherosclerotic plaque in hypercholesterolemic apolipoprotein E (Apo- E) -null mice.METHODS:Bone marrow derived DC of the mice were loaded with HSP 60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC ,DC loaded only HSP60 and PBS were injected into the ApoE-null mice at 8 weeks of age for three times at a one-week interval.8 weeks after the last injection,aorta were harvested for HE staining and anti-CD4+T cell immunostaining.Resp onses of pleenic cells to HSP60 were also evaluated.RESULTS:Compared with DC,DCHSP60 expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly,while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to p roliferation.After immunization with different injection,the numbers of CD4+ T cells in plaque were increased significantly in DCHSP60 group vs in PBS g roup (P<0.01).On the other hand,they were reduced significantly in rap-DC HSP60 group vs in PBS group (P<0.01).Plaque areas in the tolerog enic DC group were smaller than that in the PBS group (P<0.01).Stimulated by HSP60,pleenic cells in tolerogenic DC group secreted more IL-10,while in DC HSP60 group more IFN-γ secretion was observed.CONCLUSION:HSP60 specific tolerogenic DC immunization attenuate d the progression of plaque,indicating a new immune therapy for atherosclerosis.     

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of l8-month-old cattle were inoculated intratracheally with 5 X 10⁵ colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21–28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23–24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-γ responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-y responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums

Three serological tests for the diagnosis of Mycobacterium bovis infection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA using M. bovis culture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using a M. bovis specific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected with M. bovis produced cellular responses to M. bovis antigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected with M. bovis showed that antibody was first detected late in the disease.     

Influence of stress of capture on haematological values and cellular immune responses in the Australian brushtail possum (Trichosurus vulpecula)

Possums (Trichosurus vulpecula) captured for the first time (single-capture) had significantly lower blood lymphocyte and eosinophil counts, but higher neutrophil counts than possums which had been captured frequently. Single-capture possums also had depressed lymphocyte responses to concanavalin A (males and females) and pokeweed mitogen (males only). The haematological changes and the depressed lymphocyte reactivity to the T-cell mitogens were consistent with the animals being in a stressed state following capture for the first time. The haematological profile of single-capture possums changed to a profile similar to that of the frequently captured possums following housing in cages for 1 week, and high lymphocyte responses to T-cell mitogens were recorded following housing for 3-5 weeks. This information, together with body weight data, indicates that a minimum of 4 weeks adaptation to housing in cages should be allowed before possums are used in experimental studies.     

Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Clinical remission of idiopathic hypereosinophilic syndrome in a Rottweiler

Idiopathic hypereosinophilic syndrome (HES) is a rare syndrome for which Rottweilers appear to over-represent the canine cases. A 6-month-old female entire Rottweiler presented with seizures following a traumatic incident. The dog was identified as having a marked, sustained eosinophilia and investigations did not identify an underlying cause. Concurrently, the dog had chronic eosinophilic hepatitis with impaired liver function and mesenteric eosinophilic lymphadenitis. The dog went on to have spontaneous resolution of HES and normal liver function was subsequently documented. To date, the dog is still alive, more than 4 years after initial presentation. The diagnosis of idiopathic HES in Rottweilers may not carry a poor prognosis and the condition may spontaneously resolve, even in cases with documented organ damage.     

Route of BCG administration in possums affects protection against bovine tuberculosis

The Australian brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis in New Zealand. Control of bovine tuberculosis in farmed animals requires measures to reduce the transmission of M. bovis from wildlife. Possums were vaccinated with BCG intranasally by aerosol spray, orally or subcutaneously to compare the efficacy of these three routes on protection against challenge with virulent M. bovis. Possums vaccinated with BCG by the intranasal or subcutaneous routes had a marked reduction in severity of disease compared to possums which had been unvaccinated or orally vaccinated. The severity of the disease was assessed by changes in body weight and pathology. BCG vaccination by all three routes resulted in reduced dissemination of M. bovis to the spleen and liver following challenge. Intranasal and oral BCG vaccination induced lower mean peripheral blood lymphocyte blastogenic responses to bovine PPD than subcutaneous vaccination, indicating that these responses did not correlate well with protection from the disease. Given a suitable delivery system, aerosol vaccination of possums, used in conjunction with other control measures, may be a suitable method of reducing the spread of M. bovis from wildlife to domestic animals.     

The potential of oral vaccines for disease control in wildlife species

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.     

Protein requirements of preschool children consuming rice-milk,rice-toasted mung bean,and rice diets

Rice-milk and rice-toasted mung bean diets, and a high-protein rice (IR58 milled rice) were evaluated as part of a study on the protein requirements of toddlers consuming rice-based diets following the multilevel N balance method. Milk or mung bean contributed 1/3 of dietary N. At a daily energy intake of 418 kJ/kg body weight, weight losses were observed for all diets. The safe level of protein intakes obtained allowing 10 mg N/kg body weight/day for miscellaneous integumental losses and 15 mg N/kg body weight/day for growth was 1.11 g/kg body weight/day for the rice-milk diet and 1.34 g for the rice-mung bean diet. Preliminary data on four children indicate a safe level of protein intake of 1.44 g/kg/day for the IR58 milled rice. The protein quality of the high-protein rice as determined by the very short term N balance index on three children was 79–80% relative to milk.