SN/T 5184-2020Technical specifications for avian paratyphoid quarantine (English PDF)

5.1 Clinical diagnosis

Chicks with paratyphoid fever are mainly of acute septicemia, and they often die soon after hatching. No obvious symptoms are seen. Chicks are about 10 days old.

After the onset of illness, mental fatigue, fear of cold, drooping head and wings, loose feathers, like crowding in warm places, loss of appetite, polydipsia, Diarrhea, watery and thin feces are discharged, and the area around the anus is often contaminated by thin feces. Some have ophthalmia, blindness, and some have difficulty breathing. Often Death within 1 to 2 days, the mortality rate is 10% to 80%.

Adult chickens usually do not show symptoms after infection and become chronic carriers. The intestinal bacteria can be carried for 9 to 16 months. After adult chicken infection Anorexia, diarrhea, dehydration, general weakness, drooping wings and loose feathers, the mortality rate is about 10%, most of which can recover in a short period of time.

5.2 Pathological diagnosis

At necropsy, the most acute lesions are often insignificant, only the liver is congested and enlarged, the gallbladder is dilated and filled with bile. Sick chicken with a longer course After death, symptoms of weight loss, water loss, and coagulation of yolk appear. The liver and spleen have congestion, bleeding streaks, or large gray-white necrotic spots on the needle tip.

Kidney congestion. There is pericarditis, the pericardial fluid increases and contains fibrinous exudate. The small intestine has hemorrhagic inflammation, especially the duodenum is the most serious.

The cecum is dilated, and the intestinal cavity is sometimes blocked by light yellow or white cheese-like substances. Young chicks less than 10 days old often have pneumonia lesions.

5.3 Isolation and identification of pathogens

5.3.1 Reagents and materials

5.3.1.1 Non-selective enrichment solution. buffered peptone water (BP), see A.1.

5.3.1.2 Selective enrichment solution. Selenite cystine enrichment solution (SC), sodium tetrathiosulfonate brilliant green enrichment medium (TTB), RVS large Rappaport-Vassiliadis soya peptone broth, see A.2~A.4.

5.3.1.3 Non-selective agar plates. blood agar plates, nutrient agar plates, see A.5~A.6.

5.3.1.4 Selective agar plates. Bismuth sulfite agar (BS), cholestire agar (DHL), HE agar (Hektoen Enteric Agar), Xylose Lysine Deoxycholate Agar (XLD), Salmonella chromogenic medium, see A.7~A.11.

5.3.1.5 Biochemical identification reagents. triose iron agar, lysine decarboxylase medium, ornithine decarboxylase medium, urease medium, for semi-solid agar, sugar fermentation tube, micro biochemical tube, biochemical identification kit or reagent strip, see A.12~A17.

5.3.1.6 Diagnostic serum. AF multivalent O serum, O9 factor serum, O12 factor serum, Ha factor serum, Hd factor serum, Hg.m factor serum and Hg.p factor serum.

5.3.1.7 Distilled water. The distilled water used to prepare the culture medium and reagents shall meet the requirements of GB/T 6682 secondary water.

5.3.2 Equipment and equipment

5.3.2.1 Biological safety cabinet.

5.3.2.2 Fully automatic microbial identification system.

5.3.2.3 Constant temperature incubator. 25 °C ±1 °C, 30 °C ±1 °C, 35 °C ±1 °C.

5.3.2.4 Constant temperature water bath. 46 °C ±1 °C.

5.3.2.5 Homogenizer or sterilized mortar.

5.3.2.6 Microscope. 10×~100×.

5.3.2.7 Inoculation loop and inoculation needle.

5.3.2.8 Erlenmeyer flask.

5.3.2.9 Sterilized test tube. 18 mm~180 mm.

5.3.2.10 Sterilized straws. 10 mL (scale accuracy of 0.1 mL) and 1 mL (scale accuracy of 0.01 mL).

5.3.2.11 Sterilized measuring cylinder. The capacity is 100 mL and 250 mL.

5.3.2.12 Sterilized petri dishes. 90 mm to 100 mm in diameter.

5.3.2.13 Adjustable micropipette or pipette. 10 µL~200 µL, 50 µL~1 000 µL and pipette tip.

5.3.3 Disease material collection and separation

5.3.3.1 Cloaca and fresh fecal swab

After the swab is directly streaked and inoculated on the selective medium plate, the plate is placed at 36 °C ± 1 °C for 24 h, and suspicious colonies are picked for identification; at the same time, place the streaked cotton swabs in selective enrichment solution, culture for 24 h and 48 h, and transfer to selective agar plates on. The agar plate was cultured for 24 hours, and suspicious colonies were directly picked for identification.

5.3.3.2 Gallbladder contents

Swab the contents of the gallbladder on non-selective and selective agar plates. At the same time, place the swabs separately in non-selective and selective Culture for 24 h and 48 h in the sex enrichment solution, then streak and transfer to selective agar plates.

