Acute Phase Proteins (Haptoglobin and Serum Amyloid A) and Pro-Inflammatory Cytokines (IL-12 and IL-10) in Cows Vaccinated with Prototype Killed S. aureus Mastitis Vaccine and Challenged with S. aureus Mastitis Infection.

Background The economic downturn experienced by farmers and the fear of milk borne infection are of a greater public health concern. Haptoglobin, Serum Amyloid A, IL-12 and IL-10 in lactating Friesian cows vaccinated with prototype killed S. aureus mastitis vaccine and challenged with S. aureus were evaluated. Bacterin concentration at 10 8 cfu /ml of the local isolate of S. aureus was adjuvanted with KAl(SO₄)₂. Six lactating Friesian cows were grouped into A= Negative control, B = Positive control and C = vaccine group. Group C was vaccinated intramuscularly with 2ml of the monovalent vaccine, groups A and B with physiologic normal saline. Groups B and C were later challenged with the live bacterium via intramammary route . Result There was a significant increase in IL-10 concentration in group post primary booster and during bacterial There was also a significantly had significant difference was a significant difference C. During bacterial at 24 hours and day 7 PC there was a significant difference in group B. At 8 hours PC there was a significant difference in group C. For Serum Amyloid A, during PPV at 0, 3, 8, 12, 24 hours and weeks 1 and 2, the concentrations was significantly different in groups C. During PB at 0, 3, 8 and 12 hours PB there was a significant difference in groups C. During the bacterial challenge phase at 3, 8, 12, 24 hours, days 7 and 14 PC there was a significant difference in group B. At 0 hour PC there was a significant increase observed in group C. linked immunosorbent assay for Bovine SAA performed using a commercially available CUSABIO ELISA CUSABIO LTD, The assay employed the competitive inhibition enzyme immunoassay technique where the 96-well plates were pre-coated with goat-anti-rabbit antibody. Samples were added to the wells with an antibody specific for SAA and Horseradish Peroxidase (HRP) conjugated SAA. A blank well was set without any followed by addition of 50μl of Standard and Sample per well. further then blotted 50μl 50μl Stop each and optical density each using microplate reader average of the duplicate readings for each standard and were calculated and subtracted from average optical density of A standard curve was then created and the concentration read the standard curve however by the dilution factor of the

3 against mastitis infection in dairy cows.

Background
Mastitis is a disease of economic importance in the dairy sector [1,2,3]. It is caused by coliform bacteria, staphylococci and streptococci with a range of clinical appearances such as fever, feed withdrawal and reduced milk production [4,5,6,7]. Dairy cows exposed to mastitis do express a complex interaction between the cow's defence mechanism and the specific bacterial virulence factors like; toxins, polysaccharide, cell surface factors, secreted factors, gene, biofilm, capsule and membrane proteins [8,9,10]. These virulent factors hence trigger both the cellular and humoral immune responses [11,12,13].
Accordingly, cytokines responses have been found to play crucial role in the pathogenesis of most bacterial infection [14,15,16,17].
Cytokines as immunological mediators are involved in the immune build up in inflammatory conditions of the udder [18,19]. They are known for their cell signalling roles and anti-inflammatory pathways during infection as they strike a balance between the cellular and humoral immunity [20,21,17]. An experimental study following S. aureus intramammary challenge in lactating cows indicated that IL-12 is a good marker in response to S. aureus challenge and vaccination [22]. In a more related study IL-10 concentration following S. aureus infection in cows also indicated an increased in its concentration hence regarded as a good indicator of immune activation in inflammatory condition [23]. Early cytokine transcriptional alterations could be an important yardstick in unravelling their role in the pathophysiology of S. aureus mastitis infection.
Acute phase proteins are produced in the liver, synthesised by the agranulocytes, epithelial cells and found in different concentrations based on the state of animal health [24,25,26]. During any form of tissue injury, acute phase responses (APR) are initiated [27,28]. At this stage the APP either increases or reduces in concentration [29, 30, 31, 4 32] reflecting the degree of damages in the tissue [24]. In cases of clinical mastitis SAA and Hp have been indicated as APP biomarkers of inflammation in response to cytokine stimulation [33,34,35,36,37,38]. APPs are considered a powerful diagnostic measure in field or experimental mastitis studies. The pathogenesis of mastitis have been reported to involve inflammatory reactions [39,40,41] which results into increased production of Acute-Phase Proteins (APP) [31,32]. Recently, APPs are considered as important diagnostic biomarkers not only in veterinary diagnostics but also in human medicine [42,43,44].
In the present study, the responses of acute phase proteins (Haptoglobin and Serum Amyloid A) and pro-inflammatory cytokines (IL-12 and IL-10) in lactating Friesian cows vaccinated with prototype killed S. aureus mastitis vaccine and challenged with S. aureus mastitis infection were studied. These biomarkers were opted to be studied in the present study to determine the efficacy of the developed prototype killed S. aureus mastitis vaccine.

