Banu BAYRİ ÖZBİLĞE, Veli GÜLYAZ, Neslihan TAŞÇENE, Şükran YILMAZ, Yasemin GÜLTEKİN, Ayşenur ULUSOY, Mustafa HASÖKSÜZ, Gonca ÖZTAP

Enrofloxacin, Linco-Spectin ve Florfenicol antibiyotiklerinin BHK-21 hücre kültürü ve FMD 146S virus partikülü-infektif titreleri üzerine etkilerinin belirlenmesi

Abstract: Enrofloxacin, linko-spectin and florphenicol antibiotics were intended to be used in the BHK-21 An30 cells and the foot and mouth disease virus (FMDV) culture during the vaccine production process. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and cell cultures assays were performed in the BHK-21 An30 cells treated with the antibiotics that took place in this study to determine the concentration that inhibits cell proliferation and adverse effects on cell morphology. Virus cultures were performed by inoculating of FMDV serotypes (A/TUR/11, O/TUR/07, Asia-1/TUR/15) to the treated cell cultures with the antibiotics. 146S and infective tites of the obtained virus suspensions were determined. The non-toxic upper limit was determined to be 15μg /ml for enrofloxacin and 300μg/ml for linco-spectin in terms of cell morphology and cell numbers versus positive control (gentamycin, penicillin-streptomycin) and negative control (antibiotic-free medium) as a result of MTT and cell culture tests on BHK cells. It was observed that Florfenicol also inhibited cell proliferation and induced cell degeneration, even at a concentration of 5μg/ml. The mean 146S values of FMD virus cultures containing enrofloxacin and linco-spectin were determined to be 0.49, 0.46, ; 0.53, 0.47 and 0.30, 0.28µg/ml for serotype A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the infective titres were 107,04, 107,25, 106,04, 106,59 and 107,26, 107,6 pfu/ml. for serotype A/TUR/11, O/TUR/07, and Asia-1/TUR/15 respectively. In the control group used gentamycin, penicillin-streptomycin and antibiotic-free medium, the mean 146S FMD virus particles were obtained as 0.51, 0.50, 0.50; 0.52, 0.55, 0.52 and 0.36, 0.33, 0.30 µg/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the FMD infective virus titres were 107,35, 107,40, 107,11; 106,24, 106,46, 106,62, and 107,70, 107,75, 107,77 pfu/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. As a result, the infective FMDV titer and 146S results obtained in the control group (gentamicin, penicillin-streptomycin) and FMD virus cultures using Enrofloxacin (15µg / ml) and linco-spectin (300µg / ml) were very close to each other. According to these data, it was concluded that enrofloxacin and linco-spectin can be used up to the upper limit in the BHK-21 An30 cell and FMD virus cultures. However, florfenicol should not be used in cell and virus cultures.

Anahtar Kelimeler:

Antibiyotik, BHK-21 An30, hücre kültürü, kontaminasyon, şap virüsü

Determination of the effects of Enrofloxacin, Linco-Spectin and Florphenicol antibiotics on BHK-21 cell culture and FMD 146S Virus particles-infective titers

Keywords:

