Research Article

Inflammatory Mediators and Expression of L Selectin in Response to Tinospora Cordifolia and Α Tocopherol in Bovine Staphylococcal Mastitis

Jadhav R.K1, Mukherjee Reena2,*, De U.K.3
1Department of Clinical Veterinary Medicine, College of Veterinary & Animal Sciences, Udgir (MS), India
2Division of Medicine, ICAR-Indian Veterinary Research Institute, Izatnagar, India
3ICAR-Indian Veterinary Research Institute, Izatnagar, India

*Corresponding author:

Mukherjee Reena, Division of Medicine, ICAR-Indian Veterinary Research Institute, Izatnagar, India, Email: reenam1992@gmail.com

Keywords:

CD18, Ceruloplasmin, IL 8, L selectin, Mastitis, Nuc gene, Staphylococcus aureus, α tocopherol, Tinospora cordifolia

The present study was aimed to determine the IL 8 concentration in milk serum, Ceruloplasmin in plasma, and L selectin on peripheral leukocytes in lactating cows afflicted with pathogenic Staphylococcal mastitis in response to Tinospora cordifolia and α tocopherol plus Selenium (group II), Enrofloxacin (group III) and normal saline (group IV). The somatic cell count (SCC), concentration of IL 8 and Ceruloplasmin was higher in mastitic cows (P<0.05), however, the values decreased in post treated group II and III cows (P<0.05). The expression of L selectin on peripheral leukocytes was lower in mastitic cows (P<0.05). Whilst, the mean fluorescent intensity (MFI) of L selectin increased in group II post treated cows. The concentration of Vitamin E was lower in mastitic cows. The results indicate that Vitamin E plus selenium therapy decreases SCC, IL 8 and Ceruloplasmin concentration, increases L selectin counts , but does not interfere with the expression of CD 18, both the parameters are related to the reduction of inflammation and enhancement of the mammary defense. Hence combination of Vitamin E plus Selenium as an adjunct to antibiotic may be recommended for the treatment of mastitis in order to reduce the severity and duration of the course of the disease.

Bovine mastitis causes great economic losses due to lower and poor quality of milk and treatment cost [1]. Staphylococcus aureus (S. aureus) is one of the major pathogen associated with bovine mastitis as diagnosed by DNA hybridization and polymerase chain reaction for detecting virulent genes. S. aureus established in the animal system spreads rapidly to other lactating cow during milking and attributes to chronic and sub-clinical mastitis [2, 3]. Intramammary infection with S. aureus implicate to abscess formation and lodging in the mammary parenchyma. The PMNs infiltrate from the peripheral circulation into the infected gland in response to infection and mediate elimination of the infectious agents. The inflammatory process should be short lasting and regulated for the cure, however, the long lasting inflammatory cells release proteases and reactive oxygen radicals and results in injury to the mammary secretory epithelium and scarring [4]. The recruitment and migration of leukocyte into the infected udder depends on the proper and selective expression and activation of several adhesion molecules. Bovine leukocyte adhesion molecules such as L-selectin and CD 18 integrin play a key role in the migration of leukocyte to the site of infection [5, 6]. It has been observed that the PMNs bearing L selectin counts are generally lower during periparturient period, which affects leukocyte infiltration into the diseased mammary gland and its functional activities [7, 8]. Mastitis is generally treated with antibiotics [9], though the therapy does not remove the inflammatory mediators from the site of inflammation as well as it adds harmful drug residue in food chain, increasing antibiotic resistance, which is also of great concern [10].

The udder health in lactating animals is also influenced by the dietary status; supplementation of Selenium and Vitamin E in feed of lactating dairy cows was found to improve leukocyte migration, phagocytosis and intracellular killing respectively [ 11-15]. Subclinical mastitis is in increasing trend in lactating herd [16]. T cordifolia extract has been found very effective in bovine sub clinical mastitis [17], however, its synergistic effect with Vitamin E plus Selenium against Staphylococcal mastitis is not well defined. In this study we determined the effect of Vitamin E plus Selenium and polysaccharide fraction of T cordifolia therapy on SCC, IL 8, Ceruloplasmin and L selectin in lactating cows afflicted with sub clinical mastitis caused by pathogenic S. aureus.

