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SN/T 5183-2020Technical specifications for quarantine of cowhide myiasis (English PDF)

牛皮蝇蛆病检疫技术规范

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Issued by

General Administration of Customs

Level / Type

National · Recommended

Issue date

December 30, 2020

Implementation date

July 1, 2021

Scope

SN/T 5183-2020 is the English-translated version of 牛皮蝇蛆病检疫技术规范.

SN/T 5183-2020 — This document stipulates the clinical symptom examination, necropsy examination, pathogen morphology identification and enzyme-linked immunosorbent assay during the quarantine of bovine myiasis.

Attached are technical specifications such as ELISA. This document is applicable to the quarantine of live cattle and cowhide fly maggots in the entry and exit of the country, and also applies to the epidemic disease of cowhide fly maggots in cattle breeding farms. Scientific investigation and monitoring.

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Document preview — SN/T 5183-2020

National Standard of the People's Republic of China

ICS: 65.020.30
Classification: B 41

Issued by: General Administration of Customs

Foreword
1 Scope
2 Normative references
3 Terms and definitions
4 Overview of the disease
5 Materials and reagents
6 Equipment
7 Clinical Symptom Check
7.1 Scope of application
7.2 Clinical symptoms
8 Necropsy and morphological identification
8.1 Scope of application
8.2 Necropsy
8.3 Morphological identification
9 Enzyme-linked immunosorbent test (ELISA)
9.1 Scope of application
9.2 Collection and processing of serum samples
9.3 Operation steps
9.4 Calculation of results
9.5 Judgment of results
10 Comprehensive judgment of results

Foreword

This document is drafted in accordance with GB/T 1.1-2020.

This document was proposed and managed by the General Administration of Customs of the People's Republic of China.

Drafting organizations of this document. Chinese Academy of Inspection and Quarantine, Urumqi Customs, People's Republic of China, People's Republic of China Guangzhou Customs.

1 Scope

This document stipulates the clinical symptom examination, necropsy examination, pathogen morphology identification and enzyme-linked immunosorbent assay during the quarantine of bovine myiasis.

Attached are technical specifications such as ELISA.

This document is applicable to the quarantine of live cattle and cowhide fly maggots in the entry and exit of the country, and also applies to the epidemic disease of cowhide fly maggots in cattle breeding farms.

Scientific investigation and monitoring.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document.

GB/T 6682 Analytical laboratory water specifications and test methods

3 Terms and definitions

There are no terms and definitions that need to be defined in this document.

4 Overview of the disease

Bovine hypodermosis (Bovine hypodermosis) is caused by the hides of Hypodermine and Hypoderma.

Flies (H.bovis), striped flies (H.lineatum) and Chinese skin flies (H.sinense) are parasitic in the yak or cattle.

Insect disease. The main hazards of this disease are the perforation of the cowhide, the reduction of meat and milk production and quality, and the high intensity of infection can cause animal death.

The reported pathogens of cowhide myiasis belong to 39 species in 10 genera. The specific hosts are cattle, yaks, and cattle of the genus Bovine.

Cattle and buffalo, but sometimes can also infect horses and other domestic animals and humans, and can mature. In view of the serious harm of this disease to the animal husbandry, Many countries in the world initiated the control and eradication of skin myiasis in the 1920s and 1930s, and implemented legislation to eliminate skin myiasis.

Mandatory. Skin myiasis is prevalent in a wide range, and the disease is prevalent in 55 countries within the range of 18° to 60° north latitude. Mycosphagidae the geographical distribution of maggot disease is mainly in the agricultural and pastoral areas of Northeast, North China, Northwest and other regions. The cowhide myiasis is particularly serious in these agricultural and pastoral areas. In the stream the infection rate is over 80% in the walking area, and 98%-100% in severely infected areas, especially calves aged 1 to 3 years old are susceptible to infection.

The incidence of the above cattle is reduced.

5 Materials and reagents

Unless otherwise specified, the chemical reagents used are of analytical grade. The test water should meet the requirements of Grade 1 water in GB/T 6682.

Bovine Hypodermosis Antibody Test Kit. This standard uses the commercial kit Bovine Hypodermosis Antibody Test Kit as an example for operation, other ELISA test kits for bovine myiasis that have been validated and tested with reliable results can also be used.

6 Equipment

6.1 Stereo microscope.

6.2 Microplate reader.

6.3 -20 °C ordinary refrigerator, 4 °C refrigerator.

6.4 Autoclave.

6.5 Centrifuge tube, micropipette and matching tips, glass petri dishes and dissecting needles.

6.6 Constant temperature incubator.

7 Clinical Symptom Check

7.1 Scope of application

It is suitable for the quarantine of live cattle infected with bovine fly maggots during the entry and exit quarantine period.

7.2 Clinical symptoms

After cows are infected with skin flies, they generally do not show obvious clinical symptoms, but when they are severely infected, the young animals show weight loss, slow growth, and poverty.

Blood, the cow's milk production decreases, and the draught animal's ability to work is reduced. When the skin fly larva penetrates the brain, it can cause neurological symptoms, such as Retreat movement, sudden fall to the ground, paralysis or fainting, etc., severe cases can cause death. In addition, due to the formation of perforations in the skin of the third stage larvae, Bacterial infection causes suppuration and forms a fistula, often with pus and serous outflow, and the fistula gradually heals and forms a scar. But the above symptoms can only as a reference indicator of skin fly infection, it cannot be used as a basis for diagnosis. Only if tumor-like bumps and subcutaneous cellulite are found on the back of the cow the diagnosis can only be confirmed when the third stage larvae can be squeezed out.

