A selective culture medium for haemophilus parasuis and its preparation method and application

Abstract

The application relates to the technical field of pathogenic culture, in particular to a selective culture medium for Haemophilus parasuis and a preparation method and application thereof. The selective culture medium comprises pig lung 220-280g, glucose 1-3g, agar powder 13-17g, protein peptone 8-12g, sodium chloride 3-7g, chitosan 8-12g, calcium propionate 3-7g, green plum polysaccharide 13-17g, sodium hydroxide for adjusting the pH value to 7.8+ / -0.1, and deionized water added to 1L. Compared with the TSA+NAD culture medium commonly used for the separation of the HPS strain at present, the separation rate of the HPS is increased by 20%, the separation time is shortened by 4 times, meanwhile, the biological characteristics of the separated HPS strain are not affected, the selective culture medium can be used for the rapid separation and identification of the HPS strain, has good stability, can reduce the separation and identification cost, and improves the separation accuracy.

Description

Technical Field

[0001] This invention relates to the field of pathogen culture technology, and in particular to a selective culture medium for Haemophilus parasuis, its preparation method, and its application. Background Technology

[0002] Haemophilus parasuis (HPS) is a Gram-negative bacterium belonging to the Pasteuraceae family. It is small, non-motile, and morphologically diverse (from monococci to filamentous chains). It requires factor V (nicotinamide adenine dinucleotide, NAD) for growth. Infection with Haemophilus parasuis (HPS) can cause symptoms in pigs such as loss of appetite, joint swelling, difficulty breathing, nasal discharge, abortion, and ataxia, and in severe cases, death. Piglets infected with HPS can develop symptoms rapidly with a high mortality rate. Piglets that recover from acute illness may develop chronic arthritis, severely impacting their later growth rate, prolonging the fattening period, reducing feed conversion ratio, and affecting the economic benefits of the enterprise.

[0003] Haemophilus parasuis (HPS) has relatively high nutritional requirements and its growth depends on factor V, making its isolation in clinical practice relatively difficult. HPS is a parasitic bacterium in the upper respiratory tract of pigs; pathogen detection in the nasal cavity and trachea is not diagnostic. It can be isolated and identified from fibrinous exudates, the parenchyma of internal organs in diseased pigs, and lesions in the lungs of pigs with pneumonia, thus diagnosing the disease. Currently, the isolation and culture of this pathogen mainly uses TSA medium supplemented with NAD factor and fetal bovine serum. However, HPS grown in this medium grows slowly and is easily competitively inhibited by other bacteria, leading to isolation failure. Therefore, there is an urgent need to develop a selective culture medium that can improve the growth rate of HPS while inhibiting the growth of other bacteria. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a selective culture medium for Haemophilus parasuis, its preparation method, and its application. This selective culture medium is well-suited for isolating HPS strains, increasing the growth rate of HPS while inhibiting the growth of other bacteria.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first objective of this invention is to provide a selective culture medium for Haemophilus parasuis, the formulation of which is as follows:

[0007] 220-280g of pig lungs;

[0008] 1-3g of glucose;

[0009] 13-17g of agar powder;

[0010] 8-12g of peptone;

[0011] 3-7g of sodium chloride;

[0012] Chitosan 8-12g;

[0013] Calcium propionate 3-7g;

[0014] 13-17g of plum polysaccharides;

[0015] Sodium hydroxide was used to adjust the pH to 7.8 ± 0.1;

[0016] Add deionized water to a volume of 1L.

[0017] The treatment of pig lungs provides the nutrients needed for the growth of Haemophilus parasuis (HPS), including nicotinamide adenine dinucleotide (NAD); glucose and peptone provide carbon, nitrogen, and trace elements; NaCl maintains balanced osmotic pressure; chitosan inhibits the growth of Gram-positive bacteria without affecting the growth of Gram-negative bacteria; plum polysaccharide inhibits the growth of Staphylococcus aureus and Escherichia coli in the culture medium, reducing the growth of other bacteria; calcium propionate inhibits mold growth, ensuring that the culture medium is not contaminated by mold; and sodium hydroxide adjusts the pH value.

