Use Of Escherichia Coli Secondary Infection For The Immune Efficacy Assessment Of IBV Vaccines

Infectious bronchitis（IB）is an acute and highly contagious disease of poultry caused by infectious bronchitis virus（IBV）.It is popular all over the world,causing huge economic losses to the poultry industry.It mainly infected the respiratory tract,the genitourinary tract and the gastrointestinal tract.The clinical signs of IB are usually mild in laboratory conditions.While in field situations,it is usually serious because of the co-existence of other disease such as Newcastle Disease（ND）,chronic respiratory disease（CRD）and so on.It can also cause secondary bacterial diseases such as colibacillosis and salmonellosis,which aggravates the degree of damage in chickens.At present,IB is mainly prevented and controlled by immunization.However,new variants often arised because of gene mutation and genetic recombination,and vaccines usually havelow cross protection efficacy against the field strains form different serotypes complicating the prevention and control of IB.Avian pathogenic Escherichia coli（APEC）is extraintestinal pathogen,It can cause fetal death,omphalitis,septicemia,granuloma,vitelline peritonitis and panophthalmitis etc.Under normal condition,Escherichia coli（E.coli）are not not pathogenic,but can exhibit its pathogenicity in condition of malnutrition and co-infection of other pathogens such as Newcastle disease virus（NDV）,infectious bursal disease virus（IBDV）,IBV,mycoplasma and Eimeria.The main basis that assessing the efficacy of IBV vaccine come from the clinical manifestation,virus isolation and cilium stagnation test.These methods have their own advantages and disadvantages.This paper plan to establish a challenge model of secondary infection of E.coli firstly,and then applies this model to assesss the efficacy of IBV vaccine,so as to provide a new reference for the evaluation of IBV vaccine efficacy.During 2015-2017 years,42 strains of avian E.coliwere isolated from some chicken farms in Sichuan province and identified.35 of them could be serotyped and the sereotyping rate was 83.33%（35/42）.The main serotypes were O8 and O18,which accounted for 54.29%of serotyped strains.19 E.coli from 9 serotypes were selected to perform the pathogenicity test onchickens.It was found that 12 strains were highly pathogenic,which accounted for 63.16%（12/19）of tested strains;6 strains were medium pathogenic,which accounted for 31.58%（6/19）of tested strains;1 strain was low pathogenic,which accounted for 5.26%（1/19）of tested strains.A highly pathogenic E.coli strain TQ0812 belonged to the epidemic serotype in Sichuan province was selected for the eatablishment of model of secondary infection,and the optimum infection dose was determined to be 2x10⁷CFU.A total of 70 unimmunized partridge shank broilers were randomly divided into 6 groups A,B,C,D,E and F.At 34days of age,group A and C,B and D were inoculated with 1x10⁵EID₅₀ and 1x10⁶EID₅₀of TC07-2 type ZQX strain,respectively,while group E and F were inoculated with PBS.4days later,group C,D and E group were inoculated with 2x10⁷CFU of E.coli TQ0812strain respectively,while chickens from group F were not inoculated.The effect of IBV early infection on the pathogenicity of E.coli secondary infection was assessed by the analysis of clinical symptoms,pathological anatomy（pericarditis,airsacculitis and tracheal bleeding）and microscopic damage of trachea.The results showed that the clinical symptoms,pericarditis,airsacculitis and tracheal bleeding in the D group were more severe than other groups.In respect of clinical symptoms,the rspirotary symtom such as sneezing and tracheal in chickens from D group was most serious than other gropus,and it bagan to occur at 2-3 days post infection（d.p.i）of IBV,and the respiratory symptom aggravate after inoculation of E.coli.For the pericarditis,the difference between group D and C was significant（P<0.05）.For the airsacculitis,the manifestation in chickens from group D was most serious than other groups.For the tracheal bleeding,the manifestation was different among different groups（D>C>B>A>E）,the difference between group D and B,group A and E group,group C and A,group C and E were significant（P<0.05）.In summary,the secondary infection of 2x10⁷CFU of E.coli TQ0812 strain at 4 days post infection of 1x10⁶EID₅₀of IBV ZQX strain could significantly improve the pathogenicity of E.coli,and the severity of pericarditis could reflect the difference of pathogenicity caused by E.coli secondary infection comparing to the lesion in other organs.To observe whether the model of secondary infection of Escherichia coli can be used to evaluate the protection efficacy of IBV vaccine,96 one-day old partridge shank broilers were randomly divided into 8 groups a～h with 12chickens in each group andreared in separated rooms.At 20 days of age,chickens from groups a andc were immunized with H120 intranasally and intraocularly,and the b and d groups were immunized with 4/91by the same route,and the immune dose was 1x10⁵EID₅₀.Chickens from groupse,f,g,and h groups were inoculated with PBS in the same way.14dayes later,chickens from groups a,b,c,d,e and g were challenged with TC07-2 type ZQX strain.After 4 days,chickens from groupsc,d,e,and f were inoculated with 2x10⁷CFU TQ0812 by intratracheally.The protective efficacy of live vaccine against ZQX strain were assessed by the analysis of clinical symptoms,pericarditis,and tracheal hemorrhage.The results showed that the anatomy changes in group c was more serious than group d on the whole,the difference of pericarditis between group c and d was significant（P<0.05）,and the protective efficacy of4/91 was higher than that of H120.The model of secondary infection of Escherichia coli can be used to evaluate the protection efficacyof the H120 and 4/91 vaccine strains againstthe ZQX strain.To sum up,O8 and O18 are the epidemic serotypes of avian E.coli in Sichuan area.The secondary infection of avian pathogenic E.coli strain at 4 days post infection of IBV field strain could significantly improve the pathogenicity of avian E.coli.The model of secondary infection of E.coli established in this experiment can provide a new reference for the evaluation of the protection efficacy of IBV vaccine.