Year : 2017, Volume : 41, Issue : 3
First page : ( 189) Last page : ( 195)
Print ISSN : 0250-4758. Online ISSN : 0973-970X. Published online : 2017 September 1.
Article DOI : 10.5958/0973-970X.2017.00046.3

Role of TGF-β in pathology of Staphylococcus aureus induced chronic mastitis in mice

Hingade Sonal⁴, Nagdive A.^1,4, Pantawane P.^2,4, Kesavan M.^3,4, Karikalan M.⁴, Rai R.B.⁴, Sharma A.K.^4,*

⁴Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India.

¹Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India.

²Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India.

³Division of Pharmacology And Toxicology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India.

*Corresponding author: e-mail: aksharmaivri@rediffmail.com

Received:  8  June,  2017; Accepted:  21  June,  2017.

Abstract

The aim of the present investigation was to study sequential histopathological changes and the role of transforming growth factor-β (TGF-β) in Staphylococcus aureus induced chronic mastitis in mice. The lactating female Swiss albino mice were inoculated by intramammary routeof 100 pl of inoculum containing 10⁷cfu of a field strain of Staphylococcus aureus isolated from bovine mastitis case. The mammary gland tissue samples were collected at 1,5,10,20, 30, 40 and 50 days post infection (DPI) and subjected to histopathological and immunohistochemical assessment of TGF-β. The histopathological study at 1 DPI showed congestion, edema and infiltration of inflammatory cells predominantly the neutrophils in interstitium as well as in alveoli. As infection became more chronicwith passing of time, the expression of TGF-β kept upregulated correspondingly from 1.00±0.29 fold at 1 DPI to 2.86±0.06 folds at 50 DPI. The alveoli exhibited necrosis, desquamation of glandular epithelial lining in acute phase to atrophy of alveoli, increased interalveolarfibroplasia with or without suppurative changes and infiltration of macrophages, lymphocytes, plasma cells in interstitium of alveoli in chronic phase. The immunohistochemical expression of TGF-β was moreintense in cytoplasm of lymphocyte, macrophages and fibrocytesin mammary gland tissue during chronic phase. Thus, the level of TGF-β was found positively correlated both qualitatively and quantitatively with degree of chronicity and fibrosis in glandular parenchyma making the gland sclerosed and non-functional due to Staphylococcus aureus infection.

Top

Keywords

Fibrosis, Mastitis, Mice, Staphylococcus aureus, TGF-β.

Top

INTRODUCTION

Top

MATERIALS AND METHODS

Experimental animals

The adult lactating female Swiss albino mice, 8–12 weeks of age weighing 25–30 g and in apparently good healthafter weaning their pups were procured from the Laboratory Animal Resource section of ICAR-IVRI, Izatnagar. The mice were housed in polypropylene cages with 12 h light and dark cycles and provided with standard diet and water ad libitum. The experimental trial was conducted in 10–15 day of post litter lactating mice as described by Chandler¹⁷. The experimental design was approved by the Institute′s Animal Ethics Committee (IAEC).

Source and confirmation of bacteria

The highly virulent field strain of Staphylococcus aureus isolated from bovine mastitis case was further cultured on selective medium (Mannitol salt agar). The smears were also prepared from culture on glass slide, stained by Gram′s staining and examined for morphology of bacteria. Further confirmation of bacteria was done by biochemical tests and PCR. From the bacterial colony DNA was extracted using snapchill method. The isolated DNA was used for amplification of nuc gene of S. aureus as described earlier 18.

Experimental design

Twenty eight adult lactating female mice, divided into two groups of 7 (Control) and 21(treated) were anaesthetized with ketamine and xylazine prior to inoculation. Abdominal surface of the mice of both the groups were disinfected with 70% ethanol. The left 4 and 5 mammary glands were secured by forceps and inoculated each with 100μl of inoculum in PBS containing 10⁷ c.f.u. of field strain of Staphylococcus aureus through teat canal in treated group and contralateral right 4 and 5 mammary glands were kept as unaffected for comparison. The control mice were inoculated intramammary with sterile PBS 100 μl only. On 10 day of infection, the animals in treated groups were again inoculated with similar dose of the same strain of Staphylococcus aureus through teat canal as above for continuation of inflammatory changes and development of chronic changes in mammary gland. On 1, 5, 10, 20, 30, 40 and 50 day post infection (DPI) three mice each were sacrificed from the treated group along with one from control at each interval. Mammary glands tissue samples were collected in 10% neutral buffered formalin for histopathology and 100 mg mammary tissue in RNA later for RNA extraction.