[0018] Furthermore, the formulation of the selective culture medium is as follows:

[0019] 250g of pig lungs;

[0020] 2g of glucose;

[0021] 15g of agar powder;

[0022] 10g of peptone;

[0023] 5g of sodium chloride;

[0024] 10g of chitosan;

[0025] Calcium propionate 5g;

[0026] 15g of plum polysaccharide;

[0027] Sodium hydroxide was used to adjust the pH to 7.8;

[0028] Add deionized water to a volume of 1L.

[0029] A second objective of this invention is to provide a method for preparing a selective culture medium for Haemophilus parasuis, comprising the following steps:

[0030] S1. Preparation of basic nutrient solution: Dissolve the prescribed amounts of glucose, agar powder, peptone, and sodium chloride in 700 mL of deionized water. After complete dissolution, adjust the pH value to 7.8±0.1 with sodium hydroxide, then bring the volume to 800 mL with deionized water. After autoclaving, the basic nutrient solution is obtained.

[0031] S2. Preparation of selective culture medium: Take the prescribed amount of pig lung, add 200 mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -40 to -30℃ 2-3 times. Filter it through a sterile filter into the basic nutrient solution obtained in S1 and cooled to 55-58℃. Add the prescribed amount of chitosan, calcium propionate and plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0032] Furthermore, in S1, the autoclaving is performed at 121°C for 20-30 minutes.

[0033] Furthermore, in S2, the sterile filter is a 0.22μm microporous membrane.

[0034] A third objective of this invention is to provide the application of the above-mentioned selective culture medium for Haemophilus parasuis in the isolation of Haemophilus parasuis.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] 1. This invention uses porcine lung as the main component of a selective culture medium. Through cell wall disruption, freeze-thaw cycles, and aseptic filtration, it provides the necessary nutrients for *Haemophilus parasuis*, which depends on nicotinamide adenine dinucleotide (NADP), and also provides the most suitable environment for its growth, thus increasing its growth rate. The selective culture medium prepared by this invention allows for the isolation of the target strain of *HPS* within 6 hours, shortening the growth time of the *HPS* strain by four times, without affecting the biological characteristics of the isolated *HPS* strain. This allows for the rapid isolation and identification of *HPS* strains, reducing isolation and identification costs and improving isolation accuracy.

[0037] 2. This invention adds chitosan to inhibit the growth of Gram-positive bacteria without affecting the growth of Gram-negative bacteria; plum polysaccharide can inhibit the growth of Staphylococcus and Escherichia coli in the culture medium, reducing the growth of other bacteria; calcium propionate can inhibit mold growth, ensuring that the culture medium is not contaminated by mold. By adding chitosan, plum polysaccharide, and calcium propionate to the selective culture medium, the isolation rate of HPS strains can be improved while inhibiting the growth of other bacteria.

[0038] 3. The selective culture medium provided by this invention has a high isolation rate (26.67%) of Haemophilus parasuis, strong specificity and stability, which solves the problem of low isolation rate of Haemophilus parasuis sent from pig farms and provides a guarantee for the subsequent research on drug sensitivity testing and vaccines for Haemophilus parasuis. Detailed Implementation

[0039] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. Unless otherwise specified, the raw materials, reagents, and instruments involved in this invention are all commercially available products that can be purchased from the market.

[0040] The following detailed description of the technology and features of the present invention is provided through specific embodiments, but these embodiments are not intended to limit the scope of protection of the present invention.

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0042] Instruments and reagents

[0043] (1) Equipment: Suzhou Purification SW-CJ-1FD ultra-clean workbench, CHP-80S carbon dioxide incubator, STIK MJ-54A autoclave

[0044] (2) Preparation of TSA+NAD medium: Take 15g tryptone, 5g soybean papain hydrolysate, 5g sodium chloride and 15g agar and dissolve them in 1000mL of deionized water. After fully dissolving, autoclave at 121℃ for 15min. After cooling to 55℃, add 1.5mg NAD and finally filter to sterilize for later use.

