Changes of Cytokines in Serum and Whey of Dairy Cows with Subclinical Mastitis

Wang Lu-jun, Chen Yu, Liu Wen-han, Zhang Zhi-heng, Ma Tian-wen, and Gao Li

Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University,Harbin 150030, China

Abstract: In order to study the correlation of different degrees of subclinical mastitis and cytokines, the changes of interleukin-1β(IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in blood and milk and whether the changes of these indexes were caused by mastitis were analyzed.The 40 Holstein cows in lactation period were tested and grouped by somatic cell count (SCC) method in milk: Group C (SCC＜5×105 cells · mL-1), Group I (5×105 cells · mL-1＜SCC＜1.5×106 cells · mL-1), Group II(1.5×106 cells · mL-1＜SCC＜5×106 cells · mL-1) and Group III (SCC＞5×106 cells · mL-1).The contents of IL-1β, IL-6, IL-8 and TNF-α were analyzed by radioimmunoassay.The results showed that the contents of IL-6, IL-8 and TNF-α in the blood and milk of different degrees of subclinical mastitis increased with the severity of mastitis, and were higher than those in normal group.The contents of IL-6, IL-8 and TNF-α in serum and whey of dairy cows affected by different degrees of subclinical mastitis were higher than those in normal group (Group C).The content of IL-1β increased and decreased with the increase of inflammation, and it did not have the value of evaluating mastitis.In conclusion, the severities of cow subclinical mastitis and the contents of cytokines were positive correlations.These indexes could be used as the basis for judging mammary gland injury.

Key words: bovine subclinical mastitis, interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-alpha

Introduction

Mastitis is a certain degree of inflammation in the mammary gland tissues of cows, accompanied by changes in physical, chemical and microbiological milk.The occurrence of mastitis not only prolongs the mating time of estrus, reduces the production performance of dairy cows (Wolfensonet al., 2015),but also seriously affects the quality of milk, and indirectly endangers human health (Akers and Nickerson, 2011).At the same time, mastitis also causes huge economic losses per year (Baret al.,2008).There are several types of immune cells and non-antibody protective factors in the mammary gland and its secretions, which provide protections against the infection of the mammary gland by pathogenic bacteria (Targowski, 1983).Therefore,regulating the activity of mammary gland immune cells and enhancing the body's anti-infective ability have become important parts of the current study of mastitis.The natural defense function of dairy cows can be activated by pathogenic bacteria and their products as well as a variety of biological effect substances, especially the role of cytokines is particularly obvious.

Cytokine (CK) is a kind of non-immunoglobulinlike peptide substance secreted by activated lympho-cytes (such as T lymphocytes, B lymphocytes and natural killer cell) and other immune-related cells (such as mononuclear macrophages, fibroblasts, keratinocyte and vascular endothelial cells), which play an important role in cell differentiation, inflammatory response and immune regulation of animal interleukin(IL), colony stimulating factor (CSF), interferon(IFN) and tumor necrosis factor (TNF).And CK plays an important role in breast immune regulation(Riolletet al., 2000).As a member of the IL-1 family,Interleukin-1β(IL-1β) is expressed in a variety of cell lines, including systemic monocytes, macrophages,neutrophils, hepatocytes and tissue macrophages(Krummet al., 2014).Previous studies have shown that IL-1βcan induce a series of inflammatory reactions by promoting the expression of vascular endothelial-leukocyte adhesion molecules, which can make neutrophils and other inflammatory cells enter the pathological site of the body (Morzyckiet al., 1990).Interleukin 6 (IL-6) not only promotes the proliferation of multiple cells, but also promotes cell differentiation.IL-6 is the main cytokine of acute phase reaction,which can induce the expression of acute phase genes in hepatocytes.In addition, IL-6 can promote the activation and aggregation of neutrophils, the levels of IL-6 in serum can reflect the degree of tissue injury (Weiet al., 2015).Above all, IL-6 plays an important role in immune and inflammatory reactions.Interleukin 8 (IL-8) can activate neutrophils, change their appearance and promote their activation and adhesion.IL-8 enhances the activity and phagocytosis of neutrophil lysosomal enzymes and stimulates and chemotactic basophils, which release histamine(Fujishimaet al., 1993).Tumor necrosis factoralpha (TNF-α) is mainly a substance produced by activated monocytes and macrophages that enhances the phagocytic ability of neutrophils and improves the cellular toxicity of antibody-dependent cells.In addition, TNF can stimulate endothelial cells and white blood cells to release a series of inflammatory media and induce the synthesis of hepatocyte acute phase protein (Baumannet al., 1993).The purpose of this study was to investigate the correlation between the changes of cytokine content in serum and whey of cows with different types of mastitis and mastitis, so as to provide theoretical basis for the pathogenesis and clinical diagnosis of mastitis in cows.