Collection and Extraction of Polysaccharide Fraction of T.cordifolia (PFTC)

T. cordifolia was procured from the institute campus and identified at the Department of Botany, Ruhelkhand University, Bareilly, India and the voucher specimen number was provided (PVM- 018). The stems were washed, shade dried and pulverized to coarse powder. The powdered content was weighed and polysaccharide fraction was isolated as per the method described by Desai et al., 2002. The herb fraction was tested for the presence of polysaccharide by Molisch’s test.

Selection of lactating cows and Experimental Protocol

Twenty four lactating crossbred cattle were selected from one dairy farm aged between 3 to 7 years, in 1st to 4th lactation at 4 to 60 days postpartum period, maintained under identical manage mental conditions. The screening was done on the basis of CMT reaction. The selection criteria for mastitic cows being the CMT positive reaction as + point score and ++ point score, SCC more than 0.5 million cells/ ml of milk and only those cows were selected whose udder secretions found positive for S.aureus and animals with normal demeanor. The cows screened were divided in 4 equal groups. Group I, consisting of 6 healthy cows whose milk was negative for pathogenic isolate and SCC was less than 0.3 million cells/ ml of milk, eighteen mastitic cows (group II, group III, group IV) , formed the drug trial groups. Group II, cows received 150 mg of sterile PFTC per teat with a sterile antibiotic dispensing canula after diluting the drug in 10 ml sterile phosphate buffer saline, twice daily for 5 days, along with α tocopherol plus selenium (content α tocopherol 50 mg, selenium 1.5 mg/ ml) at the rate of 10 ml per cow by intramuscular route on day 0 for single application. Group III cows were treated with Enrofloxacin injection at the dose rate of 3 mg per kg body weight for 5 days by intramuscular route. Group IV, cows received 10 ml of sterile normal saline solution by intramammary route for 5 days.

Collection of milk samples and isolation of S. aureus

Two hundred ml of milk from each cow was collected by maintaining sterile conditions after discarding few streams of milk. The milk was collected on day 0 and on day 8 of treatment time period from diseased and healthy cows. SCC of the milk samples was done as per standard method [18]. The identification of S. aureus in collected milk samples was initially identified on the basis of colony morphology and odor on 5% blood agar and later by gram staining and growth on selective media like Mannitol salt agar and Baird Parker agar plates. Positive samples on selective media were biochemically characterized. The biochemical characterization of Staphylococcus aureus was performed by HiStaphTM Identification Kit (Himedia Lab Pvt. Ltd., India). SCC was done on day ‘0’ and on day 8 time periods.

Molecular Identification of S. aureus by NUC Gene

Bacterial DNA was isolated by snap chilling method from18 hour old BHI broth culture of S. aureus. The PCR product was determined by comparing with a standard molecular weight marker, and was photographed by the gel documentation system (Alpha Innotech Corporation,USA).Oligonucleotide primers used for nuc gene of S. aureus F: 5’-GCGATTGATGGTGATACGGTT-3’; R: 5’-AGGCCAAGCCTTGACGAACTAAAGC-3’ [37]

Estimation of Interleukin-8 (Il-8) in Milk Serum

Milk serum was collected by centrifugation of milk, the translucent supernatant collected and stored at -20°C. IL-8 in milk serum was quantified by using a commercial anti-human IL-8 ELISA kit (Quantikine, R and D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Cytokine level was measured before treatment and on day 8, similarly the observations were recorded in healthy cows.

Estimation of Ceruloplasmin in Plasma

Plasma Ceruloplasmin level was determined by measurement of paraphenylene diamine oxidase activity according to the method described by [35]. Level was measured before treatment and on day 8 of treatment time period.

Expression of L-Selectin in Blood

Expression of L-Selectin in blood was done by using a commercial kit (L-Selectin, Clone- DU1-29, Isotype- IgG1,VMRD, Inc. Pullman, WA, USA) as per method described by Soltys and Quinn (1999). The cellular concentration was determined by flow cytometry (Becton Dickinson Immunocytometry System, Bioscience, USA). A total of 10,000 events were counted for each sample. The expression of leukocyte adhesion molecules in terms of mean fluorescence intensity (MFI) was calculated after plotting of the fluorescence of the histograms with CellQuest software (Becton Dickinson, Bioscience, USA). For isotype control, similar procedure was followed, except instead of adding monoclonal antibodies, isotypes (FITC labelled rabbit antimouse IgG) were added in sample.

