بیان نسبی ژن‌های سیتوکین پیش التهابی در گاوهای شیری هولشتاین تحت تاثیر ورم پستان اشریشیا کلی

نوع مقاله : مقاله کامل

نویسندگان

10.22099/ijvr.2025.49388.7250

چکیده

پیشینه: ورم پستان شایع‌ترین و پر هزینه‌ترین بیماری در گاوهای شیری است که با التهاب غدد پستانی مشخص می‌شود. در گاوهای شیری، باکتری اشریشیا کلی یکی از عوامل اصلی ورم پستان و لیپوپلی ساکارید آن (LPS) یک عامل بیماری‌زای مهم است. LPS مسئول تحریک بیان سیتوکین‌های پیش التهابی است که اجزای اصلی پاسخ اولیه ایمنی ذاتی میزبان هستند و نقش مهمی در پاسخ التهابی برای از بین بردن عفونت ایفا می‌کنند. هدف: هدف این مطالعه، بررسی پروفایل بیان ژن‌های برخی از سیتوکین‌های پیش التهابی IL-6IL-8، TNF-α، IFN-γ، و (GM-CSF در سلول‌های سوماتیک شیر گاوهای سالم و گاوهایی بود که در اولین دوره شیردهی به طور طبیعی با اشریشیا کلی آلوده شده بودند. روش کار: از شش گاو سالم و شش گاو مبتلا به ورم پستان بالینی نمونه‌های شیر به صورت استریل جمع‌آوری شد. RNA کل از سلول‌های سوماتیک شیر استخراج شد، اولین رشته cDNA سنتز و PCR real-time برای ژن‌های مورد مطالعه انجام شد. به عنوان ژن مرجع، از ژن‌های β-اکتین و GAPDH برای نرمال سازی داده‌ها استفاده شد. از برنامه‌های REST و SAS برای تجزیه و تحلیل داده‌های حاصل از واکنش  real-time PCR استفاده شد. نتایج: ژن‌های سیتوکین پیش التهابی در همه گاوهای سالم و مبتلا به ورم پستان بالینی بیان شد. پروفایل بیان ژن‌های سیتوکین نشان داد که بیان ژن‌های IL-8، TNF-α، IFN-γ، و GM-CSF در گاوهای مبتلا به ورم پستان بالینی در مقایسه با حیوانات عاری از عفونت بسیار بالاتر بود (P<0.01). همبستگی متفاوتی در بیان ژن‌ها در حیوانات سالم و دام‌های مبتلا به ورم پستان مشاهده شد. در حیوانات سالم، همبستگی مثبت و معنی‌داری بین بیان mRNA ژن‌های IL-6 و IL-8 یافت شد (P<0.01). علاوه بر این، سطوح mRNA ژن GM-CFS با بیان ژن‌های IL-6 و IL-8 همبستگی مثبت معنی‌داری نشان داد (P<0.01). در گاوهایی که از ورم پستان بالینی رنج می‌بردند، یک یافته جالب وجود همبستگی مثبت معنی‌دار بین سطوح mRNA ژن GM-CFS و سطوح بیان ژن‌های IL-6، IL-8، IFN-γ و TNF-α بود (P<0.05). نتیجه‌گیری: یافته‌های این مطالعه نشان می‌دهد که ژن‌های IL-8، GM-CFS، IFN-γ، و به ویژه TNF-α می‌توانند شاخص‌های قوی از پاسخ اولیه سیستم ایمنی در غده پستانی گاوهای شیری آلوده به اشریشیا کلی باشند. با این حال، برای تایید یافته‌های این تحقیق باید مطالعات بیشتری انجام شود.