Materials and Methods

Animals

The experimental animals were selected from a dairy farm in the area of Acheng District in Harbin City and fed uniformly to whole-grain compound feeds.They were also housed under the same conditions and artificial milked.The 40 Holstein cows (n=10) in lactating period (similar in age and milk yield) were grouped by milk somatic cell counter (SCC).They were randomly divided into healthy control group(Group C), mild subclinical mastitis (Group I: 5×105cells · mL-1＜SCC＜1.5×106cells · mL-1), moderate subclinical mastitis (Group II: 1.5×106cells · mL-1＜SCC＜5×106cells · mL-1) and severe subclinical mastitis(Group III: SCC＞5×106cells · mL-1).

Sample collection and treatment

Collection and treatment of blood samples

The blood collection site of the jugular vein was disinfected before each cow was sampled.The 15 mL of blood from four groups (n=10) was collected by jugular puncture.And then, they were placed at room temperature (16℃-20℃) to make naturally solidified for 1-2 h.After the samples were placed in ice for 30 min, they were centrifuged at 16 000×g for 10 min.The serum was saved and stored at 4℃ until the analyses were conducted.

Collection and treatment of milk samples

Washed and thoroughly dried the teat, and then disinfected it with 75% alcohol before collecting milk from each cow.Stripped out and discarded the first three squirts.Samples were sampled at intervals of 5 h and sampled three times, each cow with a total of 20 mL.Finally, the milk from different milk areas of the same cow was mixed into a sterile bottle.Thecollected milk samples were placed at 0℃ and brought back to the laboratory for inspection.Milk samples were centrifuged at 1 000×g for 5 min to remove the upper milk fat, then, centrifuged at 28 600×g for 10 min to remove precipitates.The supernatants were frozen at 4℃ for further analyses.All the procedures in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University.

Determination of contents of IL-1β, IL-6, IL-8 and TNF-αby radioimmunoassay method

IL-1β, IL-6, IL-8 and TNF-αradioimmunoassay kits were all provided by Beijing Sino-uk Institute of Biological Technology (Beijing, China).According to the instruction (Table 1), all the liquid was added sequentially and placed at room temperature for 20 min.Next, the samples were centrifuged at 1 000×g for 25 min at 4℃ and the supernatants were discarded.The count per minute (cpm) of the precipitated portion of each tube was measured by automaticγimmune counter FJ-2008PS (Xi'an Nuclear Instrument).The percentage of NSB and S0was calculated by N/T and B/T.The percentage of standard and samples to be tested was calculated by B/B0.Then, the standard curves of IL-1β, IL-6, IL-8 and TNF-αwere plotted to calculate the contents of IL -1β, IL-6, IL-8 and TNF-αin the corresponding samples.

Statistical analysis

All the data were analyzed with GraphPad Prism 5.1(GraphPad Software Inc, USA).Data were presented as mean±standard deviation.Values were considered to be significant ifp＜0.05 and values were considered to be extremely significant, ifp＜0.01.

