Evaluation of MMP-9 and iNOS expressions in sheep with encephalitic listeriosis

Abstract

This study aimed to correlate Matrix Metalloproteinase-9 and iNOS expressions with the severity of histopathological findings in tissue samples taken from sheep with encephalitic listeriosis. Thus, the role of these molecules in the pathogenesis of the disease can be elucidated. After systemic necropsy, tissue samples of adult sheeps with meningoencephalitis were investigated by the culture, histopathological and immunohistochemical methods in the presence of Listeria spp. isolation from tissues was performed in accordance with the USDA-FSIS method with some modifications. Tissue samples were fixed in a 10% buffered formaldehyde solution. Following routine procedures, tissue sections at 5 μm were stained with Hematoxylin and Eosin, investigated under light microscope and photographed. Immunohistochemical staining was performed on the tissues using the avidin-biotin immune peroxidase complex method. Listeria spp. were obtained in 20 (83.3%) of 24 tissue samples with the presence of bright grey-black centred smooth colonies on Listeria Selective Agar and identified as Listeria monocytogenes through the phenotypically supportive tests. Liquefaction necrosis, purulent meningoencephalitis, perivascular cuffing, microabscesses and glial nodules were the most important histopathological findings. MMP-9 immunpositive reactions were observed in the cytoplasm of microglial cells and neurons in areas where inflammatory and necrotic areas are concentrated in medulla oblongata and pons. In perivascular cuffing areas, immune reactions in endothelial cells were detected. We detected iNOS positive reactions in the medulla oblangata and pons, especially in inflammatory cells in the microabscesses. Consequently, a positive correlation (p < 0.05) was found between MMP-9 expression and the severity of histopathological findings in sheep with encephalitic listeriosis. expression. In addition, we found that iNOS expression increased in parallel with the increase in MMP-9

Keywords

• culture
• encephalitic listeriosis
• histopathology
• immunohistochemistry
• mmp-9

Ensefalitik listeriyozisli koyunlarda MMP-9 ve iNOS ekspresyonunun değerlendirilmesi

Öz

Bu çalışmada ensefalitik listeriyozisli koyunlardan alınan doku örneklerinde gözlenen histopatolojik bulguların şiddeti ile Matriks metalloproteinaz-9 ve iNOS ekspresyonlarını korele etmeyi amaçladık.Böylece bu moleküllerin hastalığın patogenezindeki rolü açıklanabilecektir. Sistemik nekropsi sonrası meningoensefalitli erişkin koyunlardan alınan doku örnekleri Listeria spp. varlığı için kültür, histopatolojik ve immunohistokimyasal olarak incelendi. Dokulardan Listeria spp. izolasyonu bazı modifikasyonlarla USDA-FSIS yöntemine uygun olarak gerçekleştirildi. Doku örnekleri %10’luk tamponlu formaldehit solüsyonunda tespit edildi. Rutin işlemlerden sonra 5 μm kalınlığındaki kesitler Hematoksilen&Eozin ile boyandı, ışık mikroskobu altında incelendi ve fotoğraflandı. Dokulara immunohistokimyasal boya olarak avidin-biotin immunperoksidaz kompleks metodu uygulandı. Listeria spp. Listeria Selective Agar üzerinde parlak gri-siyah merkezli pürüzsüz koloniler bulunan 24 doku örneğinden 20'sinde (% 83.3) elde edildi ve fenotipik olarak destekleyici testler yoluyla Listeria monocytogenes olarak tanımlandı. Likefaksiyon nekrozu, purulent meningoensefalitis, perivasküler hücre infiltrasyonu, mikroapseler ve glial nodüller en önemli histopatolojik bulgulardı. MMP-9 immunpozitif reaksiyonları yangının ve nekrozun yoğun olduğu alanlardaki mikroglial hücreler ve nöronların sitoplazmasında gözlemledik. Perivasküler hücre infiltrasyonu alanlarında, endotelyal hücrelerde de immun reaksiyonu saptadık. iNOS pozitif reaksiyonları özellikle medulla oblongata ve pons bölgesinde yer alan mikroapselerdeki yangısal hücrelerde tespit ettik. Sonuç olarak ensefalitik listeriyozisli koyunlarda MMP-9 ekspresyonu ile histopatolojik bulguların şiddeti arasında pozitif bir korelasyon tespit ettik (p<0.05). Buna ek olarak MMP-9 ekspresyonundaki artışa paralel olarak iNOS ekspresyonun da artış gösterdiğini ortaya koyduk.