5.3.3.3 Tissues and organs

Swabs or homogenates of diseased tissues collected from individuals aseptically are directly streaked and inoculated on non-selective and selective agar plates. Of Afterwards, place the streaked swab or the remaining tissue homogenate in non-selective and selective enrichment solutions, respectively, and incubate at 36 °C ± 1 °C for 24 h and After 48 h, streak and transfer to selective agar plates.

5.3.3.4 Chicken with bacteria

A large amount of sample material is required for the diagnosis of the chicken with bacteria. Eggs and oviducts should be used for the separation of Salmonella pullorum, and typhoid fever sand should be separated the liver and gallbladder are suitable for phylum bacteria, but also the eggs and fallopian tubes. For a good separation effect, samples should be taken from various tissues including the spleen. Use less Measure the broth to homogenize the tissue, and directly streak it on the selective and non-selective agar plates. At the same time, take 10 mL of each to inoculate separately 90 mL of non-selective broth such as buffered peptone water and 90 mL of selective broth such as SC, incubate at 36 °C ± 1 °C for 24 h, then streak Transplant on selective agar plates.

5.3.3.5 The digestive tract includes cecal tonsils and intestinal contents After 1.10 grinding or homogenization, 10 mL of each was added to 90 mLSC and 90 mL TTB selective enrichment broth, and divided Do not place 36 °C ± 1 °C and 42 °C ± 1 °C for 24 h and 48 h, and then transfer to selective agar plates.

5.3.3.6 Eggshell

Take 10 g of broken eggshells and add them to 90 mL SC and 90 mL TTB selective enrichment broth, respectively, and place them at 36 °C ±1 °C Incubate at 41 °C ± 1 °C for 24 h and 48 h, and then transfer to selective agar plates by streaking.

5.3.3.7 Egg contents

Aseptically take 25 g of the contents of fresh eggs, add 225 mL of buffered peptone water or nutrient broth, stir and homogenize, and store at 36 °C ±1 °C Cultivate for 24 h and 48 h, then streak and transfer to selective agar plates. Hatched eggs, regardless of whether they are fertilized or contain small embryos Make a similar treatment.

5.3.3.8 Chicken embryo

The chicken embryos are made into a homogenate and directly streaked and inoculated on non-selective and selective agar plates. At the same time, take 1 g of tissue homogenate and then Planted in 9 mL of non-selective enrichment broth such as buffered peptone water and selective enrichment broth such as SC, cultured at 36 °C ± 1 °C for 24 h and After 48 h, streaked and transferred to non-selective and selective agar plates.

5.3.3.9 Environmental samples such as litter

Including fluff, eggshell fragments or chicken excrement samples stuck to the incubator, hatcher litter, feces or garbage samples on the floor Wait. Take 25 g samples and add them to 225 mL SC and 225 mL TTB, mix well, and place them at 36 °C ±1 °C and Incubate at 42 °C ±1 °C for 24 h and 48 h, and then transfer to selective agar plates by streaking.

5.3.4 Identification of pathogenicity

First use the inoculating needle to pick 5 typical or suspicious colonies on the plate to inoculate trisaccharide iron agar, firstly draw a line on the inclined plane, and then puncture the bottom Layer; the inoculation needle does not need to be sterilized, and directly inoculate lysine decarboxylase medium or urease medium and nutrient agar plates. At 36 °C ±1 °C Incubate for 24 h ± 2 h. It can be extended to 48 h ± 3 h if necessary. Salmonella is cultured in triose iron agar and lysine decarboxylase medium or urease the nutrient response is shown in Table 1 and Table 2, respectively.

5.3.5 Serological identification

5.3.5.1 Mark two 1 cm×2 cm areas on a clean glass slide, pick a ring of bacteria to be identified, and place 1/2 ring on the glass slide.

In the upper part of each area, add 1 drop of Salmonella AF group "O" polyvalent serum to the lower part of one area, and in the lower part of the other area Add 1 drop of normal saline as a control. Use sterile inoculation loops or needles to grind the colonies in the two areas into emulsions. Shake the slide gently 1 min to 2 min, observe against a black background. If agglutination occurs in the control, it is judged that the strain is self-coagulated and cannot be serotyped;

If there is no agglutination, any agglutination in the test area is judged as a positive reaction.

5.3.5.2 If the culture reacts positively with O1, O2, O4, O5, O6, O7, O12 factor sera, it will react with Ha, Hb, Hc If the factor serum is positive, it can be judged as avian Salmonella paratyphi.

5.4 Slide agglutination test

5.4.1 Material preparation

5.4.1.1 Avian paratyphoid multivalent staining plate antigen, strong positive serum (500 IU/mL), weakly positive serum (10 IU/mL), negative serum.

5.4.1.2 Glass plate, straw, wire ring (inner diameter 7.5 mm~8.0 mm), reaction box, alcohol lamp, needle, disinfection tray and alcohol Cotton etc.

5.4.2 Operation

Under the environmental conditions of 20 °C~25 °C, use a quantitative dropper or pipette to suck the antigen, and drop 1 drop vertically on the glass plate (equivalent to 0.05 mL), and then use a needle to pierce the chicken's wing vein or crown tip to take blood 0.05 mL (equivalent to a wire ring with an inner diameter of 7.5 mm~8.0 mm ...