[0045] Example 1: Preparation of selective culture medium for Haemophilus parasuis

[0046] S1. Preparation of basic nutrient solution: Dissolve 2g glucose, 15g agar powder, 10g peptone and 5g sodium chloride in 700mL deionized water. After fully dissolving, adjust the pH to 7.8 with sodium hydroxide, and then make up the volume to 800mL with deionized water. Sterilize at 121℃ for 30min to obtain the basic nutrient solution.

[0047] S2. Preparation of selective culture medium: Take 250g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -40℃ 3 times. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 56℃. Add 10g of chitosan, 5g of calcium propionate and 15g of plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0048] Example 2: Preparation of selective culture medium for Haemophilus parasuis

[0049] S1. Preparation of basic nutrient solution: Dissolve 1g glucose, 16g agar powder, 12g peptone and 7g sodium chloride in 700mL deionized water. After fully dissolving, adjust the pH value to 7.9 with sodium hydroxide, and then make up the volume to 800mL with deionized water. Sterilize at 121℃ for 20min to obtain the basic nutrient solution.

[0050] S2. Preparation of selective culture medium: Take 240g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw twice at -35℃. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 55℃. Add 8g of chitosan, 7g of calcium propionate and 17g of plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0051] Example 3: Preparation of selective culture medium for Haemophilus parasuis

[0052] S1. Preparation of basic nutrient solution: Dissolve 3g glucose, 13g agar powder, 8g peptone and 4g sodium chloride in 700mL deionized water. After dissolving completely, adjust the pH to 7.7 with sodium hydroxide, and then make up to 800mL with deionized water. Sterilize at 121℃ for 15min to obtain the basic nutrient solution.

[0053] S2. Preparation of selective culture medium: Take 230g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -30℃ 3 times. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 58℃. Add 12g of chitosan, 3g of calcium propionate and 14g of plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0054] Comparative Example 1:

[0055] S1. Preparation of basic nutrient solution: Dissolve 2g glucose, 15g agar powder, 10g peptone and 5g sodium chloride in 700mL deionized water. After fully dissolving, adjust the pH to 7.8 with sodium hydroxide, and then make up the volume to 800mL with deionized water. Sterilize at 121℃ for 30min to obtain the basic nutrient solution.

[0056] S2. Preparation of selective culture medium: Take 250g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -40℃ 3 times. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 56℃. Add 5g of calcium propionate and 15g of plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0057] Comparative Example 2:

[0058] S1. Preparation of basic nutrient solution: Dissolve 2g glucose, 15g agar powder, 10g peptone and 5g sodium chloride in 700mL deionized water. After fully dissolving, adjust the pH to 7.8 with sodium hydroxide, and then make up the volume to 800mL with deionized water. Sterilize at 121℃ for 30min to obtain the basic nutrient solution.

[0059] S2. Preparation of selective culture medium: Take 250g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -40℃ 3 times. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 56℃. Add 10g of chitosan and 5g of calcium propionate, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0060] Comparative Example 3:

[0061] S1. Preparation of basic nutrient solution: Dissolve 2g glucose, 15g agar powder, 10g peptone and 5g sodium chloride in 700mL deionized water. After fully dissolving, adjust the pH to 7.8 with sodium hydroxide, and then make up the volume to 800mL with deionized water. Sterilize at 121℃ for 30min to obtain the basic nutrient solution.

[0062] S2. Preparation of selective culture medium: Take 250g of pig lung, add 200mL of deionized water and place it in a cell wall breaker for cell wall breaker treatment. Then, repeatedly freeze and thaw at -40℃ 3 times. Filter it through a 0.22μm microporous membrane into the basic nutrient solution obtained in S1 and cooled to 56℃. Add 10g of chitosan and 15g of plum polysaccharide, shake well to dissolve, pour into plates, and obtain selective culture medium.

