In the veterinary clinic or experiment, the E. coli that needs to be isolated and identified is mostly limited to some strains that are pathogenic to livestock and poultry, such as ETEC strains that cause diarrhea in newborn young animals (pigs, calves, lambs) and weaned pigs. Pathogenic strains causing swine edema disease and diarrhea in young rabbits and various E. coli infections in chickens. In addition to biochemical typing and serological typing of the isolates, it should also include the identification of ETEC adhesin antigens and enterotoxins, as well as the determination of SLT-â…¡ strains of edema disease. As for the pilus identification of strains of chicken, rabbit and swine edema disease, it is still in the laboratory research stage.
1. Morphology and culture characteristics
Most strains that cause edema disease and diarrhea of ​​newborn piglets are hemolytic, forming red colonies on MacConkey agar, and dark blue-black colonies with green metallic flashes on eosin blue agar.
2. Biochemical identification
If the bacteria of the tested colonies are positive in trisaccharide iron agar (TST), the O antigen is positive or at the same time, the conventional biochemical project identification is also in line with the biochemical characteristics of the genus Escherichia. Further serological typing and toxicity factor determination.
It is known that 97% of the isolated E. coli produces glucuronidase, which can decompose glucuronide-4-methylumbellene (MUG) to generate fluorescent methylumbelliferone, which can be used as Quick check. The rapid method of Thompson (1990) can be completed within 20 minutes. The test E. coli was cultured on Columbia agar base (Oxoid, containing 5% sheep blood) and incubated at 36 ° C for 18-24h.
MUG reagent: 4-methylumbelliferyl-D glucuronide 1OOmg, distilled water 1OOml, three drops (TritoxX-1OO) 2 drops, to be completely dissolved, filtered and sterilized with O.45tzm L diameter filter membrane , Stored in sterile glass bottles, can be stored in the refrigerator for 6 months. The solution was clear in the refrigerator and slightly turbid at room temperature.
Test: Use 6mmX 50mm test tubes for cultivation. Drop 2-3 drops (about 0.5 ml) of MUG reagent in a test tube, heat to room temperature, scrape off a part of the pure culture with a sterile inoculation stick and emulsify in MUG reagent to make it a milky suspension. Incubate in a water bath at 44.5 ° C for 2Omin. This temperature can produce the strongest reaction; however, there can be a satisfactory reaction between 22 and 44.5 ° C. Use a secondary high-intensity long-wavelength (366nm) ultraviolet lamp for inspection in a dark room. Place the test tube 15cm below the light source for easy observation. Positive people emit cyan fluorescence.
With the same principle, glucuronide can also be combined with various other fluorescent-producing reagents for the detection of glucosidase acidase.
3. Serotype identification
E. coli currently has 164 O antigens (O1 to O171), 72 K antigens (K1 to K1O3) and 55 H antigens (H1 to H57), and at least thousands of serotypes have been found. Therefore, the serotype identification of the bacteria is based on the three antigens of O antigen, K antigen, and H antigen. ETEC strains must also be added to the identification of adhesin antigens. In view of the fact that ETEC and EPEC are often limited to a small number of O antigen groups, and there is a certain correlation between the former O antigen group and adhesin and enterotoxin.
Therefore, the strains of animal ETEC, swine edema disease and avian colibacillosis can be determined firstly by O antigen type, and then complete serotype identification. Anti-serum to identify O antigen must use single factor anti- () serum to avoid cross reaction. If non-single factor serum is used, the result of test tube agglutination should be used to determine.
4. Identification of ETEC virulence factors
E. coli isolates related to diarrhea of ​​young animals are identified as the bacterium after the above-mentioned biochemical and serotype (at least O antigen) identification, and then identified whether they have adhesion antigen and enterotoxin. The identification of enterotoxin can be selected according to the relationship between adhesin and enterotoxin. Generally, bovine and sheep-derived bacteria originally produce ST, while pig-derived strains produce LT and ST. There are many methods for identifying adhesins. In general laboratories use glass plate agglutination test, fluorescent antibody technology, ELISA, MRHA and its inhibition test. The methods commonly used to identify enterotoxins are rabbit ileal ligation test or piglet small intestine ligation test, rabbit skin blue spot test, and plate immune hemolysis test. Animal testing on ST is only available for intragastric administration of suckling rats. The identification of SLT-Ⅱ strains of swine edema disease is still dominated by Vero cell assay.
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