کلیدواژه‌ها

موضوعات


Aggarwal, BB; Shishodia, S; Ashikawa, K and Bharti, AC (2002). The role of TNF and its family members in inflammation and cancer: lessons from gene deletion. Inflamm. Allergy Drug Targets, 1: 327-341.
Alluwaimi, A; Farver, T and Cullor, J (2003). Transcriptional activity of IL-8 in healthy bovine mammary gland at mid and late-lactation. Pak. J. Biol. Sci., 6: 729-731.
Asuero, AG; Sayago, A and González, AG (2006). The correlation coefficient: An overview. Crit. Rev. Anal. Chem., 36: 41-59.
Bannerman, DD; Paape, MJ; Lee, JW; Zhao, X; Hope, JC and Rainard, P (2004). Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin. Vaccine Immunol., 11: 463-472.
Bhatt, VD; Shah, TM; Nauriyal, DS; Kunjadia, AP and Joshi, CG (2014). Evaluation of a topical herbal drug for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis. AYU., 35: 198-205.
Boulanger, MJ; Bankovich, AJ; Kortemme, T; Baker, D and Garcia, KC (2003). Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gp130. Mol. Cell., 12: 577-589.
Bradley, J (2008). TNF-α mediated inflammatory disease. J. Pathol., 214: 149-160.
Bradley, A and Green, M (2001). Adaptation of Escherichia coli to the bovine mammary gland. J. Clin. Microbiol., 39: 1845-1849.
Brankatschk, R; Bodenhausen, N; Zeyer, J and Bürgmann, H (2012). Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples. Appl. Environ. Microbiol., 78: 4481-4489.
Buitenhuis, B; Røntved, CM; Edwards, SM; Ingvartsen, KL and Sørensen, P (2011). In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis. BMC Genom., 12: 1-10.
Burvenich, C; Van Merris, V; Mehrzad, J; Diez-Fraile, A and Duchateau, L (2003). Severity of E. coli mastitis is mainly determined by cow factors. Vet. Res., 34: 521-564.
Chen, W; Liu, Y; Yin, J; Deng, Y; Ali, T; Zhang, J; Cheng, J; Gao, J and Han, B (2017). Cloning, expression, and immunogenicity of fimbrial-F17A subunit vaccine against Escherichia coli isolated from bovine mastitis. Biomed. Res. Int., 1 : 3248483.
Cobirka, M; Tancin, V and Slama, P (2020). Epidemiology and classification of mastitis. Animals. 10: 2212.
De Schepper, S; De Ketelaere, A; Bannerman, D; Paape, M; Peelman, L and Burvenich, C (2008). The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Vet. Res., 39: 1-23.
Diehl, S and Rincón, M (2002). The two faces of IL-6 on Th1/Th2 differentiation. Mol. Immunol., 39: 531-536.
Flavell, RA (2002). The relationship of inflammation and initiation of autoimmune disease: role of TNF super family members. Curr. Top. Microbiol. Immunol., 266: 1-9.
Fonseca, I; Silva, PV; Lange, CC; Guimarães, MF; Weller, MMDCA; Sousa, KRS; Lopes, PS; Guimarães, JD and Guimarães, SE (2009). Expression profile of genes associated with mastitis in dairy cattle. Genet. Mol. Biol., 32: 776-781.
Galligan, C and Coomber, B (2000). Effects of human IL-8 isoforms on bovine neutrophil function in vitro. Vet. Immunol. Immunopathol., 74: 71-85.
Guo, YF; Xu, NN; Sun, W; Zhao, Y; Li, CY and Guo, MY (2017). Luteolin reduces inflammation in Staphylococcus aureus-induced mastitis by inhibiting NF-κB activation and MMPs expression. Oncotarget. 8: 28481-28493.
Hamilton, JA (2008). Colony-stimulating factors in inflammation and autoimmunity. Nat. Rev. Immunol., 8: 533-544.
Hassan, RF and Torky, HA (2016). Cytokines expression associated with E. coli infection in bovine mammary glands. Alex. J. Vet., 48: 54-60.
Hisaeda, K; Hagiwara, K; Eguchi, J; Yamanaka, H; Kirisawa, R and Iwai, H (2001). Interferon-γ and tumor necrosis factor-α levels in sera and whey of cattle with naturally occurring coliform mastitis. J. Vet. Med. Sci., 63: 1009-1011.
Hucker, GJ (1933). The laboratory detection of bovine mastitis. New York Agr. Exp. Station Bul., 626: 1-24.
Huma, ZI; Sharma, N; Kour, S; Tandon, S; Guttula, PK; Kour, S; Singh, AK; Singh, R and Gupta, MK (2020). Putative biomarkers for early detection of mastitis in cattle. Anim. Prod. Sci., 60: 1721-1736.
Jiang, KF; Zhao, G; Deng, GZ; Wu, HC; Yin, NN; Chen, XY; Qiu, CW and Peng, XL (2017). Polydatin ameliorates Staphylococcus aureus-induced mastitis in mice via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB pathway. Acta Pharmacol. Sin., 38: 211-222.
Kaper, JB; Nataro, JP and Mobley, HL (2004). Pathogenic Escherichia coli. Nat. Rev. Microbiol., 2: 123-140.
Kauf, A; Rosenbusch, R; Paape, M and Bannerman, DD (2007). Innate immune response to intramammary Mycoplasma bovis infection. J. Dairy Sci., 90: 3336-3348.
Kehrli Jr, ME and Harp, JA (2001). Immunity in the mammary gland. Vet. Clin. North Am. Food Anim., 17: 495-516.
Lee, JW; Bannerman, D; Paape, M; Huang, MK and Zhao, X (2006). Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR. Vet. Res., 37: 219-229.
Leutenegger, CM; Alluwaimi, AM; Smith, WL; Perani, L and Cullor, JS (2000). Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan® polymerase chain reaction. Vet. Immunol. Immunopathol., 77: 275-287.