[0063] Experiment 1: Observation of bacterial growth

[0064] The fresh tissue samples diagnosed as Haemophilus parasuis were obtained from a large-scale pig farm, totaling 30 samples. The 30 samples were aseptically inoculated into the culture media prepared in Example 1 and Comparative Examples 1-3, and then placed in a constant temperature incubator at 37°C for incubation.

[0065] During the culture process, the growth of bacteria in the culture medium was observed at 6h, 12h, 24h, 36h and 48h, respectively, and the isolated strains were sent for sequencing. The sequencing results showed that the strains isolated by the selective culture medium of the present invention in Example 1 were all HPS strains; the sequencing results are shown in SEQ ID NO:1.

[0066] Meanwhile, the number of HPS strains and the number of miscellaneous bacteria on the culture media prepared in Example 1 and Comparative Examples 1-3 were statistically analyzed, and the results are shown in Table 1.

[0067] Table 1. Statistics on the growth status of HPS strains and miscellaneous bacteria.

[0068]

[0069]

[0070] As can be seen from Table 1, no contaminating bacteria grew in Example 1 using the selective culture medium of the present invention, while contaminating bacteria appeared in Comparative Examples 1-3. Moreover, the number of HPS strains isolated in Comparative Examples 1-3 was less than that in Example 1. It can be seen that chitosan, plum polysaccharide and calcium propionate can inhibit the growth of contaminating bacteria. At the same time, the addition of chitosan, plum polysaccharide and calcium propionate is beneficial to the growth of HPS strains.

[0071] Experimental Example 2: Separation Rate Test

[0072] The fresh tissue samples diagnosed as Haemophilus parasuis were obtained from a large-scale pig farm, totaling 30 samples. The experiment was divided into two groups: a control group and an experimental group. The control group used TSA+NAD medium, which is commonly used for the isolation of HPS strains, while the experimental group used the selective medium prepared in Example 1 of this invention.

[0073] Thirty samples were aseptically inoculated into the culture media of the experimental and control groups, respectively, and then incubated at 37°C. During incubation, bacterial growth was observed at 6h, 12h, 24h, 36h, and 48h. The number of HPS strains isolated from the control and experimental groups was counted, and the isolation rate was calculated. The HPS strains isolated from the two groups in different culture media are shown in Table 2. The isolation rate was calculated as follows: Isolation rate = Number of isolates / Total number of isolated samples.

[0074] Table 2. HPS strains isolated from different isolation media

[0075]

[0076]

[0077] As shown in Table 2, the target strain was isolated in the experimental group within 6 hours, while the target strain was isolated in the control group within 24 hours at the earliest. The experimental group isolated 8 HPS strains, while the control group isolated only 2 strains. Compared with the control group, the isolation rate of HPS strains in the experimental group was increased by 20%, and the growth time of HPS strains was shortened by 4 times.

[0078] Test Example 3: Stability Test

[0079] The selective culture medium of Example 1 of the present invention was used to conduct isolation stability tests on the above 30 samples. The test method was to use the selective culture medium of Example 1 of the present invention, perform streaking inoculation under aseptic conditions, set up 3 replicate groups, and then place them in a constant temperature incubator at 37°C for incubation. The number of HPS isolated strains in each replicate group was observed at 6h, 12h, 24h, 36h and 48h respectively.

[0080] The results showed that the number of HPS strains isolated from the three replicate groups was similar, at 8, 8, and 7 strains respectively, indicating that the selective medium of the present invention has good stability in selecting and isolating HPS strains.

[0081] Experimental Example 4: Biochemical Experiment

[0082] The eight HPS strains isolated from the experimental group in Example 2 were subjected to biochemical tests according to conventional biochemical test methods. The biochemical items and test results are shown in Table 3.

[0083] Table 3. Biochemical test results of 8 HPS strains

[0084]

[0085]

[0086] As shown in Table 3, the biochemical test results of the 8 HPS strains isolated in Experimental Group 2 were positive for xylose, mannitol, raffinose, arabinose, maltose, fructose, and glucose, and negative for sucrose. This result is consistent with the biochemical test results of the actual Haemophilus parasuis strains, indicating that the selective culture medium of the present invention has no effect on the biochemical characteristics of the HPS strains isolated from it.

