ارزیابی الگوی مقاومت آنتی‌بیوتیکی و حضور ژن حدت spvR در سالمونلا‌های جدا‌شده از کبد و تخمدان گله‌های مرغ تخم‌گذار صنعتی استان آذربایجان‌شرقی

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانش‌آموخته‌ دکترای‌حرفه‌ای ‌دامپزشکی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

2 استادیار گروه علوم‌درمانگاهی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

3 استادیار گروه پاتوبیولوژی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران. مرکزتحقیقات بیوتکنولوژی، واحدتبریز، دانشگاه آزاداسلامی، تبریز، ایران.

چکیده

عفونت با باکتری­های جنس سالمونلا موجب بیماری­های مزمن و حاد در طیور می­گردد که می­توانند باعث ایجاد خسارت اقتصادی قابل توجهی به صنعت طیور شوند. هدف از مطالعه حاضر بررسی میزان مقاومت آنتی­بیوتیکی در سالمونلاهای آلوده‌کننده گله­های مرغ تخم­گذار در سطح استان آذربایجان­شرقی و نیز بررسی حضور ژن حدت spvR در جدایه­های مذکور بود. بدین منظور از تعداد 45 گله مشکوک به سالمونلا در مجموع تعداد 200 نمونه کبد و تخمدان اخذشده و در محیط­های انتخابی و افتراقی سالمونلا کشت داده شد. پس از جداسازی باکتری سالمونلا از نمونه­های مذکور، آزمایش آنتی­بیوگرام بر­اساس روش انتشار دیسک در آگار جهت تعیین میزان حساسیت آنتی­بیوتیکی جدایه­ها انجام شد. جهت بررسی حضور ژن حدت spvR  در سالمونلاهای جدا شده نیز از آزمایش مولکولی واکنش زنجیره­ای پلی­مراز و پرایمر­های اختصاصی مربوطه استفاده شد. نتایج مطالعه حاضر نشان داد که تمامی جدایه­های مورد آزمایش نسبت به آنتی­بیوتیک­های اریترومایسین، سولتریم و تتراسایکلین مقاوم بودند. بیشترین میزان مقاومت آنتی­بیوتیکی هم به­ترتیب در برابر داکسی­سایکلین (3/94 درصد)، دانوفلوکساسین (6/92 درصد) و فلورفنیکل (7/91 درصد) مشاهده شد. همچنین بیشترین میزان حساسیت نیز مربوط به آنتی­بیوتیک­های فوزباک (7/94 درصد) و انروفلوکساسین (2/74 درصد) بود. نتایج آزمایش مولکولی نیز نشان داد که ژن spvR در غالب گله­های طیورتخم­گذار استان آذربایجان­شرقی وجود دارد (در 46/88 درصد جدایه­های سالمونلا). با توجه به نتایج حاصله، لزوم پیشگیری از اشاعه سالمونلاهای آلوده­کننده طیور تخم­گذار در راستای بهبود صنعت پرورش طیور و نیز سلامت جوامع انسانی ضروری به­نظر می‌رسد.

کلیدواژه‌ها


عنوان مقاله [English]

Evaluation of antibiotic resistant patterns and spvR virulence gene in isolated salmonellas from liver and ovary of industrial layer farms in east Azarbaijan province

نویسندگان [English]

  • amir allahyari 1
  • H. Nikpiran 2
  • Yunes Anzabi 3
1 D.V.M. Graduate, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
2 Assistant Professor, Department of Clinical Sciences, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
3 Assistant Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran. Biotechnology Research Center, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
چکیده [English]

