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

Abstract

BackgroundInfectious 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.ResultsThe 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.ConclusionsThe 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.

Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio

“…As shown in the results of analytical sensitivity tests, the detection limit of the multiplex PCR was lower than the singleplex PCR assays, when multiple parasites were simultaneously detected. This is expected, since in a multiplex PCR reaction for detection of multiple targets, all primer pairs will compete for limited reaction reagents, resulting in sub-optimal amplification efficiencies (Bilgiç et al 2013, Moustacas et al 2013.…”

“…As shown in the results of analytical sensitivity tests, the detection limit of the multiplex PCR was lower than the singleplex PCR assays, when multiple parasites were simultaneously detected. This is expected, since in a multiplex PCR reaction for detection of multiple targets, all primer pairs will compete for limited reaction reagents, resulting in sub-optimal amplification efficiencies (Bilgiç et al 2013, Moustacas et al 2013.When applied to screening of the 104 field samples, the multiplex PCR and the 4 singleplex PCR methods were proven to be more effective for detection of the 4 target myxosporeans compared to microscopic examinations. During the microscopical detection, although the early stages of myxosporeans could be observed, only samples infected by mature spores were recorded as positive, as the early stages of myxo sporeans were difficult to distinguish morphologically to species level.…”

Real-time PCR for detection of Brucella ovis and Histophilus somni in ovine urine and semen

“…The gold standard diagnosis of B. ovis infection is based on clinical examination, serology and semen bacteriology (Xavier et al, 2011;Poester et al, 2013), while for H. somni infections, only clinical examination and bacteriology are routinely performed. Semen and urine are the samples of choice for diagnosis (Xavier et al, 2010;Costa el al., 2012;Moustacas et al, 2013). These techniques are labor intensive and slow, whereas molecular techniques have been increasingly used, including conventional PCR (Xavier et al, 2010;Costa et al, 2012), nested PCR , and conventional multiplex PCR (Saunders et al, 2007;Moustacas et al, 2013).…”

Distribution of lymphocytes, immunoglobulin-containing cells, macrophages, and dendritic cells in the accessory sex glands of rams experimentally infected with Actinobacillus seminis

“…Sheep epididymitis has been mainly associated with Brucella ovis and Actinobacillus seminis infections (Burgess 1982, Moustacas et al 2013. The pathogenesis of the disease caused by A. seminis has been explored to a small extent; the presence of pathogenicity factors in A. seminis, such as adhesins (Healey et al 1991) or RTX toxins (Schaller et al 2000), is likely to contribute to the development of the pathology.…”