Diagnosis,pathogen Identification And Vaccine Development Of Duck Plague Virus-pasteurella Mixed Infection In Geese

Duck plague（DP）,also known as Duck virus enteritis（DVE）,is an acute,febrile and septic infection that occurs in ducks,geese and other birds of angoose infected by Duck plague virus（DPV）.Because of its high morbidity and mortality,it often causes great economic losses to waterfowl breeding industry.Avian pasteurellosis,also known as avian cholera,is a contagious and septic infection characterized by fever,diarrhea and breathing difficulties that occurs in birds infected by Pasteurella multocida（Pm）.It can occur all year round and is considered to be one of the important infection that seriously harm the development of poultry industry.In October 2021,a large number of adult grey geese died in a goose farm in Yinjiang County,Guizhou province.This study conducted laboratory diagnosis and preventive vaccine development for this case,in order to provide technical support for disease prevention and control in this goose farm.1.Diagnosis of duck plague-avian cholera mixed infection in geese.In order to explore the etiology of grey goose,gross lesions and tissue sections were observed,bacteria were isolated,cultured and identified,and common waterfowl viruses were detected by PCR in this study.Results:（1）The feathers of the diseased grey goose were messy,the head and neck were swollen,the palpation was undulating,and there was secretion in the corner of the eye.Necropsy showed grayish-yellow necrotic foci of different sizes on the surface of the liver,pericardial effusion,a large number of hemorrhagic spots on the surface of the coronary fat and lungs.（2）In histological sections,there was no obvious hepatic lobule segmentation,interstitial fibrous tissue hyperplasia,partial hepatic cell steatosis,and inflammatory cell infiltration mainly composed of lymphocytes;Pulmonary bronchus and capillary epithelial cell lysis,blood vessel dilation congestion,a large number of serous fluid and inflammatory cells exudate;Renal tubule epithelial cell lysis,interstitial mild congestion;Thymic lymphocytopenia,partial vasodilation congestion.（3）Isolation and culture showed that there were neat edges,smooth surface,central uplift,gray and white dew droplet colony growth on the blood plate.The growth was poor on the ordinary plate,but did not grow on the Mac Conkey medium.Gram staining showed that the isolated bacteria were Gram-negative Bacillus pumilus with strong bipolar staining.Biochemical tests showed that it could use glucose,maltose,fructose and mannitol,but could not decompose lactose,xylose,sorbitol and xylitol.It was positive for indigo matrix and nitrate reduction,but negative for V-P and M-R tests.PCR detection showed that specific primers of Pasteurella multocida KMT1 gene could amplify specific DNA bands.（4）The specific primers of duck plague virus,avian influenza virus,goose parvovirus,duck Tambusu virus,duck circovirus and duck hepatitis virus were used for PCR detection.All the viruses were negative except for the specific DNA bands of duck plague virus.These results suggested that the case was caused by mixed infection of Pm and DPV.2.Pathogen identification of duck plague-avian cholera mixed infection in geese.In order to further identify the pathogen,this study conducted homology classification,serotype identification and pathogenicity detection of Pm isolates from geese and pathogenicity identification of DPV from geese.Results:（1）The sequence size of 16S r RNA gene of the Pm Yinjiang strain was 1 449 bp,which was 98.6%-99.3%homologous to the Pm reference strain in NCBI database,and converged with the Pm Guangzhou strain from duck（entry number OL958439.1）in the same branch（99.3%）.Serological identification showed that the Pm Yinjiang strain was A type capsular strain.Drug sensitivity test showed that the isolated strain was moderately sensitive to vancomycin,sensitive to gentamicin,ampicillin,norfloxacin,ceftazidime,and so on,and no drug resistance was found.The pathogenicity test showed that the MLD of the isolated strain was less than 5 CFU when mice were inoculated with 2.1×10⁹CFU/m L bacterial solution.（2）DPV could be detected by PCR in allantoic fluid of duck embryo after blind transmission for 3 generations;After inoculating duck embryo fibroblasts（DEF）with collected DPV venom,the results showed that DEF cells were round at 9 h,and adherent DEF cells were shed or formed syncytium at 24 h.The TCID₅₀ of DPV Yinjiang isolates was2.88×10^-9.0.1m L^-1.3.Preparation of Pasteurella multocida-duck plague virus combined inactivated vaccine.In order to prepare Pasteurella multocida（Pm）-duck plague virus（DPV）combined inactivated vaccine,in this study,after measuring the bacterial growth curve and screening the inactivation conditions,the Pm-DPV combined inactivated vaccine was prepared and the effect of inducing immune response was analyzed in immunized mice.Results:（1）The detection showed that the delay stage,logarithmic growth stage,steady development stage and decline stage of Pm isolates from goose were 0～4 h,4～8 h,8～14 h and after 14 h.The optimal inactivation conditions were 0.2%formaldehyde concentration,37℃inactivation temperature and 24 h inactivation time.Pm antigen content was 2×10⁶ CFU/m L and DPV antigen content was 10³ TCID₅₀.Sterility test showed no bacterial colony growth;The mice were inoculated with 2 times（0.5 m L）and 4 times（1.0 m L）volume inoculations,and no obvious adverse reactions were observed,and the mental state was good.（2）Immunized mice showed that Pm-PDV vaccine,commercially available single Pm vaccine（Huabaiwei）and single DPV vaccine（Duck biying）could induce good humoral and cellular immune responses in mice,among which:the specific Pm and DPV antibodies induced by the Pm-PDV vaccine were superior to the commercially available single Pm vaccine or single DPV vaccine.But the induced secretion of Th1 and Th2 cytokines was lower than that of commercially available Pm single vaccine or DPV single vaccine.In summary,this study confirmed that the infected grey geese in Yinjiang County were caused by mixed DPV and Pm infection through bacterial isolation,identification and viral PCR detection.The isolated strain of Pasteurella multocida from geese was highly homologous to the isolated strain from ducks.It was A type capsular strain,and its MLD to mice was less than5 CFU.The TCID₅₀ of the isolated strain of duck plague virus from geese was 2.88×10^-9.0.1m L^-1.The prepared Pasteurella multocicide-duck plague virus vaccine can effectively induce good humoral and cellular immune responses in mice.