Table 1 Procedure of addition of interleukin series and tumor necrosis factor-α (μL)

Results

Serum concentrations of IL-1β, IL-6, IL-8 and TNF-α

According to the experimental results, no significant difference in IL-1βbetween Groups I and C was observed in serum (p＞0.05) (Table 2).However, the concentrations of IL-1βin Groups II and III were significantly higher than those in Group C (p＜0.01,Fig.1A).In comparison to Group C, the concentrations of IL-6 in GroupsⅡandⅢwere significantly higher (p＜0.01), although IL-6 in Group I was not significantly different (p＞0.05, Fig.1B).Similarly,levels of IL-8 and TNF-αin GroupsⅡandⅢ were significantly higher than those in Group C (p＜0.01,Fig.1C and D).In contrast to the observations of other cytokines, the concentrations of IL-8 and TNF-αin Group I were significantly (p＜0.05).Overall, the levels of IL-6, IL-8 and TNF-αin serum of dairy cows were higher than those of normal dairy cows, and the concentrations of IL-1βwere observed to increase withthe severity of inflammation.

Whey concentrations of IL-1β, IL-6, IL-8 and TNF-α

There was no difference in the levels of IL-1β(p＞0.05) (Table 3), while the concentrations of IL-1βin GroupsⅡand Ⅲ were significantly elevated(p＜0.01, Fig.2A).According to Fig.2B, the levels of IL-6 in GroupⅠwere significantly elevated, while the concentrations of IL-6 increased in GroupsⅡand Ⅲ (p＜0.01).There was no significant difference in the levels of IL-8 of GroupsⅠandⅡ (p＞0.05).In contrast, IL-8 was significantly increased in Group Ⅲ(p＜0.05, Fig.2C).A significant increase in the expression of TNF-αwas observed in GroupsⅠ, Ⅱand Ⅲ (p＜0.01, Fig.2D).The levels of IL-6, IL-8 and TNF-αin whey samples were higher than those collected from healthy cows, and the concentrations of IL-1βgradually increased with an increase in inflammation.

Table 2 Concentrations of IL-1β, IL-6, IL-8 and TNF-α in serum in each group

Fig.1 Concentrations of IL-1β, IL-6, IL-8 and TNF-α in serum of dairy cows in each group

Table 3 Concentrations of IL-1β, IL-6, IL-8 and TNF-α in whey in each group

Fig.2 Concentrations of IL-1β, IL-6, IL-8 and TNF-α in whey of dairy cows in each group

Discussion

Cytokine is an important part of animal immune system, which plays an important role in the process of inflammatory response and immune response of organism.The mammary gland of a cow is a complex bioreactor inside the body.Within the mammary gland,numerous protective factors, including cytokines,antibodies, hormones, enzymes and neurotransmitters,have been constructed to form a number of intricate signaling pathways.When inflammation occurs in the mammary gland of the cow, the body's resistance decreases, and the expression of cytokines and their receptors in the body occurs abnormally.Therefore,the importance of the detection of cytokines and their receptors is necessary to study the balance of cytokines in the inflammatory response of the mammary gland.

In this experiment, when the cows suffered from mastitis, the contents of IL-1β, IL-6, IL-8 and TNF-αin serum and whey were significantly higher than those in the control group, with the increase of inflammation,the contents of IL-1β, IL-6, IL-8 and TNF-αalso increased gradually.It showed that the contents of cytokines in the body increased significantly, when the cows suffered from mastitis.This was mainly because when cows had mastitis, the body's resistance decreased and the expression of cytokines and their receptors in the body occurred abnormally.

