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Molecular survey of bacterial communities associated with bacterial chondronecrosis with osteomyelitis (BCO) in broilers.

Jiang T, Mandal RK, Wideman RF, Khatiwara A, Pevzner I, Min Kwon Y - PLoS ONE (2015)

Bottom Line: Relatively little is known about the microbial communities associated with BCO.Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples.These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions.

View Article: PubMed Central - PubMed

Affiliation: Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States of America.

ABSTRACT
Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers.

No MeSH data available.


Related in: MedlinePlus

Design of the PCR primers and the amplicon library.V6 rRNA region were amplified using the two primers with barcode tags and Illumina adaptors attached at 5’ ends.
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pone.0124403.g002: Design of the PCR primers and the amplicon library.V6 rRNA region were amplified using the two primers with barcode tags and Illumina adaptors attached at 5’ ends.

Mentions: For isolation of bacterial DNA, PBS buffer was added to each sample for 10X dilution, and the samples were homogenized using a tissue tearor. Two hundred microliter of each sample was transferred into a microcentrifuge tube for DNA isolation. DNA was extracted from this cell suspension using a QIAamp DNA Mini kit (Qiagen, Valencia, CA., USA). DNA concentrations were estimated spectrophotometrically using NanoDrop (Thermoscientific, USA). Because of its taxonomic resolution [25], conserved flanking regions [26], and length [21], the hyper-variable region 6 (V6) of the bacterial 16S rRNA gene was selected for this study. PCR amplification was carried out for each sample using Illumina paired-end primers in combination with unique barcode sequence at the 5’ end of each primer (S2 Table). Gloor et al. used paired-end sequencing in combination with unique barcode sequence tags at the 5′ end of each primer to perform microbiome analysis of the V6 region of 272 clinical samples using the Illumina sequencing technology [21]. We modified the primer design to use a different barcodes and added the random 5 nt sequence “NNNNN” in the first 5 nt positions of the forward reads of Illumina sequencing to facilitate more efficient and accurate analysis of clusters during processing of image data [27,28] (Fig 2). The PCRreactionss consisted of approximately 0.1 μg of purified genomic DNA, 1× cloned Pfu polymerase buffer, 5 U Pfu polymerase (Stratagene La Jolla, CA, USA), 1 mM dNTPs (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), 1.2 μM each primer in a total volume of 50 μL. The DNA engine thermal cycler (Bio-Rad, Hercules, CA, USA) was used with the following amplification conditions: 94°C for 2 minutes; 30 cycles of 94°C sec for 30 sec, 58°C for 60 sec, 72°C for 90 sec; and 72°C for 10 minutes for final extension. PCR products of the correct size were gel-purified (Qiagen, Valencia, CA, USA). PCR products of each sample were mixed in equal quantities, based on measurement with Qubit 2.0 Fluorometer. Illumina sequencing was performed for 100 cycles in paired-end mode with Illumina HiSeq 2000.


Molecular survey of bacterial communities associated with bacterial chondronecrosis with osteomyelitis (BCO) in broilers.

Jiang T, Mandal RK, Wideman RF, Khatiwara A, Pevzner I, Min Kwon Y - PLoS ONE (2015)

Design of the PCR primers and the amplicon library.V6 rRNA region were amplified using the two primers with barcode tags and Illumina adaptors attached at 5’ ends.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400152&req=5

pone.0124403.g002: Design of the PCR primers and the amplicon library.V6 rRNA region were amplified using the two primers with barcode tags and Illumina adaptors attached at 5’ ends.
Mentions: For isolation of bacterial DNA, PBS buffer was added to each sample for 10X dilution, and the samples were homogenized using a tissue tearor. Two hundred microliter of each sample was transferred into a microcentrifuge tube for DNA isolation. DNA was extracted from this cell suspension using a QIAamp DNA Mini kit (Qiagen, Valencia, CA., USA). DNA concentrations were estimated spectrophotometrically using NanoDrop (Thermoscientific, USA). Because of its taxonomic resolution [25], conserved flanking regions [26], and length [21], the hyper-variable region 6 (V6) of the bacterial 16S rRNA gene was selected for this study. PCR amplification was carried out for each sample using Illumina paired-end primers in combination with unique barcode sequence at the 5’ end of each primer (S2 Table). Gloor et al. used paired-end sequencing in combination with unique barcode sequence tags at the 5′ end of each primer to perform microbiome analysis of the V6 region of 272 clinical samples using the Illumina sequencing technology [21]. We modified the primer design to use a different barcodes and added the random 5 nt sequence “NNNNN” in the first 5 nt positions of the forward reads of Illumina sequencing to facilitate more efficient and accurate analysis of clusters during processing of image data [27,28] (Fig 2). The PCRreactionss consisted of approximately 0.1 μg of purified genomic DNA, 1× cloned Pfu polymerase buffer, 5 U Pfu polymerase (Stratagene La Jolla, CA, USA), 1 mM dNTPs (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), 1.2 μM each primer in a total volume of 50 μL. The DNA engine thermal cycler (Bio-Rad, Hercules, CA, USA) was used with the following amplification conditions: 94°C for 2 minutes; 30 cycles of 94°C sec for 30 sec, 58°C for 60 sec, 72°C for 90 sec; and 72°C for 10 minutes for final extension. PCR products of the correct size were gel-purified (Qiagen, Valencia, CA, USA). PCR products of each sample were mixed in equal quantities, based on measurement with Qubit 2.0 Fluorometer. Illumina sequencing was performed for 100 cycles in paired-end mode with Illumina HiSeq 2000.

Bottom Line: Relatively little is known about the microbial communities associated with BCO.Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples.These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions.

View Article: PubMed Central - PubMed

Affiliation: Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States of America.

ABSTRACT
Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers.

No MeSH data available.


Related in: MedlinePlus