Antibiotic, BHK cell culture, contamination, FMD virus,

PDF

___

Akkan HA and Karaca M. (2003). Veteriner iç hastalıklarında antibiyotiklerin kullanımı. YYÜ Vet Fak Derg. 14 (2), 72-77.
Baer A and Kehn-Hall K. (2014). Viral concentration determination through plaque assays: Using Traditional and Novel Overlay Systems. J O V E. 93, 1-10.
Berg G, Harris E K, Chang S L, Busch K A. (1963). Quantitation of viruses by the plaque technique. J Bacteriol. 85, 691-700. 1963.
Campos C O, Bernuci M P, Vireque AA, Campos J R, Silva-de-Sa M F, Jamur M C, and Rosa-e-Silva ACJS. (2012). Preventing microbial contamination during long-term in vitro culture of human granulosa-lutein cells: an ultrastructural analysis. ISRN Obstetrics and Gynecology. Vol. 2012, Article ID 152781, 6 pages.
Çokçalışkan C, Türkoğlu T, Sareyyüpoğlu B, Uzunlu E, Babak A, Özbilge BB, Gülyaz V. (2016). Qs-21 enhances the early antibody response to oil adjuvant foot and mouth disease vaccine in cattle. Clin. Exp. Vacc. Res. 5:138-147.
Doronin MI, Timina AM, Lozovoy DA, Starikov VA, Mikhalishin DV, Medvedeva NN, Borisov AV. (2018). Method for fmd vırus 146S component concentratıon determınatıon wıth real-tıme reverse transcrıptıon-polymerase chaın reaction in vaccıne raw materıals. Vet Scıence Today. 2 (25).
Egerbacher M, Edinger J, Tschulenk W. (2001). Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture. Am J Vet Res. 62(5), 704-708.
Fleckenstein E, Uphoff CC, Drexler HG. (1994), Effective treatment of mycoplasma contamination in cell lines with enrofloxacin (Baytril). Leukemia. 8(8),1424-1434.
Freshney R.I. (2005). Culture of Animal Cells. A manual of basic tecnique/Fifth Edition.
Grubman MJ and Baxt B. (2004), Foot and mouth disease. Clin. Microbiol. Rev. 17(2), 465-493.
Holst-Hansen C, Brünner N. (2000), Cell and tissue culture and associated tecniques. Cell Biology 1, Julio E. Celis (eds), Academic Press
Hu JF, Gilmer L, Hopkins R, Wolfinbarger L Jr. (1989). Effects of antibiotics on cellular viability in porcine heart valve tissue. Cardiovascular Research. 23(11), 960-964.
Jamal SM and Belsham G J. (2013). Foot-and-mouth disease: past, present, and future. Vet Res. 44, 116.
Lim S, Hossain MA, Park J, Choi SH, Kim G. (2008), The effects of enrofloxacin on canine tendon cells and chondrocytes proliferation in vitro. Vet Res Commun. 32, 243-253.
Liu B, Cui Y, Brown PB, Ge X, Xie J, Xu P. (2015). Cytotoxic effects and apoptosis induction of enrofloxacin in the hepatic cell line of grass carp (Ctenopharyngodon idellus). Fish&Shellfish İmmunology. 47(2), 639-644.
Naughton GK, Eisinger M, Bystryn JC. (1983). Antibodies to normal human melanocytes ın vitiligo, J Exp Med. 158, 246-251.
Nawaz Z, Siddique A B, Zahoor M A, Bilal Aslam et al., 2019. Detectıon of foot and mouth disease vırus shedding ın milk of apparently healthy buffaloes and cattle of Punjab, Pakıstan. Buffalo Bulletin, 38(2), 255-261.
Or ZS and Fidancı UR. (2009). Şap virüsü ile enfekte ve aşılı danalarda serum proteinlerinin elektroforetik dağılımı. Ankara Üniv Vet Fak Derg. 56, 13-18.
OIE, (2017). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Terrestrial manual: Foot and mouth disease: OIE terrestrial manual, Chapter 3.1.8. (May),433-464.
Sareyyüpoğlu B, Gülyaz V, Cokçalışkan C, Ünal Y, Çökülgen T, Uzunlu E, Gürcan S, Ilk O (2019). Effect of FMD vaccination schedule of dams on the level and duration of maternally derived antibodies. Vet. Immunol. Immunopathol. Vol: 217
Perlman D. (1979). Use of antibiotics in cell culture media. Cell Culture Methods In Enzymology. 58, 110-116.
Reeves P T. (2012). Antibiotics: groups and properties. Chemical Analysis of Antibiotic Residues in Food, First Edition. John Wiley & Sons, Inc.
Saran B and Karahan ZC. (2010). Ürolojide antibiyotik kullanımı-antimikrobiyal ajanlara genel bakış. Turk Urol Sem. 1, 216-220.
Spier RE and Grıffıts JB. (1985). Animal Cell Biotechnology. Academic Press.
Uphoff CC and Drexler H.G. (2002). Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. Invitro Cell Dev Biol Anim. 38(2):86-9.
Uphoff CC, Denkmann SA and Drexler HG. (2012). Treatment of mycoplasma contamination in cell cultures with plasmocin. Journal of Biomedicine and Biotechnology. Article ID 267678, 8 pages. DOI:10.1155/2012/267678.
Yarsan E. (2012). Veteriner hekimlikte antibiyotikler: antibiyotiklere direnç ve direncin çok yönlü etkileri. tvhb.org.tr › wp-content › uploads › 2019/03 ›
Xiao Y, Chen H Y , Wang Y, Yin B, Lv C, Mo X, Yan H, Xuan Y, Huang Y, Pang W, Li X, Yuan Y A and Tian K. (2016). Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle. BMC Biotechnology. 16:56