Livak, KJ and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 25: 402-408.
Ma, J; Zhu, Y; Zhang, L; Zhuge, Z; Liu, P; Yan, X; Gao, H and Wang, J (2011). Serum concentration and mRNA expression in milk somatic cells of toll-like receptor 2, toll-like receptor 4, and cytokines in dairy cows following intramammary inoculation with Escherichia coli. J. Dairy Sci., 94: 5903-5912.
McClenahan, D; Krueger, R; Lee, HY; Thomas, C; Kehrli Jr, ME and Czuprynski, C (2006). Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli. Comp. Immunol. Microbiol. Infect. Dis., 29: 127-137.
Mitterhuemer, S; Petzl, W; Krebs, S; Mehne, D; Klanner, A; Wolf, E; Zerbe, H and Blum, H (2010). Escherichia coli infection induces distinct local and systemic transcriptome responses in the mammary gland. BMC Genom., 11: 1-16.
Noleto, PG; Gilbert, FB; Rossignol, C; Cunha, P; Germon, P; Rainard, P and Martins, RP (2023). Punch-excised explants of bovine mammary gland to model early immune response to infection. J. Anim. Sci. Biotechnol., 14: 100.
Özaktay, AC; Kallakuri, S; Takebayashi, T; Cavanaugh, JM; Asik, I; DeLeo, JA and Weinstein, JN (2006). Effects of interleukin-1 beta, interleukin-6, and tumor necrosis factor on sensitivity of dorsal root ganglion and peripheral receptive fields in rats. Eur. Spine. J., 15: 1529-1537.
Paape, M; Mehrzad, J; Zhao, X; Detilleux, J and Burvenich, C (2002). Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes. J. Mammary Gland Biol. Neoplasia. 7: 109-121.
Pfaffl, MW; Horgan, GW and Dempfle, L (2002). Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res., 30: e36.
Rabot, A; Wellnitz, O; Meyer, HH and Bruckmaier, RM (2007). Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue. J. Dairy Res., 74: 93-99.
Rambeaud, M; Clift, R and Pighetti, G (2006). Association of a bovine CXCR2 gene polymorphism with neutrophil survival and killing ability. Vet. Immunol. Immunopathol., 111: 231-238.
Remick, DG (2005). Interleukin-8. Crit. Care Med., 33: 466-467.
Riollet, C; Rainard, P and Poutrel, B (2000). Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus. Clin. Diagn. Lab. Immunol., 7: 161-167.
Riollet, C; Rainard, P and Poutrel, B (2002). Cells and cytokines in inflammatory secretions of bovine mammary gland. In: Mol, JA and Clegg, RA (Eds.), Biology of the mammary gland advances in experimental medicine and biology. (1st Edn.), Vol. 480, Boston, MA, Springer. PP: 247-258.
Safak, T; Rısvanli, A and Asci-Toraman, Z (2022). Th1/Th2 cytokine polarization in milk according to different pathogens causing subclinical mastitis in cows. J. Dairy Product. Proces. Improv., 72: 105-113.
Sbuster, D; Kehrli, M and Stevens, MG (1993). Cytokine production during endotoxin-induced mastitis in lactating dairy cows. Am. J. Vet. Res., 54: 80.
Schukken, YH; Bennett, GJ; Zurakowski, MJ; Sharkey, HL; Rauch, BJ; Thomas, MJ; Ceglowski, B; Saltman, RL; Belomestnykh, N and Zadoks, R (2011). Randomized clinical trial to evaluate the efficacy of a 5-day
ceftiofur hydrochloride intramammary treatment on nonsevere gram-negative clinical mastitis. J. Dairy Sci., 94: 6203-6215.
Shuster, DE; Kehrli Jr, ME; Rainard, P and Paape, M (1997). Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia coli. Infect. Immun., 65: 3286-3292.
Singathia, R; Sharma, DK and Gaurav, A (2023). Relative expression of Toll-like receptors, cytokines and acute phase protein by real-time PCR in milk somatic cells of subclinical mastitis affected cattle. Indian J. Anim. Res., 1: 780-784.
Smith, JA; Das, A; Ray, SK and Banik, NL (2012). Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res. Bull., 87: 10-20.
Steimle, A; Autenrieth, IB and Frick, JS (2016). Structure and function: Lipid a modifications in commensals and pathogens. Int. J. Med. Microbiol., 306: 290-301.
Vangroenweghe, F; Duchateau, L and Burvenich, C (2020). J-5 Escherichia coli vaccination does not influence severity of an Escherichia coli intramammary challenge in primiparous cows. J. Dairy Sci., 103: 6692-6697.
Vangroenweghe, F; Lamote, I and Burvenich, C (2005). Physiology of the periparturient period and its relation to severity of clinical mastitis. Domest. Anim. Endocrinol., 29: 283-293.
Vitenberga-Verza, Z; Pilmane, M; Šerstņova, K; Melderis, I; Gontar, Ł; Kochański, M; Drutowska, A; Maróti, G and Prieto-Simón, B (2022). Identification of inflam-matory and regulatory cytokines IL-1α-, IL-4-, IL-6-, IL-12-, IL-13-, IL-17A-, TNF-α-, and IFN-γ-producing cells in the milk of dairy cows with subclinical and clinical mastitis. Pathogens. 11: 372.
Waller, KP; Colditz, IG; Lun, S and Östensson, K (2003). Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis. Res. J. Vet. Sci., 74: 31-36.
Wenz, J; Barrington, G; Garry, F; Ellis, R and Magnuson, R (2006). Escherichia coli isolates’ serotypes, genotypes, and virulence genes and clinical coliform mastitis severity. J. Dairy Sci., 89: 3408-3412.