Estimation of Selenium and Vitamin E in Serum

Blood was collected before treatment and on day 10 time period for estimation of selenium and Vitamin E.The serum was separated and digestion was done as per procedure described by Kolmer et al. (1951). Serum Selenium was estimated before treatment and on day 10 by Atomic Absorption Spectrophotometry (AAS 4141, ECIL, India). Estimation of serum vitamin E was done before treatment and on day 8 as per the method described by [36].

Statistical Analysis

Somatic cell count data were analyzed using repeated measurement model with animal as subject and period as repeated measurement. The data of Ceruloplasmin, IL 8, adhesion molecules, Vitamin E and Selenium concentration, were analyzed applying One-way analysis of variance (ANOVA) to determine the level of significance between the groups, and Duncan’s Multiple Range Test (DMRT) was applied to determine the significance within the group at different time interval with a statistical software (SPSS, Version 10, South Asia, Bangalore, India).

Qualitative Analysis of the Extract

The qualitative analysis of plant extract revealed the presence of carbohydrate in the polysaccharide fraction.

Characterization of S. aureus

Colonies grown on blood agar plates were further subcultured to Baird Parker agar which revealed characteristic black colonies confirming as S aureus.

Biochemical Confirmation of S aureus

Fifty micro liter of overnight grown BHI broth cultures were inoculated into each well of the kit (HiStaphTM Identification Kit) and kept at 37˚C for 24 hours. Isolates showed characteristic color change before and after reagent addition, the isolates were positive for VP, Alkaline Phosphate, Urease, Arginine utilization and ability to ferment carbohydrate including Mannitol, Sucrose, Maltose, Trehalose, and negative for ONPG, Arabinose and Raffinose.

Molecular Identification of Staphylococcus aureus by Amplification for nuc gene

The amplified products were of nearly 279 bp when resolved in gel electrophoresis.

Effect of Treatment on SCC

The SCC ranged from 2.69±1.37 to 2.73±1.18 x 10 5 and 21.60±1.48 to 22.03±1.77 x 10 5 cells per ml of milk samples isolated from normal healthy cows and mastitic cows respectively. There were no differences in SCC in the milk sample isolated from healthy cows and in group IV, at different time intervals of the study period. The SCC in group II and group III, decreased (P<0.05) on day 8 as compared to pre-treatment values (Table 1)

Groups of cow (n = 24) Somatic Cell Count (SCC ) 1x 10 5 cells / ml of milk

 

Day 0

Day 8

Gr I 

2.69 ± 1.37x

2.73 ± 1.18 x

Gr II

21.89 ± 1.99 a, y

 5.12 ±1.09 b, y

Gr III

22.03 ±1.77 a, y

6.02 ±1.25 b, y

Gr IV

21.60 ± 1.48 a, y

19.09 ± 1.33 a, z

*Values with different superscripts in each rows (a, b) and each column (x, y, z)

differ significantly (P<0.05).

 

Effect of treatment on plasma ceruloplasmin

The plasma ceruloplasmin ranged from 0.057.09± 1.29 gm/l to 0.056±1.34 gm/ l in normal healthy cow. The Ceruloplasmin concentration was significantly higher (P<0.05) in mastitic cows on day 0 as compared to healthy control. The levels decreased on day 8 (P<0.05) in group II and III compared to pretreatment values. Whilst, lower level was observed in group II cows on day 8 compared to group III and group IV cow (P<0.05)

Effect of Treatment on IL8

IL 8 in milk serum ranged from 3.19±1.91 ρg/ml to 3.07±1.96 ρg/ ml serum in normal healthy cow. The IL 8 concentration was significantly higher (P<0.05) in the milk serum collected from mastitic cows as compared to normal healthy cows. IL8 levels decreased on day 8 (P<0.05) in both the treated groups compared to pretreatment values. Whilst, lower level was observed in group II cows on day 8 compared to group III and group IV cow (P<0.05) (Table 2).

