Sağlıklı sığırların nazal boşluk flora bakterilerinin moleküler identifikasyonu

Öz

Bu çalışmada, klinik olarak sağlıklı sığırların nazal florasını oluşturan aerobik bakterilerin belirlenebilmesi için, alınan nazal sıvap örneklerinden izole edilen etkenlerin, 16S rRNA dizi analizi ile moleküler identifikasyonlarının yapılması amaçlandı. Çalışmada 15 çiftlikteki 56 sığırdan alınan nazal sıvap örnekleri kullanıldı. Nazal sıvaplardan konvansiyonel yöntemler kullanılarak bakteri izolasyonu gerçekleştirilip, etkenlerin makroskobik ve mikroskobik morfolojileri incelendikten sonra, identifikasyonları üniversal primerler kullanarak 16S rRNA geni polimeraz zincir reaksiyonu ile çoğaltıldı. İzolasyonları yapılan 192 mikroorganizmanın sekans analizi ile 143 (%74,5)’ünün Gram pozitif ve 49 (%25,5)’unun Gram negatif mikroorganizma olduğu tespit edildi. Koagulaz negatif stafilokoklar (%36,5), Bacillus sp. (%15,6), S. aureus (%11,0), M. haemolytica (%6,3), Enterobacteriaceae sp. (%6,8), P. multocida (%5,2), M. luteus (%4,2), Streptococcus sp. (%2,6), Acinetobacter sp. (%2,6), Pseudomonas sp. (%2,1) ve Corynebacterium sp. (%1,0) en çok identifiye edilen türler olarak belirlendi. Bu çalışmada konvansiyonel mikrobiyolojik yöntemler ve moleküler identifikasyon yöntemlerinin büyük oranda birbirleri ile uyumlu oldukları görüldü. Bundan sonra yapılacak olan çalışmalarda, floradan izole edilen mikroorganizmaların virulens ve antibiyotik direnç genlerinin incelemesi yapılabilir.

Anahtar Kelimeler

• Süheyla TÜRKYILMAZ
• Zeynep ESKİN
• Bakteri
• Nazal flora
• Moleküler İdentifikasyon
• 16S rRNA
• Etlik Veteriner Mikrobiyoloji Dergisi
• 2014

Molecular identification of bacteria of flora nasal cavity of healthy calves

Öz

This study aimed to carry out the molecular identification of aerobic bacterial nasal flora of clinically healthy calves, collected by nasal swabs, by using 16S rRNA sequence analysis. Nasal swab samples from 56 calves on 15 farms were used in the study, isolation of bacteria was carried out by using conventional methods, after examining the macroscopic and microscopic morphology of the factors, their identifications were carried out by using universal primers duplicating polymerase chain reaction of 16S rRNA gene. With the sequence analysis of the isolated 192 micro-organisms, it was determined that 143 (74.5%) were Gram-positive and 49 (25.5%) were Gram-negative microorganisms. Coagulase-negative staphylococci (36.5%), Bacillus sp. (15.6%), S. aureus (11.0%), M. haemolytica (6.3%), Enterobacteriaceae sp. (6.8%), P. multocida (5.2%), M. luteus (4.2%), Streptococcus sp. (2.6%), Acinetobacter sp. (2.6%), Pseudomonas sp. (2.1%) and Corynebacterium sp. (1.0%) were the most commonly identified species. In our study, results of conventional methods microbial and molecular identification techniques were found to be similar. It was suggested that virulence and antibiotic resistance genes of the most common agents isolated from flora should be determined at the future studies.

Anahtar Kelimeler

• Bacteria
• Nasal flora
• Molecular Identification
• 16S rRNA

Kaynakça

1. Ajuwape TP, Aregbesola EA, The bacterial flora of the upper respiratory tracts of normal rabbits. Israel Vet Med Assoc. 2000, 57: 1-5.
2. Arcangioli MA, Duet A, Meyer G, Dernburg A, Bezille P, Poumarat F, Le Grand D, (2008) The role of Mycoplasma bovis in bovine respiratory disease outbreaks in veal calffeedlots. Vet J. 177, 89- 93.
3. Barbour EK, Nabbut NH, Hamadeh SK, Al-Nakhli, (1997) Bacterial identity and characteristics in healthy and unhealthy respiratory tracts of sheep and calves. Vet Res Commun, 21, 421- 430.
4. Carter GR, (1984) Isolation and identification of bacteria from clinical specimens. In: Charles C. Thomas (Eds), Diagnostic procedures in veterinary bacteriology and mycology. USA. 4th p. 19- 30.
5. Collier JR, Rossow CF, (1964) Microflora of apparently healthy and pneumonia prone herds. J Vet Res. 25, 101- 103.
6. Holth JB, Krieg NR, Sneath HAP, Stanley JT, Williams T, (2000) Bergey’s manual of determinative bacteriology USA: Lippincott. Williams and Wilkins, Philadelphia PA, USA.
7. Koneman E W, Allen S D, Janda W M, Schreckenberger P C, Winn W C, (1997) Color Atlas and Textbook of Diagnostic Microbiology. pp: 539-76. The Gram-positive cocci: Part-1: Staphylococci and related organisms. Lippincott, New York, USA.
8. Magwood SE, Barnum DA, Thomson RG, (1969) Nasal bacterial flora of calves in healthy and pneumonia-prone herds. Canadian J Comp Med. 33, 237- 243.

9. Megra T, Sisay T, Assaged B, (2006) The aerobic bacterial flora of the respiratory passageways of healthy goats in Dire Dawa Abattoir, Eastern Ethiopia. Rev Méd Vét. 157: 84-87.
10. Quinn PJ, Markey BK, Carter ME, Donnely WJ, Leonard FC, (2002) Veterinary Microbiology and Microbial Disease.1st Edn. Blackwell Publishing Professional, Iowa, pp:461-464. ISBN: 0-632-05525-1.
11. Sghir A, Antonopoulos D, Mackie RI, (1998) Design and evaluation of a Lactobacillus group-specific ribosomal RNA-targeted hybridization probe and its application to the study of intestinal microecology in pigs. Syst Appl Microbiol. 21, 291-296.
12. Shemsedin M, (2002) Bacterial species isolated from respiratory tract of camels slaughtered at Dire Dawa abattoir. Eastern Ethiopia. Debre Zeit, 1-25.
13. Suau A, Bonnet R, Sutren M, Godon J J, Gibson G, Collins MD, Dore´J, (1999) Direct rDNA Community Analysis Reveals a Myriad of Novel Bacterial Lineages within the Human Gut. Appl Environ Microbiol 65, 4799- 48
14. Şeker E, Kuyucuoğlu Y, Konak S, (2009) Bacterial examinations in the nasal cavity of apparently healthy and unhealthy Holstein cattle. J Anim Vet Adv. 8 , 2355- 2359.
15. Woo PC, Leung AS, Leung KW, Yuen KY, (2001) Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing. Mol Pathol. 54, 244-7.