Diagnosis and gene analysis of mixed infection of Pasteurella multocida and avian leukemia virus-K subgroup

【Objective】To clarify the etiology of Sanhuang chickens in a large-scale(200000 feather) breeding farm in Guizhou,scientifically used medicine to minimize economic loss,and provide reference for prevention and control of avian leukemia and purification of chickens in large-scale breeding farms.【Method】The heart,liver and spleen tissues of chickens were collected aseptically for bacterial isolation and identification,biochemical test,capsule typing,virulence gene and drug resistance gene detection,avian leukemia virus(ALV) PCR detection,gp85 gene cloning and sequencing analysis,etc.【Result】A Pasteurella multocida(Pm) strain was isolated and named as Pm-GZXF2021. Pm-GZXF2021 was type A capsulatus of Pm with the associated virulence genes of adhesin(ptfA,hsf-1,hsf-2,pfhA,fimA and tadD), superoxide dismutase(sodA),outer membrane protein(ompA,ompH,plpB and oma87) and iron uptake(exbB,exbD, tonB,Fur,hgbB and hgbA). Meanwhile,the isolate strain carried a gyrB resistance gene for quinolones and a tem resistance gene for lactamides. Drug sensitivity test showed that Pm-GZXF2021 was sensitive to minocycline,mikacin,piperacillin,gentamicin,doxycycline,levofloxacin,neomycin,cefradine,medicin and cefoperazone,but resistant to ceftazidime,oxacillin,carbenicillin and ceftriaxone sodium. In addition,all healthy Kunming mice died within 12 h after inoculation with Pm-GZXF2021 pure culture broth(1.7×10⁸ CFU/mL). By necropsy,the small intestine of Kunming mice showed jelly infiltration,and the liver was enlarged with white necrosis. ALV was detected from tissue samples of sick chickens and named ALV-GZXF2021. The ALV phylogenetic tree constructed based on the nucleotide sequence similarity of the gp85 gene showed that ALV-GZXF2021 was in the same branch as the five ALV-K reference strains and was in different branches with subgroups A,B,D,E,and J. In other words,ALV-GZXF2021 belonged to ALV-K.【Conclusion】The incidence of Sanhuang chicken group in a large-scale farm in Guizhou is caused by exogenous ALV infection of Pm and K subgroups. Therefore,the farm should take corresponding prevention and control measures,generally including strengthening feeding management,biosafety measures and vaccination,especially the establishment of breeding chickens without avian leukemia.