| Enterotoxigenic Escherichia coli(Enterotoxigenic Escherichia coli, ETEC) is one of major pathogens that causes newborn animals(calves, lambs, piglets) diarrhea. It’s popular widely in most parts of the world and its high morbidity and mortality caused serious damage to livestock. It’s of great significance to establish a rapid, simple, sensitive and specific diagnostic technique for the prevention and treatment of the disease.ETEC colonized in the intestine in the assistance of pili adhesin,then produced toxin, causing diarrhea in young animals. Many ETEC strains carried pili adhesins, such as K88, K99, 987 p and F41. Therefore, the rapid detection of fimbriae could provide important reference for the detection of ETEC.This study aimed to extract enterotoxigenic Escherichia coli fimbriae K88 ab, K99, 987 p and F41 to immunize New Zealand white rabbits to prepare mono-specific serum, then colored silica microspheres(Colored Silica Nanoparticles, CSN) as the carrier, mono-specific serum modificated to the nanoparticles surface to prepare immune colored silica microspheres(Immune Colo red Silica Nanoparticles, ICSN), establishing a new indirect agglutination technology for the rapid detection of K88 ab, K99, 987 p and F41 fimbriae.Preparation of mono-specific serum. Extracted four types fimbriae to immunize New Zealand white rabbits three times at intervals 21 d. Sterile collection rabbit blood for preparing antiserum, the results slide agglutination test showed that anti K88 ab, K99, 987 p and F41 serum agglutination titer were 1:256, 1:512, 1:128 and 1:4096. The results of cross-agglutination test showed that the K88 ab antiserum and K99 antigen agglutination titer was 1:32, what’s more, K99,987 p, F41 antiserum and K88 antigen were cross-agglutination, and agglutination titer were 1: 8,1: 16 and 1:16, respectively.By optimizing the reaction conditions, we determined to mix antiserum with cross agglutination cell hematocrit at volume ratio 1:1, and absorped cross antibody 48 h at 4℃. The results showed that K88 ab, K99, 987 P and F41 only reacted with corresponding antigen with great specificity after absorption, and agglutination titer were 1:64, 1:256, 1:32 and 1:2048, respectively.Preparation and application of ICSN. CSN prepared by reverse microemulsion method bound to mono-specific serum to prepare different color of K88 ab, K99,987 p and F41 ICSN, while using three different colors ICSN detecting antigen showed that the color had no effect on agglutination results.By optimizing the reaction conditions, we determined the reaction system and the reaction conditions for the slide agglutination test,which acquired that 20 μL ICSN was mixed with 20 μL bacteria(the concentration range of bacterial solution was 106~109CFU?m L-1) at 25℃~37℃ for 1 min. The results of specificity test showed that K88 ab, K99, 987 p, F41 ICSN had no cross agglutination reaction with ETEC C83903(K88ab+), C82529(K99+), C83915(987p+), O142(F41+), E.coli DN69-A, E.coli DN89-B, Pseudomonas aeruginosa(SD004), Bacillus erysipelatos-suis(CVCC124), Salmonella choleraesuis(CVCC503), Pasteurella(CVCC430) and physiological saline. The results of sensitivity test showed that the minimum detectable concentration of bacilli of K88 ab, K99, 987 p and F41 ICSN was 1.0~5.1×106CFU ? m L-1. ICSN stored at 4 ℃ for 30 d, but its specificity, sensitivity did not change significantly, with good stability. The detection results of the double blind samples were in accordance with the actual samples. The results of new agglutination test were intuitive and easy to judge, and the amount of antibody of 1/500 of slide agglutination test could detect antigen, thus, it was suitable for the situation that the quantity of the antibody was small and the sample is was much.This study established an indirect agglutination test which regarded CSN as the carrier, for detecting ETEC K88 ab, K99 and F41 fimbriae, and provided a rapid, simple, specific and sensitive detection method for the diagnosis, prevention and treatment of diarrhea of pup Escherichia coli. |
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