The potency of immune response to S. aureus mastitis in dairy cows is defined by the concentrations of lymphocyte, leucocytes and cytokines released during its invasion of the mammary gland [47, 48, 49, 50, 51]. Studies have indicated that S. aureus enterotoxin A (SEA), B (SEB), C (SEC) and toxic shock syndrome toxin-1 (TSST-1) usually act as superantigens (SAg) by way of activating T-lymphocytes, mononuclear cells and also initiate the release of IL-1, 2, 4, 6, 10 and12 [52, 53, 54, 55]. SEA, SEB, SEC and TSST-1 have been shown to be potential inducers of polyclonal activation of bovine T-cells, CD4+, CD8+, and monocytes with release of interferon-c (IFN-c) and tumor necrosis factor-a (TNF-a) [56, 57]. Changes in concentration of cytokine synthesis are regarded as a crucial factor in scoring the inflammatory responses to S. aureus infection.
To the very best of our knowledge this present work is the new study that demonstrates the effect of laboratory-prepared killed S. aureus vaccine from local Malaysian isolate on Friesian dairy cows. The current study had indicated that there was a significant increase in IL-10 concentration in vaccinated group at 0, 3,12 hours and week 2 post primary vaccination (PPV) with about 1, 1, 1 and 2 folds increased respectively. There was also a significant increase in IL-10 concentration in vaccinated group C at 3, 8, 12 and 24 hours during the vaccine booster phase (PB) with about 2, 2, 1 and 2 folds increased respectively. During the bacterial challenge phase at 0, 3 and 12 hours PC there was also a significant increase in IL-10 in vaccinated group with about 1, 1 and 1 folds increased respectively. Though at 24 hours PC there was a significant difference in IL-10 recorded in positive control group with about 3 folds increased. There was also a significantly increased IL-12 concentration in the vaccinated group at 24 hours, weeks 1 and 2 PPV with about 223, 3 and 2 folds increased respectively. During the vaccine booster phase at 0, 3, 8, 12 and 24 hours PB there was a significant increase of IL-12 in the vaccinated group with about 4, 8, 5, 3 and 1 folds increased respectively. The bacterial challenge phase at 12, 24 hours, days 7 and 14 PC recorded a significant increase in IL-12 concentration in the vaccinated group with about 2, 2, 5 and 5 folds increased respectively. Although, at 3 and 8 hours PC there was a significant increase in IL-12 noted in the positive control group with a surge of about 80 and 2 folds increased respectively which return to 0 fold afterwards at 12 hours PV. The extended increase in IL-10 and IL-12 in the vaccinated group across the phases of PPV, PB and PC in the present study is an indication suggesting the potency of the prototype killed vaccine in suppressing the effect of S. aureus intramammary challenge in the Frisian dairy cows. The result of the present study is in harmony with the study reported on cytokines as markers in S. aureus mastitis of bovine mammary gland which indicated that the suppression of these bacteria PPV or PC is usually accompanied by an elevated IL-10 and IL-12 concentration . There are similarities between the findings of the present study and that of D’andrea, (1993) where a different model and an in-vitro studies indicated that a negative correlation existed between IL-10 and IL-12 concentration with an explanation that IL-10 plays a down regulatory role on IL-12 which can be why it limits the duration of IL-12 in most bacterial infection . This may suggest why IL-12 concentration was downregulated by IL-10 at 0, 3 and 8 hours PPV and at 0 hours PC in the current study. There are studies where a positive strong correlation had been reported between IL-10 and IL-12 during bacterial infection . Bernthal,(2011), in his study described on the protective roles of cytokines in S. aureus infection indicated that the increase in the concentration of most cytokines is a positive sign of prompt defence response against S. aureusinfection .
The findings of the present study further supports the reports of , in a study conducted on the characterisation of cytokines in mammary glands infected with S. aureus which related that most cytokines generally pick up increasingly up to about 6 hrs PC (as seen in IL-12 in the present study) and gets to its peak by 12 hrs PC, but a gradual decline was noticed in their concentrations 24hrs to about 48hrs PC (as observed in IL-10 the present study) . Though Wang, (2015) in the same study also maintained that at 24 hrs to 48 hrs PC, elevated levels of IL-10 can be sustained but not all the time which will eventually fade away by 48 hrs PC. The findings of the current study are consistent with the study of  in an investigation on cytokines as markers in S. aureus mastitis infection of bovine mammary gland where he indicated that there exist an emergence of IL-12 at 7hrs PC ( as seen in IL-12 in the current study) and 24hrs PC (as seen in IL-12 and IL-10 in the current study) which later declined 32hrs PC .