Histopathological examination of mammary gland

For histopathological changes, the mammary glands fixed in 10% neutral buffered formalin and processed for histopathological examination¹⁹. Duplicate sections were used for Gram′s staining to demonstrate bacteria and Masson′s trichrome staining for detection of collagen.

Immunohistochemical staining for TGF-β

The paraffin embedded sections taken on APES coated slide were processed by standared immuno-histochemical staining protocol with primary antibody mouse anti TGF-β (Santa cruz, USA) in 1:50 dilution and incubated in humidified chamber overnight at 4°C. The slides were subsequently incubated with secondary antibody HRP conjugated anti- Mouse IgG (R&D, USA) for 1 h at room temperature. The Diaminobenzidine (DAB, Amresco, USA) was used as chromogen and finally sections were counterstained with Mayer′s haematoxylin. For negative control sections, PBS was used in place of primary antibody. Each slide was microscopically examined and enumerated the percentage of positively stained cells. TGF-β immunostained cells were counted in 10 representative high power fields and averaged. The cells showing cytoplasmic staining were considered as positive. The percentage of positive cell were scored as: +++ = strong staining (> 50% of cells); ++ = moderate staining (between 20 to 50% of cells); + = weak staining (between 1to 20% of cells); 0= negative²⁰.

Cytokines Expression

RNA was extracted from infected mammary gland tissues using QiaAmp mini kit (Qiagen, Germany) as per manufacturer′s recommendations. RevertAid® first strand cDNA synthesis kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA in a 20 μl reaction. Relative quantification of cytokine TGF-β in the mammary glands of all the animals were done by real time PCR. The Brilliant III Ultra-Fast SYBR Green® Q- PCR Master Mix kit (Agilent, USA) was used according to manufacturer′s instructions. Each reaction mixture contained 1p 5μl cDNA, 5 5μl of Ultra-Fast SYBR Green master mix, ROX 0.15 5μl, sense and antisense each 0.4 5μl TGFμ published primers²¹ (Forward, 52 - CCCTATATTTGGAGCCTGGA-32; Reverse, 52 - CTTGCGACCCACGTAGTAGA-32) and nuclease free water 3.05 5μl. The PCR cycling conditions were programmed (denaturation at 95°C for 3 min, followed by 40 cycles with denaturation at 95°C for 5s, and annealing and extension at 49°C for 30s). The samples were amplified using the ABI 7500 Fast Sequence Detection System (Applied Biosystem). Each sample was run in duplicate and the results were averaged. Melting curves were constructed to assess PCR accuracy. To study the relative change in gene expression, the 2-AAct method was used²². Fold change = 2-AAct, where -AAct U = (CT of target gene -CT of GAPDH) treatment - (CT of target gene - CT of GAPDH) control. The gene specific amplification was corrected for the difference in input of RNA by taking housekeeping gene GAPDH into account. The results were analyzed in comparison to the Ct value of the target gene and the reference gene. The level of significance was set at P<0.05.

Top

RESULTS

Confirmation of Bacteria

The bacteria used in the present study was confirmed to be Staphylococcus aureus based on golden yellow colour colony on Mannitol salt agar plate after overnight incubation. Gram′s positive cocci in clusters were observed. Biochemical tests showed nitrate + ve, VP + ve, methyl red -ve, malonate -ve, urease -ve, tween - ve, motility-indole-lysine -ve, triple sugar iron -ve and citrate -ve. PCR of bacterial DNA colony amplified product of 279 bp size of nuc gene.

Histopathology of mammary glands

In control group, the tissue sections of mammary gland at 1 day revealed dilated lactating alveoli containing milk secretion with at times presence of fatty vacuoles, intact alveolar and ductal epithelium (Fig. 1) with little interacinar connective tissue. The blood vessels were moderately congested. The interlobular stroma exhibited mild edema. On day 5, mammary acini were dilated with fat globules and contained small quantity of milk as compared to at day 1. On 10 day alveoli were reduced in size and lacked milk secretion. On 20 to 30 day alveoli atrophied with barely visible lumen and on 40 to 50 day the stroma made up of loose connective tissue were seen in clusters with distinct lining epithelium.