Anahtar Kelimeler

• ensefalitik listeriyozis
• histopatoloji
• immunohistokimya
• kültür
• mmp-9

Kaynakça

1. Referans1 Abdlla OA, Elboshy ME, Reisha EF, Gadlla HA, El-Khodery (2015): Tumor Necrosis Factor-α, Interleukins-12(p40), 6, and 10 levels in cerebrospinal fluid and outcome prediction in Ossimi sheep with encephalitic listeriosis. Cytokine, 73(2), 283-287.
2. Referans2 Akça D, Şahin M (2011): Kars yöresi sığırlarından alınan süt ve vajinal sıvap örneklerinden Listeria türlerinin araştırılması. Kafkas Univ Vet Fak Derg, 17(6), 987-993.
3. Referans3 al-Dughaym AM, Elmula AF, Mohamed GE, Hegazy AA, Radwan YA, Housawi FM, Gameel AA (2001): First report of an outbreak of ovine septicaemic listeriosis in Saudi Arabia. Rev Sci Tech, 20(3), 777-783.
4. Referans4 Barkallah M, Gharbi Y, Hmani M, Mallek Z, Gautier M, Gdoura R, Fendri I (2016): Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants. Mol Cell Probes, 30(3), 138-145.
5. Referans5 Berman NE, Marcario JK, Yong C, Raghavan R, Raymond LA, Joag SV, Narayan O, Cheney PD (1999): Microglial activation and neurological symptoms in the SIV model of NeuroAIDS: association of MHC-II and MMP-9 expression with behavioral deficits and evoked potential changes. Neurobiol Dis, 6(6), 486-498.
6. Referans6 Braun U, Stehle C, Ehrensperger F (2002): Clinical findings and treatment of listeriosis in 67 sheep and goats. Vet Rec, 150(2), 38-42.
7. Referans7 Brugère-Picoux J (2008): Ovine listeriosis. Small Rum Res, 76(1-2), 12-20.
8. Referans8 Campero CM, Odeón AC, Cipolla AL, Moore DP, Poso MA, Odriozola E (2002): Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis. J Vet Med B Infect Dis Vet Public Health, 49(8), 379-383.

