Synthesis of Sporidesmin Based Antigens

<p>In a program aimed at developing a chemically derived vaccine against sporidesmin-A, the toxin which causes the pasture disease facial eczema, two haptens have been prepared, coupled to protein carriers, and tested for immunogenicity. The hapten, 2-amino-5-chloro-3,4-dimethoxybenzyl alcohol has been synthesised from vanillin and the general synthetic methods developed in the course of this work have been used to prepare a large number of related vanillin derivatives for use in cross-hapten studies. The carbon-13 nuclear magnetic resonance spectra of 16 vanillin derivatives have been obtained, and two independent methods of deducing vanillin substitution patterns have been developed. 5-chloro-6, 7-dimethoxy-N-methyl-lH-indole has been prepared by a new route and its 3-oxoacetic acid derivative has been synthesized for use as a hapten. The Vilsmeier-Haack intermediate, l-methyl-3-(N,N-dimethylimonio) methyl-lH-indole chloride was isolated and a number of possible synthetic routes from it to pyrroloindoles were explored. The vanillin and indole haptens have been coupled to protein carriers to form antigens which generated a low antibody response. However, the use of mycobacterium phlei as a carrier greatly increased the antibody response but the antibodies produced did not interact strongly with sporidesmin-A.</p>

Synthesis of Sporidesmin Based Antigens

<p>In a program aimed at developing a chemically derived vaccine against sporidesmin-A, the toxin which causes the pasture disease facial eczema, two haptens have been prepared, coupled to protein carriers, and tested for immunogenicity. The hapten, 2-amino-5-chloro-3,4-dimethoxybenzyl alcohol has been synthesised from vanillin and the general synthetic methods developed in the course of this work have been used to prepare a large number of related vanillin derivatives for use in cross-hapten studies. The carbon-13 nuclear magnetic resonance spectra of 16 vanillin derivatives have been obtained, and two independent methods of deducing vanillin substitution patterns have been developed. 5-chloro-6, 7-dimethoxy-N-methyl-lH-indole has been prepared by a new route and its 3-oxoacetic acid derivative has been synthesized for use as a hapten. The Vilsmeier-Haack intermediate, l-methyl-3-(N,N-dimethylimonio) methyl-lH-indole chloride was isolated and a number of possible synthetic routes from it to pyrroloindoles were explored. The vanillin and indole haptens have been coupled to protein carriers to form antigens which generated a low antibody response. However, the use of mycobacterium phlei as a carrier greatly increased the antibody response but the antibodies produced did not interact strongly with sporidesmin-A.</p>

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, γ-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the β-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit β (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult β-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

Health Information Integration Based on Network Platform in Postpartum Maternal and Infant Health Care

Objective: The self-care ability of puerpera is poor, and their health knowledge of maternal and infant and care skills is relatively poor. The aim of our study was to investigate the effect of health information integration based on network platform in the postpartum maternal and infant health care. Methods: A total of 80 maternal women admitted to our hospital from September 2018 to March 2019 were randomly divided into a control group and a study group, with 40 patients in each group. The puerpera in control group received regular telephone visits after discharge. The puerpera in study group received health information integration based on network platform. The uterus recovery of puerpera in two groups at 42 days postpartum, as well as the lochia, bloating, nipple splitting and breastfeeding behaviors were compared. The time of jaundice regression and umbilical cord detachment of neonates in t two groups, as well as the incidence of facial eczema and umbilical inflammation were compared. Results: The uterus recovery rate and exclusive breastfeeding rate of puerpera in study group were higher than those in the control group at 42 days postpartum, and the incidences of abnormal lochia, swollen breasts, and nipple splitting were lower than those in the control group. The difference between the two groups was statistically significant (P < 0.05). The neonates in study group were lower than the observation group in terms of the days of jaundice regression, facial eczema, and umbilical inflammation. The difference between the two groups was statistically significant (P < 0.05). However, there was no significant difference in the days of umbilical cord shedding (P > 0.05). Conclusions: The health information integration based on network platform in postpartum maternal and infant health care can effectively improve maternal breastfeeding compliance and reduce the adverse symptoms of maternal and neonatal discharge.