In a more related study, the excessive production of IL-10 PC or PPV had been suggested to be associated with the recruitment of more CD8 + T suppressor cells , altering pathophysiology of the bovine mammary gland and early recruitment of neutrophils . The shift from Th2 cells production to Th1 helper cell production as a result of IL-10 elevation in S. aureus infection had been confirmed and documented as a fundamental phenomenon in immune response . The results of the current findings matches those observed by Hessle et al., 2000 in a study on bacteria being potent inducers of IL-10 production, where they indicated an elevated increase in IL-10 following S. aureus infection in cows, . The same findings in the association of IL-10 elevation following S. aureus challenge was concluded by Bannerman et al., 2004 in their study on the ability of S. aureus to elicit differential innate immune responses following intramammary infection . In accordance with the present results, previous study by Armstrong, (1996) on alveolar macrophages and peripheral blood monocytes also reported that generally high levels of IL-10 were observed in challenge quarters during experimental mastitis infection (Armstrong et al., 1996).
IL-12 (produced by macrophages) plays the special role of mediating between the innate and specific immune response  thereby bears the name maintainer of antibody production. The expression of IL-12 have also been detected in dairy cows during acute phases PC indicating their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection . Riollet, (2001) had also reported that increased concentrations of IL-12 is a norm observed in serum samples obtained from dairy of cows challenged with S. aureus . Previous studies have reported that intramammary challenge with S. aureus elicited both localised and systemic response in the vaccinated cows which also indicated that cytokines are potent inducers of acute phase responses during natural or experimental bacterial challenge. This was obtainable in the report of Bannerman, (2004) in an experimental study on S. aureus challenge in lactating cows which indicated that IL-12 were seen at 32 h PC which remained elevated through 40hrs PC . The increase in IL-12 concentration observed in positive control group in the present study at 3 hrs PC which was about 80 folds increase suggested that such an increase is directly associated with the deterioration of udder health due to S. aureus challenge, this is seen in cases where the titre values of cytokines increased greatly with increased inflammation of the mammary glands  as seen in the positive control group.
An increased concentration of cytokines is considered strong evidence in its role in inducing the migration of leukocytes and production of antibody . However, contrary opinions have been indicated by Hagiwara, where they clearly stipulated that there was no relationship between cytokine concentration and stages of mammary inflammation in mastitis cows . Taylor, (1997) studied the immunomediative role of increased cytokines from mastitic cows and stipulated that the causative agent of mastitis does not relatively determine alteration in cytokine response . There are several possible explanations for the discrepancies which could be attributed to different animal model used in the S. aureus experimental mammary challenge, gram status of the bacteria (positive or negative), types of cytokine estimated, the specificity of assay used, individual animal heterogeneity and functional impairment of white blood cells in infected udder.
Based on the data obtained and analysed from the current study, we can hypothesis that our vaccine provided protection against S. aureus mastitis in Friesian cows. This explains why the IL-12 and IL-10 levels were significantly higher in vaccinated group than in the positive control group as the major role of IL-12 is associated with regulating Th-1 and Th-2 lymphocytes, mediating between the innate and acquired immunity [69, 70] and its enhancing role in immune responses in the mammary gland during S. aureus infection. While IL-10 is associated with the immune cell signalling and T- cell shift production against S. aureus challenge in the Friesian cows.
Recently, the applications of APPs biomarkers as indicators of inflammation of the mammary gland have gained a wider acceptance in the assessment of udder health. Acute phase proteins constitute part of innate immune components whose concentration varies with respect to stimuli of external or internal origin . APPs concentration such as SAA and Hp increases during early phase of reaction of cows to bacterial challenge or infection [72, 73]. These two APPs aid in determining the progress of inflammatory conditions thereby acting as a valuable diagnostic and prognostic tool in cases of mastitis in dairy cows [74, 41].
To the very best of our knowledge, this current study is the new information on the responses of SAA and Hp to locally developed killed S. aureus vaccine of Malaysian isolate on lactating Friesian cows. The current study had indicated that there was a significant increase in Hp concentrations of vaccinated group at 12 and 24 hours PPV with about 4 and 3 folds increased respectively. During the vaccine booster phase there was also a significant increase in the Hp concentration of the vaccinated group at 8 and 12 hours PB with about 10 and 13 folds increased respectively. During the bacterial challenge phase there was a significant increase in Hp concentration of the positive control group at 0, 3, 24 hours and day 7 PC with about 17, 83, 80 and 24 fold increased respectively. Interestingly, at about 8 hours PC there was a significant increase observed in the Hp concentration vaccinated group with about 35 fold increased. The current study had also indicated a significant increase in SAA concentration of vaccinated group at 0, 3, 8, 12, 24 hours and weeks 1 and 2 PPV with about 5, 6, 3, 4, 9, 5 and 16 folds increased respectively. During the vaccine booster phase there was also a significant increase in SAA concentration of vaccinated group at 0, 3, 8 and 12 hours PB with about 29, 11, 8 and 8 folds increased respectively. During the bacterial challenge phase there was a significant increase in SAA concentration of positive control group at 3, 8, 12, 24 hours, days 7 and 14 PC with about 4, 11, 3, 6, 8 and 1 folds increased respectively. The elevated levels of Hp and SAA of vaccinated group PPV and PB in the present study are a prominent and sensitive indication that the prototype killed S. aureus vaccine has the potential to curtail inflammatory tendencies of S. aureus challenge. The reduced SAA and Hp concentrationd in the vaccinated group and increased concentrations in the positive control group PC is quite an indication that the prototype killed S.aureus vaccine had played a vital role of immune modulation and bacterial opsonisation in the vaccinated group.