Introduction: Infectious by salmonella genus bacteria causes acute and chronic diseases in poultry, and finally causes considerable economic losses to poultry industry. Poultry is one of the important hosts of the salmonella bacteria and most developed countries, is a major cause of contaminated foodborne illnesses. Non-typhoid Salmonellae have been recognized as a major cause of intestinal infections of human origin, a major source of these infections being animal products, especially poultry products (Swayne et al., 2013). The virulence spvRABCD plasmid has been found in Salmonella gallinarum and Salmonella pullorum and is also essential for the development of clinical disease (Rychlik et al., 1998). However, the results of some studies have shown that the role of virulence plasmids in the pathogenesis of salmonella is not clear. But there is evidence that spv genes can infect Salmonella typhimurium in the spleen and liver and increase the rate of bacterial proliferation in host cells (Gulig and Doyle, 1993). The aim of current study was to evaluate antibiotic resistance in infected layer flocks by salmonella and determination of spvR gene from isolated samples of east Azarbaijan province flocks.


Material and methods: From 45 suspected flocks overall 200 samples taken from liver and ovary. Samples was cultured in selective and differential growth medium of salmonella and OMPG was performed. Antibiogram test to determination of antibiotic sensitivity was done following isolation of bacteria. For detection of spvR gene in isolated salmonella the polymerase chain reaction with specific primer was done. PCR was used to determine the presence of spvR gene with specific primers. At first, 2 ml tubes were selected and with the TE band and added buffer then a sample of bacterial colonies was dissolved in each tube then dissolved for 1–2 minutes became vortex. The samples were heated on a hot plate for 22 minutes to boil and the lysis of bacteria release the DNA. The samples were then centrifuged and centrifuged at 10,000 rpm for 12 minutes, then the supernatant was removed (clear fluid) and placed on 2 mL tubes on the nanodrag and if the marker of 260-280 was between 1.8-2 indicates good purity and good DNA concentration. Samples were then transferred to an -80°C freezer to be ready for the PCR step. The spvR-F and spvR-R was used in this study. The results of study was analyzed statistically by SPSS version 22.

Results and discussion: The 200 samples studied, a total of 26 Salmonella isolates were isolated and the rest of the samples were negative for Salmonella bacterial culture test. The results of statistical analysis showed that 13% of samples were positive for Salmonella and the rest were negative. The 23 samples (88.46%) has the spv virulence gene. The results indicated all isolates was resistant to Erythromycin, Tetracycline, and Trimethoprim-Sulphamethoxazole, and the highest antibiotic resistance was against Doxycycline 94.3%, Danofloxacin92.6%, Florfenicol 91.7%. The highest sensitivity regard to Fosfomycin 94.7%, and Enrofloxacin 74.2%. Results of molecular tests indicated the spvR gene was existed in layer flocks of east Azarbaijan province. Researchers have shown that the spvR gene is present in 100% of isolated salmonella. In Brazil 82.7% of the samples taken after food poisoning has spvR gene. It was indicated that the Salmonella was isolated from 45.52% of poultry samples and in 14.69% of cases spvR was present. In Chaharmahal va Bakhtiari province, 160 (52.45%) out of 305 samples were infected with Salmonella and the frequency of spvB, spvC, spvR genes was 45.7, 76.6, and 69.14 percent, respectively (Daruoshi et al., 2015). Three spvB, spvC, and spvR genes were present in 60 serotypes of salmonella isolated from different sources and their frequency was 43.3, 73.3, and 46.6%, respectively. The results of researchers at a slaughterhouse in Kerman showed that out of 1001 poultry samples collected, 68 were infected with Salmonella. Overall, 88.6% of the samples also confirmed the presence of the spv gene. The results of the present study showed that 23 cases (88.5%) has spv virulence gene. This is in line with the results of previous studies in this area. Amini (2010) found that 88.6% of spv genes were present in isolated salmonellas. In Brazil, 82.7% of samples the spv genes was present (Geimba et al., 2004). However, Dariush et al., (2015) Only 14.69% of spv virulence genes were present (Daruoshi et al., 2015).
Conclusions: The resistance rate against various antibiotics in layer flocks of east Azarbaijan was high and the virulence spvR gene exist in infected salmonellas of layer flocks.

کلیدواژه‌ها [English]

  • Salmonella
  • layers
  • spvR gene
  • antibiotic sensitivity
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