Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses,and contact rates between possums and carcasses

Abstract

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses.

METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5–1 g with 0.2 mL liquid culture containing approximately 5 x 10⁷ cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present.

The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A ‘contact’ was recorded if these possums came within 40–50 cm of proximity loggers fitted to possum carcasses.

RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter.

In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4–6.7) days after placement.

CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.     

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.     

Water sorption properties of extruded zein films

Water interactions in extruded zein films were investigated through moisture sorption isotherms. Sorption isotherms of zein products were affected by composition and structure morphology. Zein powder showed moisture sorption hysteresis, which was not observed in extruded samples. Extruded samples held less moisture than zein powder, while films containing oleic acid showed further reduction in moisture uptake. Brunauer, Emmett, and Teller (BET) and Guggenheim, Anderson, and De Boer (GAB) models fitted well the moisture sorption isotherms of zein products. Monolayer values estimated by BET and GAB models were consistent with predictions based on zein structural models. Water vapor permeability (WVP) of zein films was affected by the relative humidity of testing environment. Higher relative humidity resulted in higher WVP.     

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection

AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG.

METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 10⁷ colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 10⁶ cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7–8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings.

In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10–11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group.

RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis.

CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.     

Fourier transform infrared spectra of drying oils treated by irradiation

Drying oils, such as linseed oil and tung oil, have the potential as coating materials to improve barrier properties of biobased packaging films. Oil drying is a chemical reaction in which polyunsaturated fatty acids undergo autoxidation. During drying, oils polymerize and form water-resistant films. However, drying rates tend to be too slow for practical applications. Metal driers are used in the paint industry to accelerate drying, but often driers are not safe for food contact. The objective of this work was to investigate the effect of ionizing radiation on the oxidation or drying rate of drying oils. The effect of irradiation dose on the drying rate of linseed and tung oils was monitored by FTIR spectroscopy. The peak at 3010 cm (-1) was found to be a useful index of oxidation rate. The decrease in peak intensity with time was fitted with exponential functions of the form Abs = Abs 0 exp (- t/ k), where Abs 0 is the initial absorbance and 1/ k is the rate constant for the oxidation process. Values for k were 9.91 ( R (2) = 0.98), 6.59 ( R (2) = 0.95)n and 6.44 ( R (2) = 0.97) for radiation levels of 0, 50, and 100 kGy, respectively. The k values suggested that the oxidation rate increased as the radiation dose increased from 0 to 50 kGy. A further increase to 100 kGy had only a limited effect.