The findings of the current study are consistent with the reports of , who studied the acute phase proteins in serum from dairy cows with clinical case of mastitis, where they concluded that SAA and Hp have both been identified as a major acute phase protein in cattle associated with mastitis infection and that their elevation and detection in serum samples is always a clear indication of acute phase response (APR) to opsonisation, infection or challenge. In a more detailed report by  who conducted a study on the early pathogenesis and inflammatory responses in experimental bovine mastitis due to gram positive bacteria stipulated that increased in SAA and Hp is a tool of measuring the severity of mastitis infection in both experimentally and naturally challenged cows in dairy farms.
In accordance with present results, previous study by  had also reported that SAA and Hp in serum of bovine mastitic cases increases during response to inflammation, thereby suggesting that the demonstration of these APPs in the serum is a key diagnostic value in differentiating mastitis cows from non-mastitis ones. Increased levels of SAA and Hp have been associated with the occurrence of acute or sub-acute mastitis in a similar study conducted by  where the results were in harmony with the present study thereby indicating that increased APPs is associated to sub-clinical and clinical forms of mastitis.
There are similarities between the response of SAA and Hp in the current study and those described by [76, 77] in a study conducted on APPs as a diagnostic tool for bovine sub-clinical mastitis, where the report indicated that SAA and Hp were on the increased during post infection period thereby advocating that both APPs are always on the rise in response to inflammatory disease such as mastitis in cows. The findings of the present study also corroborates with the reports of  who carried out a study on Hp and SAA in serum during acute and chronic experimentally induced S. aureus mastitis where they concluded that cows challenged with S. aureus developed a high concentration of SAA in serum during an acute phase of the challenge and this is in harmony with findings observed in SAA response in the present study.
Furthermore, a study on the acute phase response in naturally occurring coliform mastitis related that increased levels of both SAA and Hp were observed during an experimental challenge of dairy cows  which tends to be in harmony with the findings in the present study. The observed increased SAA in the current study also agrees with study of  who measured the acute phase protein concentrations in serum samples from healthy cows, cows with clinical mastitis and cows with extra mammary inflammatory conditions where they reported that the SAA concentration in bovine serum with clinical mastitis had an elevation indicative of its response to bacterial invasion.
This simultaneous elevation in serum concentrations of both SAA and Hp as seen in the current work is in agreement with observations made in several studies on APP in dairy cows. Eckersall in their study on the acute phase proteins in serum from dairy cows with clinical case of mastitis had indicated a significant positive strong correlation between Hp and SAA . In contrast to our findings, however, only serum Hp concentration elevated in mastitis cows as was reported by , whereas both SAA and Hp levels have increased extensively PC in our findings. A study on the local immunization and impacts on the response of dairy cows to mastitis was conducted by , where their study concluded that only serum Hp was a better marker in assessing impact of local immunization PC in dairy cows. This is in contrary to other studies and the present study where both SAA and Hp have proven to be potential biomarkers of inflammatory tendencies caused by S. aureus in dairy cows. In another study by Pyörälä, they conducted a research on the acute phase proteins in naturally occurring bovine mastitis caused by different pathogens and they concluded that Hp is never a better tool to be used as bovine mastitis marker as SAA proved to be more reliable and sensitive .
A possible explanation for the discrepancies in the findings of different authors as it relates to SAA and Hp reliability as markers of bovine mastitis could be attributed to different geographical locations where such studies were conducted, samples used for the detection of APPs, type of APP assayed and the fact that lower APPs in serum can be as a result of leakages via the (disrupted) blood milk barrier into the milk during mastitis . It also seems possible that the inconsistency in these results from different authors might be partly explained by the different methods used in measuring these variables, variation in duration and severity of mastitis in experimentally or naturally infected cows and possibly due to different animal models used in the study of mastitis infection .
A very important and relevant point outlined by the present study was the fact that the vaccinated Friesian cows showed evidence of lower degree of Hp and SAA responses in PC group as compared to the positive control group. Friesian cows in the positive control group had evidence of increased Hp and SAA indicating the existence of bacterial proliferation in the mammary gland of the positive group more than the vaccinated group. Based on these findings in the present study, we can hypothesise that the prototype killed S. aureus mastitis vaccine conferred immunity against S. aureus challenge mastitis infection in vaccinated lactating Friesian cows. Where lower concentrations of APPs were measured in the vaccinated group as compared to the positive control group PC.