SN/T 5185-2020Technical specifications for swine paratyphoid quarantine (English PDF)

4.1 Clinical symptoms

4.1.1 Acute septicemia

Sudden increase in body temperature (41 °C~42 °C), lack of energy, and loss of appetite. Difficulty breathing, purple-red skin on the roots of the ears, chest and under the abdomen Color spots, or diarrhea in later stage. Sometimes death occurs within 24 hours after the onset of clinical symptoms, but most of the disease has a course of 2 to 4 days, and the fatality rate is very high.

4.1.2 Subacute and chronic

Sick pigs have elevated body temperature (40.5 °C~41.5 °C), lack of energy, chills, love to lay grass, get together, and have sticky or purulent secretions in their eyes.

A small number of corneal opacity, severe cases develop into ulcers, and even the eyeball is corroded. Sick pigs have poor appetite, diarrhea after initial constipation, yellowish feces or Yellow-green, foul smelling. Rough coat, anemia, long-term diarrhea, feces containing cellulose, necrotic tissue or feed clot. Some skin appears Mi Diffuse eczema. The growth is stunted, and it is a typical stiff pig.

4.2 Pathological changes

4.2.1 Acute

Mainly changes in sepsis. The spleen is often enlarged, dark and blue in color, firm like rubber, and the splenic medulla is not softened. Mesenteric lymph nodes Swollen, other lymph nodes are also swollen, marble-like. The liver and kidney also have different degrees of swelling, congestion and bleeding, and the liver parenchyma can sometimes be See yellow-gray necrotic spots. The whole body mucosa and serosa have different degrees of bleeding spots, and acute catarrhal inflammation can be seen in the gastrointestinal mucosa.

4.2.2 Subacute and chronic type

Mainly necrotizing enteritis. The intestinal wall of the cecum and colon is thickened, and the mucous membrane is covered with a layer of diffuse necrosis and fermented bean curd-like substance, which is like bran.

The bottom is a red ulcer with irregular edges. The mesenteric lymph nodes were cord-like swelling, and some were caseous. splenomegaly. Liver is sometimes visible Yellow-gray necrotic spots.

5.1 Apparatus and equipment

5.1.1 Incubator (37 °C ±1 °C and 42 °C ±1 °C).

5.1.2 Autoclave.

5.1.3 Glass grinder or tissue homogenizer.

5.1.4 Desktop high-speed centrifuge.

5.1.5 Class II biological safety cabinet or ultra-clean workbench.

5.1.6 Water bath.

5.1.7 Refrigerator (2 °C~4 °C and -20 °C).

5.1.8 Sterile straws.

5.1.9 Fully automatic microbial biochemical identification system.

5.2 Medium and reagents

5.2.1 Water. Comply with the first grade water specification in GB/T 6682.

5.2.2 Buffered peptone water (BPW), see A.1 for the preparation method.

5.2.3 Selenite Cystine (SC) Enrichment Solution, see A.2.for the preparation method.

5.2.4 Sodium Tetrathiosulfonate Brilliant Green (TTB) Bacteria Enrichment Solution, see A.3 for the preparation method.

5.2.5 CHROMagar Salmonella is a chromogenic medium agar.

5.2.6 Bismuth sulfite (BS) agar, see A.4 for the preparation method.

5.2.7 Hektoen Enteric (HE) agar, see A.5 for the preparation method.

5.2.8 Xylose lysine deoxycholate (XLD) agar, see A.6 for the preparation method.

5.2.9 DHL agar, see A.7 for the preparation method.

5.2.10 Nutrient agar, see A.8 for the preparation method.

5.2.11 Triose iron (TSI) agar, see A.9 for the preparation method.

5.2.12 Lysine decarboxylase test medium, see A.10 for the preparation method.

5.2.13 Peptone water, indigo matrix reagent, see A.11 for the preparation method.

5.2.14 Urea agar (pH 7.2), see A.12 for the preparation method.

5.2.15 Potassium cyanide (KCN) medium, see A.13 for the preparation method.

5.2.16 o-nitrophenol beta-D galactoside (ONPG) medium, the preparation method is shown in A.14.

