Pathological Changes And Pathogen Identification Of Three Cases Of Bovine Enterotoxemia

Bovine enterotoxemia is an acute,fatal infectious disease of cattle caused by Clostridium perfringens(C.perfringens).The disease can cause acute death in cattle within minutes to hours and there is no effective treatment.The pathological changes in cattle with disease onset are important for the diagnosis of the disease.It is clear from textbooks and early literature that C.perfringens type D is the main pathogen causing bovine enterotoxemia,but some scholars in recent years have suggested that C.perfringens type A is the main prevalent strain causing bovine enterotoxemia.In this study,pathological observations and isolation and culture of the pathogen were performed in three cases of bovine enterotoxemia,and their toxin genotypes were identified by multiplex PCR,and finally the isolates were amplified and sequenced for 16 S rRNA and some plasmid genes for genetic evolutionary analysis.Pathological changes: three cases of cattle with rapid onset and death,ranging from ten minutes to more than ten hours;cattle with respiratory distress,weakness of limbs,falling to the ground,rapid struggling with paddle-like limbs,and death by corneal recoil.Autopsy of the dead cattle showed that the corneal arches were reverted,the abdominal circumference was enlarged,the anal mucosa was turned out;the blood was not coagulated,and hemorrhagic spots were seen in many tissues and organs throughout the body;severe hemorrhagic enteritis,hemorrhagic spots appeared in the intestinal mucosa,and the intestine was filled with blood-like contents.The liver was enlarged and had a smeared blackish-red color with earthy foci of varying sizes scattered on the surface in long-standing cases.Histopathology showed severe hemorrhagic necrotizing enterocolitis,acute inflammatory splenomegaly,hemorrhagic necrotizing lymphadenitis,toxic nephritis,and metaplastic hepatitis.Pathogen isolation and identification: three strains of C.perfringens colonies were successfully isolated after anaerobic culture on sheep blood plate,the colonies were white translucent round with hemolytic ring-like;the colonies were taken for smear preparation and Gram stained,the organisms were single or paired Gram-positive macrobacteria,biochemical experiments showed "burst fermentation" phenomenon in purple cow milk medium;all were identified by multiple PCR as C.perfringens type A.16S rRNA and plasmid gene analysis: The 16 S rRNA gene lengths of the three C.perfringens strains were 1397 bp,1397 bp and 1389 bp,respectively,and were in the same branch as the reference British strain(accession no.JQ739656.1),Chinese Jiangsu strain(accession no.JF764797.1),Japanese strain(accession no.AB689162.1),Korean strain(accession no: OK272362.1),and the Canadian strain(accession no.MH699325.1)had the highest 16 S rRNA gene homology of 99% and were located on the same branch with the closest kinship,indicating a possible common ancestral origin.The three C.perfringens strains with partial plasmid gene sizes of 918 bp,907 bp and 918 bp were amplified and located in the same branch as the reference Korean strain(accession no.CP009598.1),Australian strain(accession no.MG456816.1)and Japanese strain(accession no.JN689220.1),which are the closest relatives;NM2023027,NM2023037 and the reference strains of Japanese(Accession No.AB444205.1),Finnish(Accession No.CP075954.1)and Chinese Jiangxi(Accession No.CP102303.1)are located in the same branch and are the closest relatives.This study elucidated the pathological changes,pathogenic toxin genotypes and genetic evolutionary characteristics of pathogenic genes in three cases of enterotoxaemia in cattle,and provided a reference for the diagnosis and prevention and control of bovine enterotoxaemia.