Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

Abstract

As a result of the work performed, the conditions for setting up multiplex PCR with electrophoretic detection for the diagnosis of horse strangles were determined, allowing the identification and differentiation of S. equi subsp. equi in one reaction. It was found that the developed PCR protocol for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection in a “multiplex” format has a high specificity and does not lead to amplification of PCR products with DNA of closely related microorganisms, saprophytic microflora, and bacterial pathogens. The sensitivity of the protocols for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection was assessed. Diluted DNA samples of two S. equi subspecies were used as objects of research: S. equi subsp. equi and S. equi subsp. zooepidemicus. DNA samples were diluted by two-fold dilutions, starting from a concentration of 5 ng (which corresponds to 2 million 170 thousand copies in the genomic equivalent) to 1.19 * 10-6 ng (which corresponds to 0.71 copies in the genomic equivalent). DNA detection limit for S. equi subsp. equi was 66 copies in genomic equivalent or 152 fg, DNA of S. equi subsp. zooepidemicus – 132 copies in genomic equivalent or 305 fg.

Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP)

“…Subsequently, abscesses are formed on the submandibular lymph nodes and will develop to become firm and painful, which can cause tightening in the equine’s esophagus; hence, the term Strangles. These abscesses rupture a week to 10 days post onset of clinical signs but can take up to 4 weeks in some cases [ 85 , 86 , 87 ], with uncomplicated cases recovering at about 1–2 weeks post rupture [ 78 ]. Severe complications can occur, including guttural pouch empyema, purpura haemorrhagica, myositis, internal abscessation and further spread of the bacteria internally [ 79 , 85 , 88 ].…”

“…Culturing was originally used for detection, but multiple studies reported the inadequacy of this method due to the variability of clinical sensitivity [ 45 , 96 ], which will be discussed further below. Treatment and prognosis are conditional to the stage of the disease; however, most horses will fully recovery with minimal supportive care [ 79 , 86 ]. As with R. equi infections, antibiotic use is debated amongst scientists [ 86 , 98 ].…”

“…Treatment and prognosis are conditional to the stage of the disease; however, most horses will fully recovery with minimal supportive care [ 79 , 86 ]. As with R. equi infections, antibiotic use is debated amongst scientists [ 86 , 98 ]. Whilst studies state antibiotics reduce the duration of the disease and degree of discharge and secretions [ 98 , 99 , 100 ], others state incorrect use, such as incorrect therapeutic regimen, results in failed treatment and subsequent reinfection [ 21 , 83 , 101 ].…”

“…Whilst studies state antibiotics reduce the duration of the disease and degree of discharge and secretions [ 98 , 99 , 100 ], others state incorrect use, such as incorrect therapeutic regimen, results in failed treatment and subsequent reinfection [ 21 , 83 , 101 ]. Yet, many countries have access to a vaccine, and despite low efficacy, it can reduce the severity of the disease [ 85 , 86 , 102 ]. Due to limited prevention and treatment options, as well as the high transmissibility of Strangles, control of an outbreak is strenuous.…”