Dataset Information

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

ABSTRACT:

Background

Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections.

Results

The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples.

Conclusions

The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

SUBMITTER: Moustacas VS

PROVIDER: S-EPMC3614447 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Publications

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

Moustacas Valéria S VS Silva Teane M A TM Costa Luciana F LF Xavier Mariana N MN Carvalho Custódio A CA Costa Érica A ÉA Paixão Tatiane A TA Santos Renato L RL

BMC veterinary research 20130321

<h4>Background</h4>Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagno ...[more]

PMID: 23514236

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Project description:Background and aimHistophilus somni is a Gram-negative bacterium belonging to the Pasteurellaceae family that can cause bovine histophilosis. Histophilus may act as a commensal or opportunistic bacterial cattle pathogen. Comparing genomes of the pathogenic strain 2336 with the non-pathogenic preputial 129Pt isolate revealed some putative virulence factors. The study of the complete genomes of H. somni strains circulating in Russia has never been conducted before. This study aimed to identify genetic features of the H. somni strains isolated in Russia and evaluate the possibility of using strains for vaccine development.Materials and methodsThree strains of H. somni were isolated from different sources. Strain 188-VIEV was isolated from a vaginal swab sample of cattle with endometritis. 532-VIEV and 551-VIEV were cultured from the cryopreserved bull semen samples imported from Canada. Histophilus somni strain ATCC 700025 provided by ATCC (American Type Culture Collection) was also used in the study. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN, USA). The whole-genome sequencing of the four strains was performed using Illumina Miseq. The comparison of the resulting sequences with the complete genomes of H. somni 2336 and 129Pt, and detection of the resistance genes and virulence factors, was performed using the ResFinder and Virulence Factor Database web services.ResultsThe genome size of the samples varied from 1.9 to 2.3 Mb. The number of coding sequences varied from 1795 to 2256. The average sequence density was 90%. The total guanine-cytosine (GC) content was 36.8%-37.2%, which coincided with data previously obtained for H. somni. Three out of four studied strains encoded putative virulence factors such as filamentous hemagglutinin homologs, lipooligosaccharide biosynthesis proteins, and proteins involved in iron transport and utilization. The Ser83Ile substitution was identified in the DNA topoisomerase II (gyrA) in H. somni strains 532-VIEV and 551-VIEV cultured from bull semen which led to resistance to fluoroquinolones. The gene (AAC-6-Ia + APH-2'') encoding a bifunctional aminoglycoside modification enzyme was detected in strain 551-VIEV.ConclusionStrains with virulence genes identified could be candidates for designing vaccines and potentially represent antigen sources. The results show that antibiotic-resistant H. somni can be spread with semen used for artificial insemination.

Project description:Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the Pasteurellaceae family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. IMPORTANCE The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.

Dataset Information

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

Background

Results

Conclusions

Publications

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

Similar Datasets

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