Results
Interleukin 10 (IL-10) The means of IL-10 concentrations of groups A, B and C are presented in Fig. 1. During the acute phase PPV at 0 hour the concentrations of IL-10 in groups A, B and C were 3.00 ± 0.038 ng/ml, 3.866 ± 0.038 ng/ml and 5.08 ± 0.038 ng/ml respectively with a significant difference (P < 0.05) in groups C having about 1 fold increased. The concentrations of IL-10 at 3 hours PV indicated a significant difference (P < 0.05) in groups C (with about 1 fold increased) and was 4.28 ± 0.014 ng/ml as compared to negative control which was 3.38 ± 0.014 ng/ml. At 8, 24 hours and week 1 PPV there was no significant increase (P > 0.05) between the groups. At 12 hours PPV there was a significant difference (P < 0.05) in group C ( with about 1 fold increased ) and was 4.79 ± 0.05 ng/ml as compared to negative 5 control which was 3.19 ± 0.05 ng/ml. At week 2 PPV there was a significant difference (P < 0.05) in group C with 2 folds increased (5.40 ± 0.04 ng/ml ) as compared to negative control (2.66 ± 0.04 ng/ml). During the vaccine booster phase at 3, 8, 12 and 24 hours PB there was a significant difference (P < 0.05) in groups C (3.86 ± 0.02 ng/ml, 10.77 ± 0.03 ng/ml, 6.78 ± 0.03 ng/ml and 4.06 ± 0.02 ng/ml ) as compared to the negative control (2.37 ± 0.02 ng/ml, 5.35 ± 0.03 ng/ml, 6.15 ± 0.03 ng/ml and 2.08 ± 0.02 ng/ml) with 2, 2, 1 and 2 folds increased respectively. During the bacterial challenge phase at 0, 3 and 12 hours PC there was a significant difference (P < 0.05) in group C (5.35 ± 0.03 ng/ml, 12.16 ± 0.03 ng/ml, 12.30 ± 0.03 ng/ml) as compared to the negative control (3.48 ± 0.03 ng/ml, 9.46 ± 0.03 ng/ml, 11.78 ± 0.03 ng/ml) with about 1, 1 and 1 folds increased respectively. At 24 hours PC there was a significant difference (P < 0.05) in group B (15.66 ± 0.05 ng/ml) as compared to the negative control (5.85 ± 0.05 ng/ml) with 3 folds increased. There was no significant increase (P > 0.05) at 8 hours, days 7 and 14 PC in all the treatment groups. The means of IL-12 concentration of Groups A, B and C are presented in Fig. 2. During the acute phase PPV at 0, 3, 8 and 12 hours there was no significant difference (P > 0.05) between the groups. The concentrations of IL-12 at 24 hours PPV indicated a significant difference (P < 0.05) in groups C ( with about 223 folds increased ) and was 422.5 ± 58 ng/ml as compared to negative control which was 1.89 ± 58 ng/ml. At weeks 1 and 2 PPV there was a significant difference (P < 0.05) in group C (with about 3 and 2 folds increased) which were 531.6 ± 0.2 ng/ml and 279.8 ± 0.14 ng/ml as compared to the negative control which was 196 ± 0.23 ng/ml and 169.8 ± 0.14 ng/ml respectively.
During the vaccine booster phase at 0, 3, 8, 12 and 24 hours PB there was a significant 6 difference (P < 0.05) in groups C (108.9 ± 14.8 ng/ml, 135.9 ± 8.6 ng/ml, 261.   The means of Hp concentrations of groups A, B and C are presented in Fig. 3. During the acute phase PPV at 0 hour the concentrations of Hp in groups A, B and C were 25381 ± 19617 ng/ml, 26217 ± 19617 ng/ml and 25158 ± 19617 ng/ml respectively with no significant difference (P > 0.05) between the groups. At 3 and 8 hours PPV there was no significant difference (P > 0.05) between the groups. At 12 and 24 hours PPV there was a significant difference (P < 0.05) in group C (with about 4 and 3 folds increased respectively) and were 21794 ± 1601 ng/ml and 65958 ± 2795 ng/ml as compared to the negative control which were 5236 ± 1601 ng/ml and 19842 ± 2795 ng/ml respectively. At weeks 1 and 2 PPV there was no significant increased (P > 0.05) in groups B and C.
During the vaccine booster phase at 0, 3 and 24 hours PB there was no significant difference (P > 0.05) between the groups. At 8 and 12 hours PB there was a significant 7 difference (P < 0.05) in group C (with about 10 and 13 folds increased respectively) and were 149307 ± 3372 ng/ml and 201804 ± 3710 ng/ml as compared to the negative control which were 14418 ± 3372 ng/ml and 15062 ± 3710 ng/ml respectively.
During the bacterial challenge phase at 0, 3, 24 hours and day 7 PC there was a significant difference (P < 0.05) in group B (with about 17, 83, 80 and 24 fold increased respectively) and were (25588 ± 513 ng/ml, 28673.4 ± 205 ng/ml, 101089 ± 6751 ng/ml and 41228 ± 4147 ng/ml respectively) as compared to the negative control group which were 1463 ± 513 ng/ml, 342.7 ± 205 ng/ml, 1264 ± 6751 ng/ml and 1714 ± 4147 ng/ml respectively. At 8 hours PC there was a significant difference (P < 0.05) observed in group C (with about 35 fold increased) and was 58790 ± 395 ng/ml as compared to the negative control group which was 1650 ± 395 ng/ml. At 12 hours and day 14 PC there was no significant increase (P > 0.05) observed between the groups. 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 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 [58]. 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 [59]. 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 [22]. 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 [60].
The findings of the present study further supports the reports of [61], 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) [61]. 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 [62] 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 [62].
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 [52], altering pathophysiology of the bovine mammary gland and early recruitment of neutrophils [63].
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 [64]. 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, [23]. 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 [22]. 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 lactating cows which indicated that IL-12 were seen at 32 h PC which remained elevated through 40hrs PC [22]. 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 12 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 [66] 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 [61]. 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 [67]. 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 [68]. There are several possible explanations for the discrepancies which could be attributed to different animal model 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 13 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 [71]. 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]. The findings of the current study are consistent with the reports of [72], 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 [73] 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 [74] 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 [75] 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 subclinical 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 [78] 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 [79] 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 [80] who  [72]. In contrast to our findings, however, only serum Hp concentration elevated in mastitis cows as was reported by [81], 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 [82], 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 [83].
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 [80].
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 [31]. 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.