8 Necropsy and morphological identification

8.1 Scope of application

It is suitable for the diagnosis of the condition of dead cattle, and also for the quarantine of inbound and outbound cowhide sheets.

8.2 Necropsy

Focus on inspection of esophageal mucosa, back subcutaneous, rumen serosa, omentum, esophageal serosa and other parts. Three-stage larvae cause skin swelling in animals from a tumor-like mass. There are small holes in the animal's bulge, and dry pus scabs are common around the holes, and larvae can be seen in the hoof knot tissue sacs in the holes. As found in the third period the larvae can be diagnosed. Take the suspicious cowhide fly maggot body, place it in a glass petri dish, turn the body with the aid of a dissecting needle, under a stereo microscope Observe whether it has relevant morphological characteristics.

8.3 Morphological identification

8.3.1 Morphology of the second stage larva

It is pale yellowish white, 11 mm to 15 mm long and 3 mm to 6 mm wide. The ventral surface is slightly protruding, and the back is flat. The appearance of the mouth hook is not obvious, only seen Two small yellow-brown dots are separated from each other. The spines on the front and back edges of the ventral sections are not clearly demarcated, and the air valve does not form a kidney shape, resembling a granular pomegranate.

Stacked arrangement.

8.3.2 Morphology of the third stage larva

The body length is 19 mm~25 mm, the width is 8 mm~11 mm, and the ratio of length to width (2.3~2.1). 1.The back is flat and the belly is raised. side There are verrucose protrusions, the color varies with the degree of maturity, from light yellow to light brown, tan to black, with 11 sections (3 chests, 3 chests, 8 sections of the abdomen). The pseudo-head has a pair of head sensors, which are separated from each other. The mouth hook degenerates into black dots. No thorns in the last two segments of the ventral surface, with a concave rear valve it is deep and funnel-shaped, and there are many thick spines on the surface of the valve plate (hole) (see Appendix A).

9 Enzyme-linked immunosorbent test (ELISA)

9.1 Scope of application

It is suitable for the detection of whether the live cattle in and out of the country are infected with bovine fly maggots.

9.2 Collection and processing of serum samples

Animal blood was collected from the jugular vein, serum was separated, and stored in a refrigerator at 4°C.

9.3 Operation steps

9.3.1 Reagent preparation

See Appendix B.

9.3.2 Coating

Use the "skin fly antigen reaction plate" in the Bovine Hypodermosis Antibody Test Kit, where the coating antigen is purified striated skin Extract from the first stage larva of fly.

9.3.3 Dosing

9.3.4 Incubation

After mixing gently, cover and incubate at 37°C for 1 h.

9.3.5 Washing

Discard the liquid in the wells, add washing solution to wash, 300 µL per well, and repeat the washing 3 times.

9.3.6 Enzyme-labeled antibody reaction

Add horseradish peroxidase HRP-labeled anti-ruminant IgG, 100 µL per well, cover, and incubate at 37°C for 30 min.

9.3.7 Secondary washing

Discard the liquid in the wells, add washing solution to wash, 300 µL per well, and repeat the washing 3 times.

9.3.8 Color rendering

Add 100 µL of substrate solution TMB to each well, cap, and incubate for 20 min in the dark at 18 °C ~ 26 °C.

9.3.9 Terminating the reaction

Add 100 µL of stop solution to each well and mix gently to stop the reaction.

9.3.10 Determination

Measure the OD450 value with a microplate reader.

9.4 Calculation of results

9.4.1 Control

9.4.2 Sample

9.5 Judgment of results

9.5.1 Determining the establishment of the test

9.5.2 Judgment of results

When the negative and positive control test results are valid, the sample result is judged, and the judgment standards are as follows.

--Negative. S/P <= 45%;

--Suspicious. 45% < S/P < 55%;

--Positive. S/P >= 55%.

The suspicious sample should be tested for the second time. If the result of the repeated test is still suspicious, a new sample should be collected for testing.

If the result is still suspicious, it is finally judged as positive.

10 Comprehensive judgment of results

If a worm body is found, the diagnosis shall be confirmed based on morphological observation. If the serological test is positive, it is judged that the sample is positive for cowfly maggot infection; blood If the cleansing test is negative, it is judged that the sample is negative for the infection of bovine fly maggots.

Appendix A

(Informative)

Morphology of the third stage larvae of two species of skin flies A.1 The third stage larva of the cowhide fly maggot the third stage larva of the cowfly maggot is shown in Figure A.1.

A.2 The third stage larva of the larvae the body is hypertrophy, and the color varies with the degree of maturity, ranging from light yellow to light brown, yellowish brown to black. Flat back, ventral protruding from the beginning, there are 11 nodules. The mouth hook degenerates into black dots. The last section of the ventral surface has no thorns, and the posterior valve is shallow and flat (see Figure A.2).

B.1 Dilution of lotion Concentrated lotion (20×) may precipitate salt when stored in cold storage. Return to room temperature and shake well to dissolve the precipitated salt before use.

Dilute 1.20 with distilled water as working fluid. The working solution should be stored at 2 °C ~ 8 °C, and it can be used stably within 3 days.

B.2 Enzyme-labeled antibody dilution Enzyme-labeled antibody concentrate (100×) should be diluted 1.100 with the No. 1 dilution buffer provided with the kit before use.

Under the condition of 18 °C ~ 26 °C, the diluted enzyme-labeled antibody can be used within 8 hours.

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This preview omits tables, figures, formulas and parts of the technical clauses. The complete document — 7 pages — is available in the English PDF.

Referenced standards

Normative references

GB/T 6682Water for analytical laboratory use - Specification and test methods

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