SN/T 5193-2020Technical specifications for rabbit coccidiosis quarantine (English PDF)

7.1 Sample collection

7.1.1 Collection of samples from liver and intestinal lesions

Cut liver nodules and intestinal mucosal nodules, each of about 20 g, put them into a clean ziplock bag, and mark the number.

Record the rabbit's age, breed, breeding and sanitary conditions, etc., and store at 4°C for microscopic examination.

7.1.2 Collection of live rabbit feces samples

Live rabbits were quarantined for 30 days, and rabbit feces were collected twice on the first day of isolation and the 12th day of isolation.

Before sampling, put a layer of plastic wrap under the rabbit cage. Take samples from the fresh-keeping film after 3 hours. Regardless of the age of the rabbits, take the individual rabbits as the unit collects about 20 g of fresh feces. Put it into a clean ziplock bag, mark the number, and record the age, breed, and health of the rabbit the conditions, medications, breeding and sanitary conditions, etc., should be stored at 4°C for microscopic examination.

7.2 Morphological identification

7.2.1 Microscopic examination of liver and intestinal lesions

Microscopic examination of the liver tissue showed atrophy of liver cells, proliferation of bile duct epithelium, showing papillary protrusions, and degeneration and necrosis of epithelial cells. Liver nodules A large number of coccidian oocysts can be seen on the tablet.

Intestinal mucosa necrosis and shedding, and coccidian oocysts can be seen on the intestinal mucosa smear.

7.2.2 Treatment and microscopic examination of live rabbit feces

7.2.2.1 Weigh 3 g of each stool sample, mash it with a glass rod and stir it into a suspension with 50 ml of normal saline.

7.2.2.2 Filter the fecal suspension through a 60-mesh small copper sieve into the second container, and pour the filtrate in the second container into a centrifuge tube.

7.2.2.3 Centrifuge at 3 000 r/min for 3 min, and discard the supernatant.

7.2.2.4 Add 10 ml of saturated sodium chloride solution to the precipitate and mix well.

7.2.2.5 Centrifuge at 3 000 r/min for 3 min.

7.2.2.6 Use a fishing net to get the surface floating liquid and shake it off in a container with water.

7.2.2.7 Transfer the oocysts into a petri dish that has been added with 2.5% potassium dichromate solution, mix well, the height of the liquid level is less than 1 cm, and mark it.

Place it in a shaking table at 28 °C for sporulation culture for 48 h~72 h, observe the sporulation situation every 12 h, and the sporulation rate reaches 80% or more.

The oocysts are stored in a refrigerator at 4 °C.

7.2.3 Morphological characteristics

Place the oocysts on a glass slide and observe under a 100x optical microscope. The oocysts are oval or approximately round, and a few are oval or pears.

Shape, morphology depends on the species of insects, the membrane wall is thicker, generally there are two layers, the eggshell-like structure can be clearly seen, colorless, light yellow, golden yellow Wait. After positioning the oocysts under a 100x optical microscope, further observe the shape and structure of the oocysts under a 400x optical microscope.

See Appendix D for the model diagram of Eimeria rabbit oocysts and the morphological characteristics of 11 species of rabbit coccidia oocysts.

7.3 PCR detection

7.3.1 PCR primers

The primer sequences used in this method are as follows.

7.3.2 Sample preparation

Take 25 mg of rabbit coccidia oocysts (or lesion tissue) and place them in a centrifuge tube to extract according to the method of extracting tissue DNA in Appendix E for DNA, commercial tissue DNA extraction kits can also be used.

7.3.3 PCR reaction system

Take 2 µL of the extracted sample DNA as a template and add it to the PCR reaction solution prepared in Table 1.At the same time set up a negative and positive control, Set up ddH2O as a blank control.

7.3.4 PCR reaction program

Pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 1 min, 32 cycles; finally 72 °C Extend for 7 min; store at 4 °C.

7.3.5 Agarose electrophoresis

Take 5 µL of the PCR amplification product and perform 1.5% agarose gel electrophoresis. After the electrophoresis is completed, use a gel imager or UV transmission the instrument observes the result.

7.3.6 Judgment of results

7.3.6.1 Conditions for the establishment of the test

The positive control amplifies the band of interest, and the rabbit coccidia species pointed to by the closest homology sequence after sequencing and comparison are compared with the positive control.

The species of rabbit coccidia are the same, and the negative control has no corresponding fragments. Otherwise, the test is invalid.

7.3.6.2 Suspicious determination

The sample has amplified bands, and it is determined that the sample is suspected of rabbit coccidia infection.

7.3.6.3 Negative judgment

The sample has no amplified band, and the sample is judged to be negative for rabbit coccidia infection.

7.3.6.4 Positive judgment

The sample has amplified bands. After the amplified products or electrophoretic bands are purified, they are directly sequenced or T vector cloned and sequenced.

Perform BLAST analysis and comparison between the sequence obtained by sequencing and the sequence in the Genbank database. If the sequence with the closest homology points to A certain rabbit coccidia, the sample can be determined to be positive for the rabbit coccidia infection; if the closest sequence of homology points to a sequence that has nothing to do with various rabbit coccidia Column, the sample is judged to be negative for rabbit coccidia infection.

8.1 Suspicious determination

During PCR detection, the sample has amplified bands, and the sample is judged to be suspicious.

8.2 Confirmation of diagnosis

Coccidian oocysts were found in liver tissue or intestinal mucosa under microscope, and the type of infection was identified based on morphology. Samples with suspicious oocyst morphological characteristics DNA is extracted for PCR amplification, and the infection status is determined based on the PCR results and the sequencing comparison analysis results.