Study On The Variation Of IgA And IgM Concentration In Milk And The Mechanisms Of The Affecting Factors

The experiments, composed of four parts, were firstly studied on the variations of the subclasses of immunoglobulin in serum and whey of bovine immune milk, normal milk and colostrum, and the analysis of the main proteins in whey by SDS-PAGE, secondly, studied the affecting factors, including parity, lactation stage, milk production, somatic cell counts score (SCS) and the SNP in the 5'flanking regions of bovine pIgR gene. Finally, studied the pIgR gene expression after immune stimulating, and their associations with specific IgA concentration, revealed the mechanism of IgA secretion and transportation. The more details were as followings:EXPERIMENT 1: A new method for immune milk production was used by implanting Antigen-releasing device (ARD). The results showed that total immunoglobulin concentration in serum of test group animals was higher than control group animals, whereas total IgG concentration in whey was up to peak at days 9, 17 and 32, respectively. These results correspond with the release of ARDs at days 0, 14 and 28. There were no significant changes (p <0.05) in total IgG and IgA on day 1, compared with that on day 5, with the exception of total IgM. The total immunoglobulin concentration in whey of colostrum was descended with the day in milk increased until maintaining at the low level. The main proteins of the colostrum, normal milk and immune milk were analyzed by SDS-PAGE. The results showed as follows: Five bands in SDS-PAGE, lactoferrin (Lf), bovine serum albulin (BSA), immunoglobulin G (H chain),β-lactoglobulin andα-lactoalbumin were detected. The IgG (L chain) was showed in colostrum at 0-3 d and immune milk at 32 d. Each whey protein of colostrum decreased from 0 d to 7 d until to the very low level. This difference was significant from 0 d to 3 d (p < 0.05). All whey proteins of immune milk in 15 d and 32d were higher than that of in mature milk.EXPERIMENT 2: The concentrations of IgA and IgM in milk from normal lactating cows were measured to determine whether the IgA and IgM concentration in milk is affected by the lactation number of cows, the lactation stage, milk production and milk somatic cell score (SCS). IgA and IgM in 284 milk samples were randomly chosen from more than 1,600 animals and then measured by means of ELISA test. The IgA and IgM concentration (means±standard deviations; logarithmic form) were 2.371±0.286 and 1.499±0.383, respectively. The mean IgA concentration (log) of more than fourth lactation cows were higher than the first, second and third lactation cows, whereas significantly differences in IgM concentration was observed among cows of the third lactation cows and the fourth lactation cows. The IgA concentration was low at early and middle lactation stage, but IgM increased when the day in milk increased. From cows with production of 20 kg to 25 kg and more than 30 kg was low in IgA concentration but it didn't affect the IgM concentration. Both milk IgA and IgM concentration (log) increased in a proportion to the increase in the SCS. The correlation analysis and path analysis demonstrated that the cumulative effects, estimated account for 0.320 and 0.358 for IgA and IgM respectively, indicating that these factors were not sufficient to explain variability in IgA and IgM concentration. Other factors such as hormonal and receptors are discussed in this study.EXPERIMENT 3: The pIgR transports dimeric IgA and pentamer IgM across the epithelial cell layers into the milk of mammary gland, the objective of this study was to detect the polymorphism in the 5'upstream region of bovine pIgR gene and their associations with IgA and IgM concentration n blood and milk. Based on the pIgR gene 5'flanking region sequence (NW₁74143) downloaded from the Map Viewer database of NCBI, 25 dairy cows genomic DNA samples were used for SNP detection. The 12-plex SNPstream genotyping system were used for total 189 dairy cows SNP genotyping and association between the polymorphisms and the IgA and IgM concentrations in milk and blood were detected. We found five SNP loci, and SNP1 and SNP2 were the A/G variation and SNP3, SNP4 and SNP5 were the T/C variation. Statistical analysis indicated that the SNP1 was significantly associated with milk IgA and blood IgM concentration (p < 0.05), whereas SNP3 and SNP5 significantly affected blood IgM concentration (p < 0.05), other SNPs and diplotypes had no effect on the IgA and IgM concentration (p > 0.05). Duncan's multiple-range test showed that the cows with genotype AB of SNP1 had much significantly difference on milk IgM and blood IgA concentration compared with the cows with genotype BB (p < 0.05), the cows with genotype AB were significantly higher milk IgM and blood IgA concentration than cows with genotype BB (p < 0.05). The different genotypes of SNP3 and SNP5 had significantly effect on blood IgM concentration (p < 0.05). Therefore, these findings implied that the polymorphisms of 5'flanking region in bovine pIgR gene had effect on IgA and IgM concentration in milk and blood; these results could provide references for further research on transportation mechanism of IgA and IgM into milk.EXPERIMENT 4: Forty eight KunMing white mice were divided into four groups, 12 mice per each group; they were injected different vaccines as following at days 4 after parturition, A group (CK) isotonic Na chloride, B group Lipase + isotonic Na chloride, C group ISCOM, D group Lipase + ISCOM. The samples, including blood, milk and mammary gland issues, were collected at days 8 and days 12 after parturition (days 4 and days 8 after injecting). The specific IgA concentration in mouse plasma and milk was determined by indirect ELISA. The results showed as follows: the specific IgA concentration in mouse plasma was not significant among four groups (p < 0.05), whereas that in mouse milk of B and D group at days 12 was higher than that at days 8. And the results of Real Time PCR also demonstrated that the pIgR gene expression in mammary gland at days 12 of group B and D was higher than that at day 8. Thus, the pIgR gene over-expression could increase the specific IgA level in milk. The results present in this study supported the previously researches that pIgR mediates the IgA transmembrane and secreted into the mammary secretions, such as saliva and milk, and the amounts of pIgR was the determinant factors affecting IgA level in milk.