Identification of new isolates of the horse strangles causative agent in Northern Siberia

Infectious and invasive diseases cause significant damage to the economy and decrease the productivity of horse herd farming. Equine strangles ( Streptococcus equi ) is the most economically damaging. Specific prevention of the disease widespread in Asia, Russia and CIS poses a serious problem. In this work, for the first time in the Far North, we isolated and identified three new Streptococcus equi strains prospective for the diagnostics and development of strangles vaccines. The study aimed to culture, identify morphologically, culturally, biochemically, and genotypically new isolates of the equine strangles causative agent for the development of vaccines. A survey of 6-10-month old Yakut and Kazakh foals ( Equus ferus caballus ) was performed in the Republic of Sakha (Yakutia) regions (the farms in Namskiy, Khangalasskiy, Amginskiy, Megino-Kangalarskiy districts and in Yakutsk) and in Kazakhstan in 2015-2017. In total, 63 collected biospecimens included 45 nasal discharges (27 from diseased and 18 from healthy foals), 7 submandibular lymph node abscesses, and 11 parenchymal organs. The infectious agents were isolated and identified by 16S rDNA genotyping using PCR and based on biochemical traits. Morphological and cultural properties were studied using meat peptone broth (MPB) added with 1 % glucose and 10 % horse blood serum and on meat peptone agar (MPA) with 1 % glucose and 10 % horse blood serum or 5 % defibrinated horse blood. Pus swabs and preparations of liquid and agar cultures stained by the Gram procedure were investigated with a light microscope. Biochemical properties were studied by plating on MPA with 40 % bile, 6.5% saline MPA, agar with sodium azide, and Giss’s medium with glucose, lactose, mannitol, maltose, sucrose, sorbitol, and dulcite. The isolates were biochemically assigned to species using API 20 Step strips (an API test system, bioMerieux, France). The virulence of the isolates (LD50) was assessed on white mice challenged subcutaneously with 0.2-0.5 cm3 of 1-day suspensions (1×103 to 1×109 CFU per mouse). Genotyping was performed with specific primers Seel-F 5ʹ-CGGATACGGTGAT-GTTAAAGA-3ʹ and Seel-R 5ʹ-TTCCTTCCTCAAAGCCAGA-3ʹ. The Streptococcus equi 16S rRNA gene was sequenced for six isolates of strangles streptococcus, of which three we suggest for the development of strangles vaccines. Polymerase chain reaction with specific primers serves as the most reliable and fastest method for identifying strangles streptococcus. Based on genotyping data and the cultural, morphological and biochemical properties, the Streptococcus equi H-5/1 isolate belongs to the Streptococcaceae family, Streptococcus genus, Streptococcus equi ssp. equi and corresponds to the typical characteristics of the species. The nucleotide sequence of the 16S rRNA gene fragment of the isolate H-5/1 after sequencing was deposited in the NCBI GenBank database (MW486609). The Streptococcus equi H-5/1 strain was deposited in the All-Russian State Collection of Microorganism Strains Used in Veterinary Medicine and Animal Husbandry (VGNKI, registration number VKSHM-B-141P, certificate of deposit dated May 22, 2018), and patent for invention No. 2703485 (“A strain of bacteria Streptococcus equi used for the production a vaccine against strangles”) dated 10/17/2019 was received. The new Streptococcus equi strains we described here hold promise in the developing strangles vaccines. Note, Enterococcus faecales , Streptococcus piogenes , toxigenic and mold fungi Aspergillus and Mucor genera were also isolated from foals with clinical sings of equine strangles. Our findings attract attention to these microorganisms possibly involved in the development of equine strangles in young horses, which should be accounted in diagnostics of this pathology.