Group of cows

 Plasma Ceruloplasmin concentration (gm/L)

 Interleukin 8 milk serum (ρg /ml)

 

Day 0

Day 8

Day 0

Day 8

Gr I

0.057.09±1.29 w

0.056±1.34 w

3.19 ±1.91 w

3.07 ±1.96 w

Gr II

 0.132.01±1.52 x, a

0.097.00±2.19x, b

9.27 ±1.37 x ,a

5.43 ±1.38 x, b

Gr III

0.129.00±1.07x,a

0.096 ±1.39x,b 

9.58 ±1.29x

6.21 ±1.52 y, b

Gr IV

0.130.00±1.56 x

0.117 ±1.23 y

9.26 ±1.19x

8.62 ±1.37z

*Values with different superscripts in each rows (a, b) and each column (w, x, y, z) differ significantly (P<0.05).

Effect of Treatment on L selectin

The expression of L-selectin in peripheral PMNs isolated from normal healthy cows ranged from 4.78 ±1.27 to 4.81 ±1.33 mean fluorescent intensity (MFI).Expression of L selectin was lower in mastitic cows (P<0.05) before treatment. However, the expression increased in group II cows on day 8 (P<0.05), as compared to group III and group IV (Table 3).

Group of cows

 L selectin MFI

 

Day 0

Day 7

Gr I

4.78±1.27 x

 4.81±1.33 x

Gr II

2.82±1.72 y, a

3.82±1.22 y ,b

Gr III

 2.71±1.57y

2.91±1.47z

Gr IV

2.69±1.64 y

2.77±1.77 z


*Values with different superscripts in each rows (a, b) and each column (w, x, y, z) differ significantly (P<0.05)

Effect of Treatment on Concentration of Vitamin E and Selenium

The Vitamin E concentration ranged from 4.72±1.01 to 4.70±1.12 μg/ ml and selenium ranged from 0.069±0.76 to 0.070±0.99 μg/ml of serum in blood collected from normal healthy cows. The Vitamin E and Selenium concentration was lower (P<0.05) in serum collected from mastitis cows before treatment. The selenium concentration increased significantly in group II cows on day 8 (P<0.05), whereas nonsignificant increase in serum vitamin E concentration was observed in response to therapy on day 8 (Figure 1 and 2).

Figure 1:Concentration of Vitamin E (μg / ml) in response to the treatment with Tinospora cordifolia and Vitamin E plus Selenium (group II), Enrofloxacin (group III), sterile normal saline (group IV) mastitic cows and in group I normal healthy cows (mean ± SE).

Figure 2: Concentration of Selenium (μg / ml) in response to the treatment with Tinospora cordifolia and Vitamin E plus Selenium (group II), Enrofloxacin (group III), sterile normal saline (group IV) mastitic cows and in group I normal healthy cows (mean ± SE)

Mastitis is one of the major causes of economic loss in dairy cattle. S. aureus represents a major agent of contagious bovine mastitis, leading to chronic, sub clinical and clinical mastitis [19, 20]. Once established in the udder impairs alveolar function, reduces milk yield and has a deleterious effect on milk composition, including increased milk SCC [21] . In the present study the animals were observed to be healthy without increase in the rectal temperature. The udder and teat were normal except the change in the physical characteristics on the milk. The milk SCC was higher in mastitic cows before treatment (P<0.05). The SCC increases many folds in mastitis cows due to the migration of polymorphonuclear neutrophils (PMN) into mammary glands in response to bacterial infection [22]. Antibiotics are used to treat mastitis but antibiotic therapy in lactating cows is not very much effective and it also contaminates the milk [23].

In the present drug trial we recorded, reduction of SCC, Ceruloplasmin and IL 8 in milk serum and enhancement of L selectin in cows treated with PFTC plus vitamin E and selenium. Reduction in SCC indicates the clearance of bacterial load from the infected udder, which could be due to the enhanced leucosis and increased phagocytic activity of the milk PMNs. [24] demonstrated antibacterial effect of T. cordifolia against pathogenic microbes like Klebsiella pneumoniae, Escherichia coli, Micrococcus luteus, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus and Lactobacilus acidophilus. [6] Observed reduction in total bacterial count and increased phagocytic activity of milk PMNs with the treatment of T. cordifolia extract in bovine sub clinical mastitis. The chemical component of Tinospora cordifolia such as polysaccharide, glycosides, alkaloid and steroids are found to have anticomplimentary and immunostimulating activities [25]. The immune competence of the phagocytic cells can be modulated by number of specific and non-specific mediators. [26] Isolated 1-4-αD-glucan from polysaccharide fraction of T. cordifolia and studied its effect on macrophages activation. Siddalingappa and co-workers (2012) studied the anti-inflammatory activity of aqueous extracts of Tinospora cordifolia in murine model, the author further explained that the analgesic and antiinflammatory activity of the herb extract could be due to the inhibition of the PG3 synthesis.

