Isolation And Identification Of Bovine Acinetobacter Towneri And Establishment And Application Of PCR Detection Method

Acinetobacter is a Gram-negative Brevibacterium.When the body's resistance decreases,it can cause infection of various organs of the human body,and even cause bacteremia;it can also cause pneumonia in cattle,sheep,and horses,and cats.Necrotizing fasciitis and fish death.In recent years,Acinetobacter has been continuously isolated from sick cattle,such as Acinetobacter calcoaceticus isolated from lungs of dead beef cattle,Acinetobacter baumannii isolated from bronchial pneumonia yak,and Acinetobacter baumannii isolated from milk of cows suffering from endometritis.In 2018,we isolated a dominant strain of Acinetobater towneri(A.towneri)from a group of nasal swabs of beef cattle with respiratory diseases,and conducted research on the pathogenicity of the bacteria on mice and PCR detection methods.1.Separation and identification of A.towneriIn order to determine the cause of major diseases such as increased body temperature and dyspnea in a beef farm,a disease sample was isolated to obtain a dominant strain ZZC3-9.After biochemical identification,16S r RNA sequencing and evolutionary tree analysis,the strain was A.towneri.The logarithmic growth period of the bacterium is6h-18h,and the stable period is 18h-30h.2.Study on pathogenicity of A.towneri in miceAfter intraperitoneal injection of A.towneri,the mice were challenged with symptoms such as depression,coarse hair,shortness of breath,increased eye secretions,etc.within 2hours.Pressing1.0×10⁹CFU/m L for 0.2 m L of bacteria can cause the death of mice,and the splenic congestion of the dead mice is enlarged.Its half lethal dose is 2.89×10⁹CFU/m L.Histological observation showed that myocardial fibers showed point necrosis;hepatic cells were slightly vacuolized degeneration;spleen had diffuse necrosis;white and red pulp cells showed a large number of nuclear fragmentation;there was a small amount of inflammatory cell infiltration in the lung interstitial.3.The establishment and application of A.towneri PCR detection methodPrimers were designed according to the sequence of A.towneri highly conserved gene Omp A(F:5'-GACTGAGCCCGTAGAAGA-3';R:5'-ACAGTTGATTACCCACCC-3'),and successfully constructed through specificity,sensitivity and annealing temperature tests A.towneri's detection system:according to 2×Master Mix 10?L,dd H₂O 11?L,template strain 2?L,upper and lower primers 1?L each;the reaction procedure is 96?7min;95?30 sec,50?30 sec,72?30sec,35 cycles of extension at 72?for 10min;401bp specific band can be obtained.The minimum detection limit is 8.7×10⁵CFU/m L.A total of 78 beef cattle nasal swabs with respiratory diseases were tested.The positive sample rate of A.towneri was 7.7%(6/78)and the negative sample rate was 92.3%(72/78);The detection rate of A.towneri was as high as 98.7%.4.A.towneri,A.baumannii,A.johnsonii multiple PCR detection method establishment and applicationSpecific primers constructed for the A.towneri conserved gene Omp A,and refer to the specific primers reported in the literature A.baumannii(Ptk gene),A.johnsonii(ITS gene)constructed PCR detection methods for the above three bacteria.According to 2×Master Mix 25?L,dd H₂O 8?L,the upstream and downstream primer amounts of A.johnsonii,A.baumannii and A.towneri are 1?L,0.5?L and 4?L respectively,and the template amount of bacterial solution is 1?L and 1?L respectively And 4?L;the reaction program is 94?5 min;94?1 min,50?1 min,72?1 min,35cycle;72?extension for 10 min;specific bands of 365bp,208bp and 401bp can be obtained respectively.The detection rates of 45 beef cattle nasal swabs with respiratory diseases were A.towneri,A.baumannii,A.johnsonii,the detection rates were 6.7%(3/45),11.1%(5/45),4.4%(2/45);compared with the identification methods using traditional bacterial isolation A.towneri,A.baumannii,A.johnsonii,the agreement is as high as 100%,93.3%and 100%,respectively.Conclusion:1)A.towneri was isolated from cattle with respiratory syndrome for the first time,and the bacterium has certain pathogenicity to mice,mainly damaging organs such as spleen,lung,heart and liver.2)For A.towneri's Opm A gene,A.towneri's single PCR detection method and its triple PCR detection method with A.baumannii and A.johnsonii were successfully constructed.3)After verification with traditional bacterial separation and identification methods,the construction of A.towneri's single-plex PCR and multiple detection methods is suitable for the detection of nasal swab samples of beef cattle clinical respiratory diseases,with the characteristics of rapid detection and high efficiency.