The infection group inoculated with S. aureus showed varying degrees of inflammatory changes as evidenced by distended interstitium with edema and infiltration of neutrophils, few mononuclear cells including lymphocytes and plasma cells at 1 DPI. The mammary acini were dilated and showed intact lining epithelium with presence of milk secretion admixed with large number of neutrophils in the lumina (Fig. 2). The blood vessels in the interstitium as well as in mammary lymph node increased in number and were severely engorged. On 5 DPI, mammary gland section showed severe inflammation with necrosis of lining epithelium of acini, distended interalveolar and interlobular stroma with edema and heavy infiltration of neutrophils and mononuclear cells. The alveoli contained necropurulent exudates with many bacteria, and the epithelial lining in such acini were not distinct (Fig. 3). Few acini contained milk secretion with fat globules. At 10DPI, mammary gland showed necrosis and desquamation in lumen of gland. At 20 DPI, majority of acini showed atrophy of glands. However, some of the acini showed little milk secretions and in interstitium infiltration of neutrophils, lymphocytes and other large mononuclear cells including plasma cells and fibrocytes. The interstitial fibrous connective tissuewas increased and variable degree of vascular congestion was noticed in one case where necrosis of parenchyma with presence of dead and degenerated neutrophils and surrounded by fibrocellular reaction (Fig. 4) which consisted of lymphocytes, macrophages and plasma cells. At 30 DPI parenchyma showed increased fibrosis and atrophy of non secreting acini. By 40 DPI, destruction and replacement of glandular tissue with pus along with small congested vessels and chronic inflammatory changes with presence of macrophages and mature fibrous connective tissue (Fig. 5) with collagen formation was observed (Fig. 6). In one case the active acini with hyperplastic epithelial lining without secretion were observed. At 50 DPI the mammary tissue was almost replaced by extensive fibrocellular reaction with few atrophic slit-like mammary acini in the area. The infiltrating cell mainly composed of large mononuclear cells including plasma cell (Fig. 7). Duplicate section stained by Masson′s trichome revealed extensive mature connective tissue with collagen. The sections of mammary gland stained by Modified Brown and Brenn staining showed presence of clusters of Gram positive cocci in parenchyma consistently upto 20 DPI.

Immunohistochemical Study

The immunohistochemical technique were employed to determine the positive staining for TGF-β in mastitic tissue section. TGF-β is primarily secreted by eosinophils, macrophages, lymphocytes, fibroblasts, epithelial cells and mast cells. At 1 to 10 DPI there was no detectable TGF-β reactive cells. But at 20 and 30 DPI due to increase infiltration of mononuclear cells showed mild to moderate intensity of TGF-β reactive cells predominantly in lymphocyte accumulate in interstitium. On 40 DPI 1 case showed mild and 2 case showed moderate reaction in cytoplasm of lymphocyte and macrophages. But on 50 DPI 1 case showed moderate and 1 case showed higher intense reaction in cytoplasm of lymphocyte, macrophages and fibroblasts as there is increase in fibrocellular reaction in mammary parenchyma (Fig. 8, Table 1).

Cytokine expression in mammary gland

As shown in (Fig. 9) the Staphylococcus aureus induced the production of TGF-β in the mammary gland which was up regulatedwith increasing time interval (1DPI- 1.00 ± 0.29, 5 DPI-1.25 ± 0.036, 10 DPI 1.49± 0.083, 20 DPI 1.77± 0.036, 30 DPI 2.55± 0.076, 40 DPI 2.77 ± 0.031, 50 DPI - 2.86± 0.06 respectively). Them-RNA fold up regulated from 1 fold expression to 1.77 fold upto 20^th day and by 2.55 fold to 2.86 fold expression from 30 to 50 DPI significantly.

Top

DISCUSSION

Top

ACKNOWLEDGEMENTS

Top

Figures

Fig.1.: Mammary gland of control mouse showing dilated lactating alveoli containing milk secretion. HE ×100; TopBack

Fig.2.: Mammary gland of 1std.p.i showed dilated acini containing milk secretion admixed with neutrophils. HE ×200; TopBack

Fig.3.: Mammary gland alveoli containing necropurulent exudates on 5th DPI.HE ×200; TopBack

Fig.4.: Mammary gland at 20th DPI showing groups of atrophied acini in fibrosed stroma.HE ×200. TopBack

Fig.5.: Mammary gland at 40th DPI showing chronic inflammatory changes with infilltration of macrophages and mature fibroblasts.HE ×400; TopBack

Fig.6.: Mammary gland at 40th DPI showing extensive fibrous tissue and macrophages infiltration.MST ×200; TopBack

Fig.7.: Mammary glandular tissue at 50th DPI replaced by fibrous connective tissue. Note infiltration of mononuclear cells. HE ×400; TopBack

Fig.8.: Fibrosed mammary gland at 50thDPIshowing positive staining for TGF-â in cytoplasm of macrophage lymphocytes and macrophages.IHC ×400. TopBack

Fig.9.: Expression levels of cytokine TGF-beta in mammary glands at various intervals. TopBack

Table