As an inflammatory and immune-derived cytokine,IL-1βparticipates in various inflammatory reactions in the body.Under normal circumstances, the amount of IL-1βin the blood was very low.However, under the guidance of physicochemical and biological factors,IL-1βcould be expressed and released by monocytemacrophage and lymphocyte, and played a variety of biological effectsin vivo(Shariefet al., 1993).IL-1βis one of the most influential mediators of the early inflammatory response; therefore, it can reflect the extent of tissue damage earlier and sensitively.After the inflammatory reaction of the mammary gland of the cow, the neutrophil infiltration around the inflammatory area and the release of cytokines were manifested, at the same time, the microcirculation was damaged.With the increase of inflammation,the production and release of IL-1βalso gradually increased, and the content decreased, when there were obvious clinical symptoms, but it was still higher than that of the control group.The main reason was that IL-1βwas the main inducer of the acute phase reaction,and the content in the body was gradually reduced after the inflammation occurred (Itoet al., 2015).Inaddition, the secretion of TNF-αin the late stage of inflammation also inhibited the secretion of IL-1β.

IL-6 is an important component of the inflammatory cytokine network and closely related to the occurrence and development of infectious diseases in the body.Previous studies have shown that the occurrence and clinical symptoms of mastitis in dairy cows are related to cytokines.The acute phase reaction of mastitis was stimulated and mediated by cytokines, such as IL-6 and TNF-α(Hagiwaraet al., 2001; Lehtolainenet al.,2004).After the inflammation in the mammary glands of cows, the inflammation stimulated the macrophages and epithelial cells in the breast and increased the content of IL-6 in the milk.At the same time,inflammation activated the body's immune function(lymphocytes in the blood were activated), and the secretion synthesis function of inflammatory response factors such as IL-6 was enhanced, therefore, the content of IL-6 in the blood also increased.Bochniarzet al.(2017) showed that the contents of IL-6 in whey and serum of acute mastitis cows had a linear relationship with the degree and recovery of mastitis,which could be an indicator for the treatment of mastitis.

IL-8 is a chemokine for polymorphonuclearneutrop hil (PMN), basophil and T-lymphocyte.It can not only improve the binding capacity of PMN in inflammatory sites, but also promote the production and release of various inflammatory mediators, which plays an important role in the inflammatory response.This study showed that IL-8 content gradually increased with the increase of inflammation.This was mainly because endotoxin levels in serum and whey increased,when mammary gland inflammation occurred (Corlet al., 2008), and endotoxin stimulated inflammatory cells to release cytokines such as IL-1βand TNF-α(Cavaillon, 2018).Under the stimulation of these cytokines, many cells, such as monocytes and PMN,could produce IL-8 (Fitzgeraldet al., 2007).IL-8 could cause chemotaxis, deformation and degranulation of PMN by binding with its surface specific receptor,so as to release elastase, tissue protease and so on,eventually cause severe tissue damage.

Tumor necrosis factor (TNF) is initially named for its ability to cause hemorrhage and necrosis in tumor tissues, and is divided into TNF-alpha (TNF-α)and TNF-beta (TNF-β).The relationship between inflammation and TNF-αis very close.When the pathogenic microorganism enters the breast tissue, it can activate mononuclear-macrophages to produce a large amount of TNF-αand PMN is the main target of TNF-α.Under the action of TNF-α, PMN not only could make the breast tissue produce and release more cytokines, but also participate in the inflammatory response and intensify the occurrence of inflammation(Lehtolainenet al., 2004).The results indicated that a large amount of secreted TNF-αmight be the cause of the aggravation of mastitis.This was consistent with the report that the release of IL-6 and TNF-αincreased, when cows had mastitis, and the content was positively correlated with the severity of clinical symptoms (Slebodzińskiet al., 2002).The level of TNF-αin whey and serum could be an important indicator of the severity of mastitis disease in the future.It was concluded that the contents of IL-1β,IL-6, IL-8 and TNF in serum and whey of dairy cows were closely related to the inflammatory reaction of mammary gland and the immune state of cow body.

Conclusions

The contents of IL-6, IL-8 and TNF-αin blood and milk of subclinical mastitis in different degrees increased with the increase of mastitis severity, which were higher than those in the control group.They could be used as one of the indexes to determine breast injury in dairy cows.The content of IL-1βincreased first and then decreased with the increase of inflammatory degree, so it did not have the value of evaluating subclinical mastitis.

Acknowledgement

Wang Lu-jun and Chen Yu contributed equally to this work.