5.2.17 Sodium malonate medium, see A.15 for the preparation method.

5.2.18 Gram-negative bacteria identification card.

5.2.19 Salmonella O and H diagnostic serum.

5.3 Sample collection

Sampling shall be carried out in accordance with the provisions of GB/T 18088.Aseptically collect fresh feces or rectal cotton swab samples from surviving animals; dead or with Liver, gallbladder, spleen and other diseased tissues, cecal tonsils and intestinal contents of bacteria.

5.4 Sample storage

The samples were stored in a refrigerator at 4 °C and sent to designated laboratories within 24 hours under refrigerated conditions.

5.5 Bacteria isolation

5.5.1 General

The detection of pathogens should be carried out in the secondary biosafety laboratory, and the sample storage and waste disposal should be in accordance with the requirements of GB/T 19489 get on.

5.5.2 Non-contaminated samples

Take the contents of tissues, organs and gallbladder, and select 2 selective agar plates (CHROMAGA Chromogenic Medium Agar, HE Agar, XLD agar, DHL agar, BS agar, etc.) for streaking inoculation and culture, and homogenizing the tissues and organs at a volume of 1.10 Inoculate BPW, incubate at 36 °C ±1 °C for 18 h to 24 h, then transfer the TTB enrichment solution and SC enrichment solution in a volume of 1.10, and divide After culturing at 42 °C ±1 °C and 36 °C ±1 °C for 24 h and 48 h, then select 2 kinds of the same selective agar plates for streaking culture, If the colony characteristics in Table 1 appear, it can be judged as a suspicious colony, and the suspicious colony is selected for further identification. If no suspicious colonies are found, it is judged as negative for bacterial isolation.

5.5.3 Contaminated samples

Contaminated samples such as rectal cotton swabs, fresh feces, cecal tonsils, intestinal contents, etc. (non-liquid samples are best to be homogenized first Solution), inoculate BPW in a volume of 1.10, incubate at 36 °C ± 1 °C for 18 h to 24 h, and the other steps are the same as 5.5.2.

5.6 Biochemical test

5.6.1 Triose iron agar and lysine decarboxylase test

Pick more than 2 typical or suspicious colonies from the selective agar plate, inoculate trisaccharide iron agar, and mark the inclined plane first, and then Bottom puncture; do not sterilize the inoculation needle, directly inoculate lysine decarboxylase test medium and nutrient agar plates, and incubate at 36 °C ± 1 °C Raise for 18 h to 24 h, if necessary, it can be extended to 48 h. Reaction of Salmonella in Triose Iron Agar and Lysine Decarboxylase Test Medium the results are shown in Table 2.

5.6.2 Secondary biochemical test

According to the preliminary judgment result in 5.6.1, select colonies from the nutrient agar plate corresponding to the suspicious Salmonella to inoculate peptone water (for Do indigo matrix test), urea agar (pH7.2), potassium cyanide (KCN) medium, culture at 36 °C ± 1 °C for 18 h-24 h, necessary Time can be extended to 48 hours. In individual cases, mannitol and sorbitol test or ONPG test should be supplemented. The results are determined according to Table 3.Picked colonies the nutrient agar plate should be stored at 2 °C~5 °C or room temperature for at least 24 h, in case of need for re-examination.

5.6.3 Salmonella biochemical group identification (if necessary)

The Salmonella biochemical group identification can be selectively carried out according to needs, as shown in Table 4.

5.6.4 Fully automatic microbial biochemical identification system

The biochemical identification kit or the automatic microbial biochemical identification system can be selected. According to the preliminary judgment result of 5.6.1, from the nutrient agar Pick suspicious colonies on the plate, prepare a bacterial suspension with appropriate turbidity with normal saline, and use a biochemical identification kit or fully automated microbial growth Chemical identification system for identification.

5.7 Serological test

5.7.1 Antigen preparation

Generally, 1.2% to 1.5% agar culture is used as the antigen for the slide agglutination test. O When the serum does not agglutinate, inoculate the strain in Check again on the medium with a higher amount of agar (such as 2% to 3%); if the O agglutination reaction is prevented due to the presence of Vi antigen, the lawn can be picked up and made into a concentrated bacterial solution in 1 mL of saline.