Capsular polysaccharide conjugate vaccines against contagious bovine pleuropneumonia: Immune responses and protection in mice.

The immunogenicity of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) vaccines was investigated in BALB/c mice. Groups of mice were vaccinated with either (1) unconjugated capsular polysaccharide (CPS), (2) CPS covalently conjugated to ovalbumin via a carbodiimide reaction, (3) CPS non-covalently bound to latex microspheres, (4) CPS non-covalently complexed with rabbit anti-CPS IgG, and (5) whole inactivated, ultrasonically disrupted (WID) MmmSC. Only mice immunized with the CPS-ovalbumin conjugate exhibited a significant (P<0 small middle dot001) antibody response against CPS. Mice immunized with WID vaccine exhibited a high ELISA antibody titre against non-CPS (protein) antigens only. Mice given WID vaccine were immune against challenge with live MmmSC, and exhibited a significantly reduced degree of mycoplasmaemia (both in incidence and duration) as compared with non-vaccinated controls (P<0 small middle dot001). Mice immunized with the CPS-ovalbumin conjugate did not exhibit a reduction in mycoplasmaemia. The bactericidal activity of rabbit MmmSC-antiserum in an in-vitro growth inhibition test was related to the CPS antibody titre. This was not observed with antisera from the vaccinated mice. None of the mouse antisera exhibited growth inhibiting activity, irrespective of a high CPS or protein antibody titre (CPS-ovalbumin or WID vaccine groups, respectively). Thus, it would seem that protection against an MmmSC-induced mycoplasmaemia in the mouse is based upon cell-mediated rather than humoral immunity. The results suggest that conjugation to ovalbumin significantly increases the antibody response to CPS in the mouse; the lack of bactericidal activity of mouse anti-CPS as compared with rabbit anti-CPS in vitro suggests either that the titre of growth inhibiting antibodies is lower in the mouse or that the mechanism of growth inhibition differs between antibodies of the two species.

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures.

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

Age estimation: the state of the art in relation to the specific demands of forensic practise.

Age estimation in cadavers, human remains and living individuals may clarify issues with significant legal and social ramifications for individuals as well as for the community. In such cases methods for estimating age should fulfil the following specific demands: (1) they must have been presented to the scientific community, as a rule by publication in peer-reviewed journals, (2) clear information concerning accuracy of age estimation by the method should be available, (3) the methods need to be sufficiently accurate and (4) in cases of age estimation in living individuals principles of medical ethics and legal regulations have to be considered. We have identified and summarized the methods that essentially fulfil these specific demands. In childhood and adolescence morphological methods based on the radiological examination of dental and skeletal development are to be recommended. In adulthood, the accuracy of most morphological methods is much reduced. Here a biochemical method based on aspartic acid racemization in dentine provides the most accurate estimates of age, followed by special morphological dental and skeletal methods. The choice of method has to take account of the individual circumstances of each case. Most methods require either the consultation of specialised and trained scientists or an adequate calibration by the "user". Very few attempts have been made to find common standardisation, calibration and evaluation procedures or to develop means of quality assurance for methods of age estimation. Efforts in these directions are necessary to guarantee quality standards and adequate answers to the important legal and social issue of age estimation in forensic medicine.

Quality assurance in age estimation based on aspartic acid racemisation.

Estimates of the age of living and dead individuals, obtained in order to answer legal or social questions, require minimum quality standards in order to guarantee data quality. We present an outline strategy (with recommendations) for the attainment of quality assurance in age estimation based on aspartic acid racemisation. The strategy is based on a definition of minimum standards for laboratories, including documentation of procedures, methodology and levels of expertise, and the formulation of guidelines for intralaboratory and interlaboratory quality control.

A review of the methodological aspects of aspartic acid racemization analysis for use in forensic science.

Accurate age determination of adult cadavers and human remains is a key requirement in forensic practice. The current morphological methods lack accuracy and precision, require specialist training and are costly. The use of aspartic acid racemization (AAR) in human dentine provides a simple, cost-effective solution and the method can achieve accuracies of +/- 3 years at best. Currently, there are differences in AAR methodology between laboratories which produce different results on the rate of racemization in teeth. These inconsistencies must be resolved if the technique is to be successfully applied to age determinations in forensic cases. This paper reviews the differences in protocol which have been used, discusses how each method will affect the results obtained from AAR analysis and gives recommendations for optimization of the methological protocol as a first step towards international standardization.

Predicting protein decomposition: the case of aspartic-acid racemization kinetics.

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.

Hydroxyproline interference during the gas chromatographic analysis of D/L aspartic acid in human dentine.

The proportion of D- to L-enantiomers of aspartic acid in metabolically isolated proteins has been used by forensic scientists to estimate age at death. We have demonstrated the interference of a derivative of hydroxyproline (N-TFA isopropyl Hyp ester) with the N-TFA isopropyl L-Aspartic (Asp) acid ester during gas chromatography of amino acids. This has serious implications for the accurate quantification of the D- to L-Asp ratio extracted from collagenous proteins. Having demonstrated the potential for this co-elution in amino acid standards, acid-soluble dentine proteins and non-mineralised collagen, we argue that this problem can be overcome either by high resolution separation or by analysis of the (Hyp-poor) non-collagenous protein fraction.