A Novel Real-Time PCR Assay for the Rapid Detection of Virulent Streptococcus equi Subspecies zooepidemicus—An Emerging Pathogen of Swine

Abstract

Streptococcus equi subspecies zooepidemicus, a zoonotic bacterial pathogen caused a series of outbreaks with high mortality affecting swine herds in multiple locations of the USA and Canada in 2019. Further genetic analysis revealed that this agent clustered with ATCC 35246, a S. zooepidemicus strain associated with high mortality outbreaks in swine herds of China originally reported in 1977. Rapid and accurate diagnosis is absolutely critical for controlling and limiting further spread of this emerging disease of swine. Currently available diagnostic methods including bacteriological examination and PCR assays do not distinguish between the virulent strains and avirulent commensal strains of S. zooepidemicus, which is critical given that this pathogen is a normal inhabitant of the swine respiratory tract. Based on comparative analyses of whole genome sequences of the virulent isolates and avirulent sequences, we identified a region in the SzM gene that is highly conserved and restricted to virulent S. zooepidemicus strains. We developed and validated a novel probe-based real-time PCR targeting the conserved region of SzM. The assay was highly sensitive and specific to the virulent swine isolates of Streptococcus equi subspecies zooepidemicus. No cross reactivity was observed with avirulent S. zooepidemicus isolates as well as other streptococcal species and a panel of porcine respiratory bacterial and viral pathogens. The PCR efficiency of the assay was 96.64 % and was able to detect as little as 20 fg of the bacterial DNA. We then validated the diagnostic sensitivity and specificity of the new PCR assay using a panel of clinical samples (n = 57) and found that the assay has 100% sensitivity and specificity as compared to bacteriological culture method. In summary, the PCR assay will be an extremely valuable tool for the rapid accurate detection of virulent swine S. zooepidemicus isolates and directly from clinical samples.

Laboratory tools for the direct detection of bacterial respiratory infections and antimicrobial resistance: a scoping review

“…We found that available research is highly human-centered, with very few species of animals considered, specifically cats, 109 cattle, 5 , 16 , 33 , 169 , 170 goats, 90 horses, 34 , 149 pigs, 45 , 77 , 85 , 92 , 95 , 119 , 146 , 165 and sheep. 60 , 145 One of the 99 bacterial pathogens reportedly identified, B. bronchiseptica , was the only organism reported in human and animal studies.…”

“…Of the included studies, all but 3 ONT studies reported numeric values of LOD. The LOD units included bacteria, 34 , 55 cfu, 14 , 29 , 127 , 156 genes/copies/genome equivalents, 6 , 37 , 170 , 174 cumulative criterion unit, 90 femtograms, 40 , 77 , 98 nanograms, 95 picograms, 120 , 121 , 161 and organisms. 71 Although these units are highly variable, they align with the MIQE recommendation that LOD can be expressed as relevant to the experiment.…”

Identification of the Novel Streptococcus equi subsp. zooepidemicus Sequence Type 525 in Donkeys of Abruzzo Region, Italy

“…Moreover, the isolation and identification of SEZ strains in other animal species provide an opportunity to examine the potential contribution of this agent in causing disease. For example, an important outbreak, caused by a SEZ clustered with ATCC 35246, inducing high mortality in swine herds, was recently reported in multiple locations in USA and Canada [ 28 ]. The SEZ strain ST236 was the dominant strain type recovered from cases of mastitis in goats and sheep in Spain, and none of the caprine and ovine isolates had been previously recovered from horses [ 29 ].…”

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

“…Particularly, molecular biology technologies have attracted much more attention for the merits of accurate, fast and easy-operation [17,18]. With advantages of high sensitivity, specificity and the nucleic acid-based molecular diagnosis, molecular biologybased methods have become the preferred option for the accurate detection of pathogens gradually [19][20][21]. As well-established nucleic acid analysis approaches, polymerase chain reaction (PCR) and isothermal nucleic acid amplification have become the mainstream for the development of pathogen detection methods [22][23][24].…”