Identification And Attenuation Of Corynebacterium Pseudotuberulosis And Its Toxoid Immunity Test In Goats

Corynebacterium pseudotuberulosis?C.p?is a zoonotic pathogen that can infect a variety of animals in the aquaculture industry.Among them,the pathogen infects sheep and goat is the most common and can cause caseous lymphadenitis?CLA?,which has been reported at domestic and foreign.When the flock is infected with Corynebacterium pseudotuberculosis,it is not lethal for many animals,and it is a chronic infectious disease.The diseased flock can show symptoms,such as asymptotic weight loss,rough hair,local caseous abscess lesion,in addition to affecting the quality of fur,meat products and dairy products,in addition to reducing its economic value,it can also threaten public health security,it has caused great losses to the development of the world's aquaculture.In recent years,while the rapid development of animal husbandry in China,CLA has also occurred in many provinces,and this disease in goat was more popular than sheep,we can also infer that CLA will be more severe.However,there is no effective vaccine to prevent the occurrence of the CLA in China.Collecting some CLA samples of goats from two farms in Baoji and Tongchuan,Shaanxi.Two strains of suspected C.p were identified by bacterial isolation and culture,microscopy,biochemical tests,molecular biology identification and animal pathogenicity tests.In addition,subculturing the C.p strain of LX1 and TC11,injected the mice with different generations of LX1 and TC11.Posterior,in the immune test,less virulent strain of C.p was injected in mice as a vaccine,feasibility of developing attenuated vaccines by studying the weak method of subculturing.At last,formaldehyde in a volume of 5%was added to the important exotoxin phospholipase D?PLD?of C.p for 48h at normal temperature,mice and goats were immunized,the immunoprotective effect of the toxoid preparation was observed.At the same time,5 toxoid-immunized goats were randomly selected and collected the serum at 30 d,90 d and 150 d for monitoring the antibody by indirect ELISA.The results were as follows:1.The isolated strains LX1 and TC11 were cultured for 48h at 37°C,all of them did not grow on macconkey agar medium,the growth was abundance on nutrient agar medium,they grew on blood agar medium with milky white,dry,easy to scrape off and opaque,the diameter of the colonies was 1mm to 2mm.Gram staining was positive,spherical,cuboid or rod-shaped.The biochemical test and PCR identification of 16S rRNA confirmed that the isolated strains were Corynebacterium pseudotuberculosis,and both isolates could cause death or injection of cheese in the injection site.Drug susceptibility test results showed that the two strains were sensitive to the antibiotics of sulfonamides,?-lactams,quinolones,macrolides,polymyxin was resistant.2.In the 7th day after inoculation of the LX1 and TC11 strains with the 50th,100th,150th and 200th generation strains,2 mice were dead after injecting the LX1 primary strain,and the average diameter of the abscess lesions at the injection site of the non-dead mice was 8.7 mm,none of the mice injected into the subculture strain died.The average lesion diameter of the lesions were 4.6mm,2.6 mm,1.4 mm,and 1 mm.There were 2 mice died after injected with the TC11 primary strain,and the average lesion diameter of the alive mice was 9.3 mm,the mice injected with the subculture strain did not die,and the average diameter of the lesions were 5.4 mm,2.8 mm,1.8 mm and 1.4 mm.The 200th generation strain of LX1 was inoculated into mice for 14 days,and then injected the primary strain,there were no deaths in experimental groups,the average abscess diameter was 2.0 mm,the control group died 4 mice,the average abscess diameter of the alive mice was 7.8 mm.3.In the toxoid immunoassay,10 mice in the experimental group were not dead,1mouse was infected,the protection rate was 90%?9/10?,and 4 mice in the control group died,the remaining 6 cases were all onset.In the pre-experiment of the flock,within 90d of the experimental group,1 case of CLA occurred,the pathogen was staphylococcus aureus,3cases of CLA occurred in the control group,and 2 strains of C.p were isolated.In the large-scale immunization test of sheep,the goat in the experimental group had 8 cases of CLA,and 1 strain of C.p was isolated,the infection rate of C.p was 0.9%?1/114?,there were 23 goats with CLA,and 9 strains of C.p were isolated.The infection rate of C.p was7.9%?9/114?.The serum OD₄₅₀ test results showed that the antibody level was highest in30th day after immunization.And then showed a downward trend,approaching the positive serum threshold at 150 d.The above results confirmed that the method of subculturing may weaken the virulence of Corynebacterium pseudotuberculosis,which lays a theoretical foundation for the development of attenuated vaccine against Corynebacterium pseudotuberculosis.And the toxoid vaccine of Corynebacterium pseudotuberculosis can provide certain immune protection for mice and goats,but there is a problem that the immunization effective period is short,and it is necessary to study the prolonged immunization effective period.