Development of a vaccine against enterococcosis for farm birds and assessment of its specific effectiveness

Enterococcosis in poultry is a disease which affects the organs of locomotor system and is accompanied by lameness, ataxia, spondylitis, necrosis of the femoral head and bacterial chondronecrosis. The main pathogen of this disease on the territory of the Russian Federation is bacteria of the species Enterococcus cecorum (EC). The disease can occur among young herds for replacement (mainly cockerels) at the age from 1 to 7 weeks; commodity broiler aged 3-5 weeks; and parent stock during peak production. In this work we represent for the first time the results of the development and tests of the domestic means of specific prevention from enterococcosis in poultry. The experimental series of vaccine, tested in industrial environment on poultry, was produced on the basis of the selection of production-control enterococcus species and the measurement of the optimal immunizing dose and adjuvant. The proposed medicine possesses areactogenicity and high specific effectiveness when used for poultry of different age groups. The aim of the work is the development of means of specific prevention from enterococcosis of poultry and evaluation of its effectiveness. The study of the epizootic situation on enterococcosis in poultry on the territory of the Russian Federation in 2017-2018 showed that 11 poultry enterprises in the Belgorod, Vladimir, Yaroslavl, Kaluga, Chelyabinsk, Tver and Penza regions, as well as in the Republics of Mari El and Udmurtia, were enterococcosis positive. All in all, 647 samples of parenchymal organs and tissues obtained from birds of Cobb 500 cross with typical clinical morphological manifestation of enterococcosis were examined. Strains E. cecorum Nos. 414, 425, 426, 837, 838, 839, 1096, 1481, 1517, 1647, 1865 were selected during the complex bacteriological diagnosis of breeding material. It was determined that 72.73 % of enterococci are resistant to ampicillin and penicillin, 45.45 % to vancomycin, 27.27 % to levofloxacin, linezolid, tetracycline, 18.18% to norfloxacin, rifampicin, chloramphenicol and ciprofloxacin, and 9.09% to doxycycline. The largest number of species are sensitive to gentamicin and levofloxacin (72.73 %); doxycycline, linezolid, rifampicin, chloramphenicol (54.55 %), respectively. All the studied strains led to the death of 100 % of laboratory mice within 24-96 hours after intraperitoneal infection. The LD50 of enterococcal cultures was in the range of 1.7×107-9.4×108 microbial cells. When determining the antigenic properties of EC species in the agglutination reaction, it was confirmed that they are all homologous to each other, i.e. belong to the same serotype. Evaluation of the level of antibodies in doubly immunized white mice with vaccines from strains No. 414 and No. 1517 showed that they have the highest antigenicity, inducing immunity in the titer of 1:26.66±9.23, while the antigenicity of other strains was 1:21.33±9.23 and less. Based on this result, strains No. 414 and No. 1517 were subsequently used for control and production. Evaluation of the immunogenic activity of the experimental medicine on white mice showed that the vaccine ensures the safety of 90 % of infected animals, while mortality among the mice of the control group was 100 %. To ensure high efficiency of the developed means, 1.5 billion microbe cells EC are needed, and the optimal amount of a single dose is 0.2 cm3. Formalin (0.3%) was used as an inactivant and polyethylene glycol 6000 (PEG-6000) as an adjuvant at the rate of 10 % v/v. Phosphate-buffered saline (PBS) was used as diluent, the pH level was set to 7.2 with a 20 % sodium hydroxide solution. The vaccine provoked the formation of immunity 12-14 days after a double intramuscular injection, which lasts at least 4 months. Clinical trials on chickens of Cobb 500 cross proved the safety and the high specific effectiveness of the vaccine for poultry. Double vaccination of replacement herds in poultry led to a 4.6 % increase in uniformity and a 0.13 % decrease in total waste. The analysis of production indicators of vaccinated laying hens showed a 1.81 % decrease in total mortality and a 1.7 % increase in egg productivity of. After the first vaccination of the parent livestock, the average antibody level in the bird was 1:5.60±2.00 (n = 25), and 14 days after the second vaccination, the titer reaches 1:43.52±15.67, which exceeds the value of the protective level of antibodies (1:26.66±9.23). The results obtained allow us to talk about the possibility of further implementation of the medicine developed on the basis on Enterococcus cecorum strains in practical use.