[0087] Test Example 5: Pathogenicity Test

[0088] The eight HPS strains isolated from the experimental group in Example 2 were purified and cultured. The purification method was to inoculate suspected single colonies onto the selective medium of this invention and culture them for 24 hours until the colonies were uniform in morphology, size, and color. Then, for each strain, 10 eight-week-old Balb / c mice were selected and divided into a control group and an injection group. The control group was injected with blank medium, while the injection group was injected intraperitoneally with HPS bacterial solution (concentration of 1.8 × 10⁻⁶). 8 The mice were observed for 10 consecutive days (cfu / mL) and mortality was recorded.

[0089] The results showed that the HPS bacterial suspension isolated using the selective culture medium of the present invention caused the mice to die, with a mortality rate of 100%, while the mortality rate of the control group mice was 0; indicating that the selective culture medium of the present invention has no effect on the pathogenicity of the HPS strains isolated from it.

[0090] SEQ ID NO:1: Nucleotide sequence obtained from sequencing of HPS strain.

[0091] The preferred embodiments of this patent have been described in detail above. However, this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, other variations or modifications can be made. It is neither necessary nor possible to exhaustively list all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the protection scope of the claims of this invention.

Claims

1. A selective culture medium for Haemophilus parasuis, characterized in that, The formula of the selective medium is as follows: Pig lung 220-280 g; Glucose 1-3 g; Agar powder 13-17 g; Proteose peptone 8-12 g; Sodium chloride 3-7 g; Chitosan 8-12 g; Calcium propionate 3-7 g; Prunus mume polysaccharide 13-17 g; Sodium hydroxide is used to adjust the pH value to 7.8±0.1; Deionized water is added to 1 L; The treatment method of the pig lung in the selective medium is as follows: the pig lung of the formula amount is added with 200 mL of deionized water and then placed in a wall-breaking machine for wall-breaking treatment, and then repeatedly frozen and thawed at-40~-30℃ for 2-3 times and filtered through a sterile filter.

2. The selective medium according to claim 1, characterized in that, The formula of the selective medium is as follows: Pig lung 250 g; Glucose 2 g; Agar powder 15 g; Proteose peptone 10 g; Sodium chloride 5 g; Chitosan 10 g; Calcium propionate 5 g; Prunus mume polysaccharide 15 g; Sodium hydroxide is used to adjust the pH value to 7.8; Deionized water is added to 1 L.

3. A method for the preparation of a selective culture medium for Haemophilus parasuis according to claim 1 or 2, characterized in that, The method comprises the following steps: S1, preparing a basic nutrient solution: the formula amount of glucose, agar powder, proteose peptone and sodium chloride is dissolved in 700 mL of deionized water, and then the pH value is adjusted to 7.8±0.1 by using sodium hydroxide after being fully dissolved, and then the volume is adjusted to 800 mL with deionized water, and then high-pressure sterilization is performed to obtain the basic nutrient solution; S2, preparing a selective medium: the formula amount of pig lung is added with 200 mL of deionized water and then placed in a wall-breaking machine for wall-breaking treatment, and then repeatedly frozen and thawed at-40~-30℃ for 2-3 times and filtered through a sterile filter into the basic nutrient solution cooled to 55-58℃ obtained in S1, and then the formula amount of chitosan, calcium propionate and prunus mume polysaccharide is added and shaken to dissolve, and then poured into a flat plate to obtain the selective medium.

4. The production method according to claim 3, characterized by, In S1, the high-pressure sterilization is high-pressure sterilization at 121℃ for 20-30 min.

5. The preparation method according to claim 3, characterized in that, In S2, the sterile filter is a 0.22 μm microporous filter membrane.

6. Application of the selective medium for Haemophilus parasuis in isolating Haemophilus parasuis according to claim 1 or 2.

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