Veterynarna biotehnologija – Veterinary biotechnology, 2022, 40, 121-131 [in Ukrainian]. https://doi.org/10.31073/vet_biotech40-11
Institute of Veterinary Medicine NAAS
National University of Life and Environmental Sciences of Ukraine
STUDYING THE PECULIARITIES OF NEURAMINIDASE PRODUCTION OF THE SWINE ERYSIPELAS CAUSATIVE AGENT
Introduction. Erysipelothrix rhusiopathiae causes significant economic losses in pigproductionin Ukraine, EU and other countries. The study of the virulence factors of erysipelas causative agent, formation of specific immunity and its intensity does not lose relevance today because of antigenic variation and due to the direct impact on the effectiveness of preventive vaccination. Control of vaccine strains quality is the actual topic for research and neuraminidase is one of the main factors of the virulence of E. rhusiopathiae and its producing peculiarities have to be investigated.
The goal of the work wasto study the peculiarities of neuraminidase production of the swine erysipelas causative agent.
Materials and methods. The strains of the bacterium E. rhusiopathiae 27, 93, 149, 251, 419, 1689, 1893, 1933, M-2 VK, BP-2 var IVM, K, Ш and 5 pathogenic field isolates stored in the museum of the Institute of Veterinary Medicine were used. S.aureus ATCC 33592 was used as a positive control. Hottinger meat-peptone broth media (BHIB, BHIB/S), soy hydrolyzate (TSB, TSB/S), Feist medium, Feist medium with serum (SF/S) manufactured by HiMedia, India, were used for research according to the manufacturer’s recommendations. Preservative-free sterile bovine serum (Leucopol, Ukraine) was added to the medium after sterialization of 10% of the total volume of the medium. Lectin-mediated haemagglutination reaction was used to detect neuraminidase production according to standard protocol.
Results of research and discussion. Neuraminidase production was found in all studied strains and isolates of E. rhusiopathiae. As a result of the conducted researches it is established that neuraminidase has a low level of production before the beginning of the stationary phase of growth. The highest level of production was found between 16 and 24 hours from the beginning of incubation. The accumulation of the enzyme allows to perform the hemagglutination reaction with reliable test results. This feature distinguishes E. rhusiopathiae from some other neuraminidase-producing bacteria, such as hemolytic staphylococci. Table 1 shows the results. The best results of optimizing lectin-mediated hemagglutination using strains of E. rhusiopathiae K were obtained by incubating the reaction mixture for 30 minutes at 37±0.2°C.
As a result of the conducted researches it is established that the necessary concentration of neuraminidase accumulates in the culture fluid after 16 hours from the incubation beginning.
To determine the optimal pH for neuraminidase activity, a range of pH values from 5.0 to 8.0 was investigated. The optimal pH level was tested using 11 museum strains and pathogenic field isolates of E. rhusiopathiae. It was found that the optimal pH was within 7.1±0.1. Beyond these indicators, the sensitivity of the reaction decreases.
Conclusions and prospects for further research. The optimal nutrient media and reaction conditions for the qualitative detection of neuraminidase in hemagglutination were selected. A pH of 7.1±0.1 is the optimal range for producing a detectable amount of neuraminidase, as it is within the highest enzymatic activity of all E. rhusiopathiae strains and isolates studied.
The BHIB/S medium, according to the research results, provided the best accumulation of neuraminidase, and the addition of bovine blood serum in amount of 10% increases the production of neuraminidase and provides reliable test results.
Future studies will focus on obtaining new data and studying the virulence factors of the causative agent of swine erysipelas in order to improve approaches for the selection of strains for the production of a highly immunogenic vaccine.
Keywords: swine erysipelas, neuraminidase, virulence factor, nutrient media.