Conclusion
In conclusion, IL-12, IL-10, SAA and Hp have the potential to be used as biomarkers to evaluate the vaccine efficacy. Therefore, in this study it can be concluded that the developed prototype killed S. aureus mastitis vaccine using local isolates was able to stimulate responses of acute phase proteins and pro-inflammatory cytokines and the pattern of responses post challenged with mastitis infection showed evidence of protection pattern.It can be concluded that mastitis infection in dairy cows can be protected via vaccination.

Ethical approval
The laid guidelines of the Institutional Animal Care and use committee (IACUC) of the Universiti Putra Malaysia was strictly adhered to and the experimental trial was approved by IACUC with approval code as UPM/IACUC/AUP-R072/ 2016. To this effect a written informed consent letter was obtained to use the animals for the present study.

Sources of Experimental animals
The experimental lactating Friesian dairy cows were sourced from the Faculty of Veterinary Medicine, University Putra Malaysia Farm.

Bacterial Segregation/Identification
Mastitis positive milk samples were smeared on blood agar (OXOID ® England) and subjected to gram staining. Catalase test and analytical profile indexing (API) were further used for bacterial profiling. The S.aureus corresponds fully with strain HC1340 with Accession CP012011.1.

Experimental Design
Six healthy lactating Friesian dairy cows weighing about 300-350kg body weight between the ages of 5 -7 years were selected randomly for the purpose of this study. The cows were divided into 3 main groups with 2 animals each. Group A was the Negative control, Group B the Positive control and Group C is the 10 8 cfu/ml of the killed bacteria vaccine group. Groups A and B were inoculated intramammary (IM) with 2ml of physiologic normal saline while Group C was vaccinated with 10 8 cfu/ml of the killed bacteria vaccine IM.

Euthanasia/ Animal Slaughter Post Experiment
Experimental animals were not anaesthetized but rather slaughtered post experiment by the institution based certified veterinary animal slaughter officer working on the farm. The carcass were properly discarded by burying.