9. Referans9 Çeribaşı S, Kızıl Ö, Karahan M (2013): Listeriyozisli koyunlarda klinik, patolojik ve mikrobiyolojik bulgular. F Ü Sağ Bil Vet Derg, 27(1), 1-5.
10. Referans10 Dağ S, Akça D, Karaman M, Çelebi Ö, Özen H, Büyük F, Şahin M (2013): Investigation of immunoperoxidase technique in smears prepared from vaginal secretions in use of early diagnosis of listerial abortions in cattle. Kafkas Univ Vet Fak Derg, 19(1), 1-6.
11. Referans11 Dhama K, Karthik K, Tiwari R, Shabbir MZ, Barbuddhe S, Malik SVS, Singh RK (2015): Listeriosis in animals, its public health significance (food-borne zoonosis) and advances in diagnosis and control: A comprehensive review. Vet Quart, 35(4), 211-235.
12. Referans12 Dreyer M, Aguilar-Bultet L, Rupp S, Guldimann C, Stephan R, Schock A, Otter A, Schüpbach G, Brisse S, Lecuit M, Frey J, Oevermann A (2016): Listeria monocytogenes sequence type 1 is predominant in ruminant rhombencephalitis. Sci Rep, 6, 36419.
13. Referans13 Dreyer M, Thomann A, Böttcher S, Frey J, Oevermann A (2015): Outbreak investigation identifies a single Listeria monocytogenes strain in sheep with different clinical manifestations, soil and water. Vet Microbiol, 179(1-2), 69-75.
14. Referans14 Food And Drug Administration (FDA) (2015): Testing methodology for listeria species or L. monocytogenes in environmental samples. Version 1, Pp.2-11.
15. Referans15 Guldimann C, Bärtschi M, Frey J, Zurbriggen A, Seuberlich T, Oevermann A (2015): Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex. BMC Microbiology, 15(1), 134.
16. Referans16 Haligur M, Aydogan A, Ozmen O, Ipek V (2019): Immunohistochemical evaluation of natural cases of encephalitic listeriosis in sheep. Biotech Histochem, 94(5), 341-347.
17. Referans17 Headley SA, Bodnar L, Fritzen JT, Bronkhorst DE, Alfieri AF, Okano W, Alfieri AA (2014): Histopathological and molecular characterization of encephalitic listeriosis in small ruminants from northern Paraná, Brazil. Braz J Microbiol, 44(3), 889-896.
18. Referans18 Henke D, Rupp S, Gaschen V, Stoffel MH, Frey J, Vandevelde M, Oevermann A (2015): Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration. Infect Immun, 83(6), 2409-2419.
19. Referans19 İlhan F, Ulusoy Y, Halıgür M (2012): Matrix metalloproteinase expression in sheep with listerial meningoencephalitis. Res Vet Sci, 92(2), 269-272.
20. Referans20 Kieseier BC, Paul R, Koedel U, Seifert T, Clements JM, Gearing AJ, Pfister HW, Hartung HP (1999): Differential expression of matrix metalloproteinases in bacterial meningitis. Brain, 122(Pt 8), 1579-1587.
21. Referans21 Liang FR, Wang QQ, Jiang YL, Yue BY, Zhou QZ, Wang JH (2020): Characterization of matrix metalloprotease-9 gene from Nile tilapia (Oreochromis niloticus) and its high-level expression induced by the Streptococcus agalactiae challenge. Biomolecules, 10(1), 76.
22. Referans22 Loeb E (2004): Encephalitic listeriosis in ruminants: Immunohistochemistry as a diagnostic tool. J Vet Med A Physiol Pathol Clin Med, 51(9-10), 453-455.
23. Referans23 Madarame H, Seuberlich T, Abril C, Zurbriggen A, Vandevelde M, Oevermann A (2011): The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion. Neuropathol Appl Neurobiol, 37(7), 753-767.
24. Referans24 McClain D, Lee WH (1988): Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J Assoc Off Anal Chem, 71(3), 660-664.
25. Referans25 Morin DE (2004): Brainstem and cranial nerve abnormalities: Listeriosis, otitis media/interna, and pituitary abscess syndrome. Vet Clin North Am Food Anim Pract, 20(2), 243-273.
26. Referans26 Nau R, Djukic M, Spreer A, Ribes S, Eiffert H (2015): Bacterial meningitis: an update of new treatment options. Expert Rev Anti Infect Ther, 13(11), 1401-1423.
27. Referans27 Nightingale KK, Schukken YH, Nightingale CR, Fortes ED, Ho AJ, Her Z, Grohn YT, McDonough PL, Wiedmann M (2004): Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment. Appl Environ Microbiol, 70(8), 4458-4467.
28. Referans28 Oevermann A, Botteron C, Seuberlich T, Nicolier A, Friess M, Doherr MG, Heim D, Hilbe M, Zimmer K, Zurbriggen A, Vandevelde M (2008): Neuropathological survey of fallen stock: Active surveillance reveals high prevalence of encephalitic listeriosis in small ruminants. Vet Microbiol, 130(3-4), 320-399.
29. Referans29 Oevermann A, Di Palma S, Doherr MG, Abril C, Zurbriggen A, Vandevelde M (2010): Neuropathogenesis of naturally occurring encephalitis caused by Listeria monocytogenes in ruminants. Brain Pathol, 20(2), 378-390.
30. Referans30 Orsi RH, Wiedmann M (2016): Characteristics and distribution of Listeria spp., including Listeria species newly described since 2009. Appl Microbiol Biotechnol, 100(12), 5273-5287.
31. Referans31 Ortatatlı M, Çiftçi MK, Tuzcu M (2001): Bir koyun sürüsünde görülen purulent ensefalitis. Vet Bil Derg, 17(2), 97-102.
32. Referans32 Özyıldız Z, Dinçel GÇ, Terzi OS, Özsoy ŞY, Kul O (2018): Immunohistochemical investigation of the damage to and repair of myelin, and astrocyte activity in small ruminants resulting from with natural meningoencephalitic listeriosis. Ankara Üniv Vet Fak Derg, 65 (3), 283-290.
33. Referans33 Precht C, Diserens G, Vermathen M, Oevermann A, Lauper J, Vermathen P (2018): Metabolic profiling of listeria rhombencephalitis in small ruminants by 1H high‐resolution magic angle spinning NMR spectroscopy. NMR in Biomedicine, 31(12), e4023.
34. Referans34 Rosenberg GA (2002): Matrix metalloproteinases in neuroinflammation. Glia, 39(3), 279-291.
35. Referans35 Shihab PK, Al-Roub A, Al-Ghanim M, Al-Mass A, Behbehani K, Ahmad R (2015): TLR2 and AP-1/NF-kappaB are involved in the regulation of MMP-9 elicited by heat killed Listeria monocytogenes in human monocytic THP-1 cells. J Inflamm (Lond), 12, 32.
36. Referans36 Sulik A, Chyczewski L (2008): Immunohistochemical analysis of MMP-9, MMP-2 and TIMP-1, TIMP-2 expression in the central nervous system following infection with viral and bacterial meningitis. Folia Histochem Cytobiol, 46(4), 437-442.
37. Referans37 Şahin M, Beytut E (2006): Abortions in sheep due to Listeria ivanovii in the Kars region. Turk J Vet Anim Sci, 30(5), 503-506.
38. Referans38 Wesley IV, Larson DJ, Harmon KM, Luchansky JB, Schwartz AR (2002): A case report of sporadic ovine listerial menigoencephalitis in Iowa with an overview of livestock and human cases. J Vet Diagn Invest, 14(4), 314-321.
39. Referans39 Yamada K, Yoshino K, Sekikawa K, Madarame H, Yagita H, Nakane A (2000): Effect of a matrix metalloproteinase inhibitor on host resistance against Listeria monocytogenes infection. FEMS Immunol Med Microbiol, 29(3), 187-194.