The acute phase response is associated with infection or tissue injury. High concentration of acute phase protein in milk from infected quarters has been detected in mastitis. Ceruloplasmin (Cp) is recognized as an important acute phase reactant in the cows and high concentration has been observed in mastitis. In the present study the ceruloplasmin and IL-8 levels were significantly reduced in Gr II and Gr III on day 8, which could be due to the reduced bacterial load and reduction of the inflammation, as the PFTC and α tocopherol possess antibacterial and anti-inflammatory properties.

The nutritional status of the cow is directly related to overall health and the ability of an animal to fight diseases. In our trial the selenium concentration in Gr II increased significantly on day 10 as compared to day 0, however the Vitamin E concentration increased non-significantly in the same group post treatment. It has been observed that supplementation of micronutrients like Selenium and Vitamin E in greater quantity than required in feed greatly benefit the cows health, it also helps in reducing somatic cell count, severity and duration of clinical mastitis [27,28]. Selenium and Vitamin E deficiency attributes to defective leukocyte function, like poor leukocyte migration, phagocytosis and intracellular killing [11-13]. Stickel and coworkers (1997) observed reduction in inflammation with Vitamin E treatment in mice. S. aureus mastitis has been shown to reduce the effectiveness of the immune response including suppression of effective mechanism in mammary gland. CD 18 and L selectin (LAM) are sensitive indicators of leukocyte activation. Inability to express adhesion molecules on leukocytes result in poor migration of neutrophils at the site of infection, which could lead to acute infection [29]. L-selectin works as cytokine like molecule in priming host defense and is mediated through CD18 [6]. β2- integrin facilitates diapedesis of neutrophils through the endothelium of mammary gland. Both the adhesion molecules acts together to promote leukocyte activity [30]. L-selectin also regulates the accumulation of leukocytes at the site of infection and control inflammation [31]. CD18 enhances neutrophil migration, chemotaxis, cellular activation, phagocytosis and cell mediated killing [32]. [7] Observed poor expression of adhesion molecules on neutrophils during periparturient period. In our study, lower expression of L selectin could be observed in mastitic cows. Although L selectin increased in group II cows treated with PFTC and Vitamin E plus Selenium (P< 0.05). Many researchers have studied the nature of adhesion molecules during mastitis and reported up-regulation of CD18 and down regulation of L selectin on peripheral PMNs in Streptococcal, Staphylococcal and E.coli mastitis [33, 31] In the present study enhanced expression of L selectin in post treated cows could bedue to the synergistic effect of PFTC and Vitamin E plus selenium treatment. Poor serum Selenium concentration was recorded to increase the prevalence of mastitis with high SCC [34].

This study represents an initial investigation into the synergistic effect of Vitamin E plus Selenium and PFTC on the activity of SCC, ceruloplasmin, IL 8 and LAM in mastitic cows. The results indicate the enhanced cellular defense of the diseased mammary gland and subsequently reduction in inflammation. The present drug trial determines the potential benefits of the combination therapy in intramammary infection in lactating cows as well as standardization of non antibiotic agents to reduce antibiotic residue from milk.

First author thanks Indian Council Agricultural Research, New Delhi for providing Junior Research Fellowship.The authors also thank the Director and Head of Division of Medicine, for providing the facilities.

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Citation: Jadhav RK, Reena M, De UK (2017) Inflammatory Mediators and Expression of L Selectin in Response to Tinospora Cordifolia and Α Tocopherol in Bovine Staphylococcal Mastitis. Int J Vet Dairy Sci 1:005

Published: 31 July 2017

Reviewed By : Dr. Emmanouil Kalaitzakis,

Copyright:

© 2017 Mukherjee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.