Prototype Killed Mastitis Vaccine Preparation
The S. aureus isolates were grown in Brain heart infusion but with a little modification as described by [45,46], autoclaved at 121 0 C for 15 minutes to achieved sterility and incubated at 37 0 C for 24hrs. The broth was however killed using 0.5% formalin at 37 0 C for 24hrs. Aluminium potassium sulfate adjuvant was suspended in normal saline to achieve 1% (Mohi-ud-din et al., 2014). An adjustment of 10 8 cfu/ml of the killed bacteria was finally prepared. The vaccine was sub-cultured on a Mannitol agar, MacConkey agar, Sabouraud dextrose agar and blood agar to ascertain effective killing and noncontamination of the vaccine by bacteria and fungi [45]. There was no evidence of growth on the entire agar used which was an indicative of the killed vaccine sterility.

Enzyme linked immunosorbent assay for IL-10 and IL-12
Enzyme linked immunosorbent assay for Bovine IL-10/IL-12 was performed using a competitive inhibition enzyme immunoassay kit from the CUSABIO BIOTECH CO., LTD, China. A 96-well plates were pre-coated with goat-anti-rabbit antibody. Samples were added to the wells with an antibody specific for IL-10/IL-12 and Horseradish Peroxidase (HRP) conjugated IL-10. A Blank well was set without any solution followed by addition of 50μl of Standard and Sample per well. Then 50μl of HRP-conjugate was added to each well (not to Blank well), 50μl antibody was further added to each well. This was then incubated for 1 hour at 37°C followed by aspiration and washing using a Wash Buffer (200μl). The plate was then blotted and 50μl of Substrate A and 50μl of Substrate B were added to each and incubated for 15 minutes at 37°C. Finally, 50μl of Stop Solution was added to each well and optical density of each well was determined within 10 minutes using a microplate reader set to 450 nm.

Bovine Serum Amyloid A (SAA) ELISA Assay
Enzyme linked immunosorbent assay for Bovine SAA was performed using a commercially available CUSABIO ELISA kit from CUSABIO BIOTECH CO., LTD, China. The assay employed the competitive inhibition enzyme immunoassay technique where the 96-well plates were pre-coated with goat-anti-rabbit antibody. Samples were added to the wells with an antibody specific for SAA and Horseradish Peroxidase (HRP) conjugated SAA. A blank well was set without any solution followed by addition of 50μl of Standard and Sample per well.
Then 50μl of HRP-conjugate was added to each well (not to blank well), 50μl antibody was further added to each well. This was then incubated for 1 hour at 37 °C followed by aspiration and washing using a Wash Buffer (200μl). The plate was then blotted and 50μl of Substrate A and 50μl of Substrate B were added to each and incubated for 15 minutes at 37 C. Finally, 50μl of Stop Solution was added to each well and optical density of each well was determined within 10 minutes using a microplate reader set to 450 nm. The average of the duplicate readings for each standard and sample were calculated and subtracted from the average optical density of blank. A standard curve was then created and the concentration read from the standard curve was however multiplied by the dilution factor of the samples.

Bovine Serum Haptoglobin (Hp) Assay
Enzyme linked immunosorbent assay for Bovine Hp was performed using a commercially available CUSABIO ELISA kit from CUSABIO BIOTECH CO., LTD, China. The assay employed the competitive inhibition enzyme immunoassay technique where the 96-well plates were pre-coated with goat-anti-rabbit antibody. Samples were added to the wells with an antibody specific for Hp and Horseradish Peroxidase (HRP) conjugated Hp. A blank well was set without any solution followed by addition of 50μl of Standard and Sample per well.
Then 50μl of HRP-conjugate was added to each well (not to blank well), 50μl antibody was further added to each well. This was then incubated for 1 hour at 37 °C followed by aspiration and washing using a Wash Buffer (200μl). The plate was then blotted and 50μl of Substrate A and 50μl of Substrate B were added to each and incubated for 15 minutes at 37 °C. Finally, 50μl of Stop Solution was added to each well and optical density of each well was determined within 10 minutes using a microplate reader set to 450 nm. The average of the duplicate readings for each standard and sample were calculated and subtracted from the average optical density of blank. A standard curve was then created and the concentration read from the standard curve was however multiplied by the dilution factor of the samples. A written informed consent letter was obtained to use the animals for the present study.

Consent to Publish
All members of the author list have attested their consent for the article to be published.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interest
There is no conflict of interest declared by the authors.

Research Funding
This research was funded by Geran Putra Universiti Putra Malaysia (Grant: 9490500 and 9490503). The funder provided logistics which was adequately utilised in carrying out the experimental process and purchase of ELISA kits used.

Supplementary Files
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