Bovine intra-mammary challenge with Streptococcus dysgalactiae spp. Dysgalactiae to explore the effect on the response of Complement activity.

Recently published work as described by the authors highlighted the extent of Complement activity in bovine milk. Localised mastitis infection occurring in the mammary glands of dairy cows is readily detectable by the levels of somatic cells in milk. Thus, it is opportune to monitor Complement activity in milks in association with the animal's innate immune response to mammary infection. Preliminary screening of milk samples taken randomly showed that milk with a high somatic cell count (SCC) reduced growth of the Complement-sensitive strain E. coli O111 to a greater extent (P < 0·05) than when the marker microorganism was grown in milk heated for the purpose of inactivating Complement. A follow-up study set out to determine the effect on Complement activity when a sub-clinical mastitis infection was induced in the mammary gland of four lactating dairy cows. The effect of Str. dysgalactiae spp. dysgalactiae inoculation into selected individual udder quarters of the mammary glands of each animal was followed by monitoring of SCC levels in the milks from the segregated udder samples during subsequent milking. At 72 and 96 h post inoculation (PI), the SCCs for the challenged quarter were increased compared to normal values. At the same time, the bactericidal sequestration assay identified increased E. coli O111 inhibition that can be directly linked to greater Complement activity in those quarter milks affected by induced inflammation. Thus, it can be identified that the high SCC milks were more effective in limiting E. coli O111 growth. Milks from the unchallenged quarters in all four cows were significantly less effective at reducing growth of the assay strain (P < 0·05). An ELISA assay targeting specific activation components of the Complement pathways confirmed that greater bacterial inhibition observed during the bactericidal sequestration assay was attributable to higher Complement activity in the milk samples from the affected quarters, i.e., with higher SCC. The induced infection was confirmed as self-limiting in three of the affected animals and their SCC returned to normal levels within 14 d PI, while the fourth cow required brief antibiotic intervention.

Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.