Vibrio parahaemolyticus capable of causing vibrio vitreovibriosis of prawns and application thereof

Abstract

The invention belongs to the technical field of pathogenic microorganism screening, and particularly relates to vibrio parahaemolyticus capable of causing prawn vibrio vitreovibriosis and application of the vibrio parahaemolyticus, and the pathogenic bacteria can be used for preparing inactivated vaccines or used as the pathogenic bacteria to screen medicine components with the effect of preventing and treating the prawn vibrio vitreovibriosis or a culture method. The preservation number of the vibrio parahaemolyticus provided by the invention is CGMCC (China General Microbiological Culture Collection Center) The pathogenicity of the vibrio parahaemolyticus with high pathogenicity and strong lethality is about 1000 times stronger than that of the vibrio parahaemolyticus which is collected by the world animal hygiene organization and causes acute hepatopancreatic necrosis, and the strain is popular in main prawn culture areas and causes serious harm to the prawn fry culture industry in China. The vibrio parahaemolyticus provided by the invention can be used as a positive control for preparing a diagnosis product for the prawn vibrio vitreovirus disease; or preparing an immune vaccine after inactivation.

Description

Technical Field

[0001] This invention belongs to the field of pathogenic microorganism screening and control technology, specifically relating to a Vibrio parahaemolyticus that causes vibrio infection in shrimp larvae and its application. Background Technology

[0002] Shrimp farming has always accounted for a large proportion of my country's aquaculture industry and is an important component of it. With the increasing scale and intensification of shrimp farming and the booming shrimp import and export trade, shrimp farming has brought people abundant protein products. However, at the same time, the continuous deterioration of the farming environment and the frequent occurrence of diseases have also brought unprecedented challenges to the shrimp farming industry.

[0003] In early 2020, large-scale outbreaks of empty intestines and stomachs, transparent body color, and acute mortality occurred in farmed Litopenaeus vannamei larvae in Guangdong and Fujian provinces of my country. Local farmers termed this disease "translucent post-larva disease" (TPD). Because it is caused by Vibrio infection and primarily affects larvae, it is also known as "translucent post-larva vibriosis" (TPV). From April to May 2020, TPV rapidly spread to major shrimp farming areas in northern my country, showing an explosive trend. This disease led to persistently high larvae discharge rates at shrimp nursery farms in Guangdong, Guangxi, and Fujian (Chen Honglin, 2020), with some areas experiencing over 90% of nursery farms affected. The TPV epidemic resulted in a shortage of shrimp larvae for farming in parts of Jiangsu, Shandong, and Hebei provinces.

[0004] In typical TPV cases, affected juvenile shrimp exhibit empty intestines and stomachs, a lighter-colored hepatopancreas, and a translucent to semi-transparent body color from the cephalothorax to the abdominal segments. TPV typically occurs in juvenile shrimp at the PL5-PL7 stage, with rapid onset; from the observation of some individuals showing symptoms to a cumulative mortality rate exceeding 90%, it often takes no more than 3 days. The causative agent of TPV is *Vibrio hygroscopicis* (…). Vibrio spp.causing TPV, V TPV or V TPD It is also pathogenic to adult shrimp, but slightly less so. If diseased shrimp are infected with... V TPV Infections with multiple pathogens, including those mentioned above, can still result in a rapid onset of illness and a high mortality rate.

[0005] Epidemiological surveys show that the disease led to the closure of more than 80% of shrimp hatcheries and seedling nurseries in major coastal shrimp farming areas across the country in 2020; in 2021, the prevalence of the disease in major coastal and inland shrimp farming areas in my country was higher than 20%. Summary of the Invention

[0006] The purpose of this invention is to provide a Vibrio parahaemolyticus that causes vibrio vulnificus disease in shrimp and its application. This pathogen can be used to prepare inactivated vaccines or as a pathogen to screen drug components, probiotics, probiotic fermentation products or aquaculture methods that have the effect of preventing and treating vibrio vulnificus disease in shrimp.

[0007] This invention first provides a Vibrio parahaemolyticus bacterium that causes vibrio infection in shrimp larvae ( Vibrio parahaemolyticus Vp-JS20200430001 was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 33883.

[0008] The present invention also provides an application of the aforementioned Vibrio parahaemolyticus as a pathogen in the construction of a vibrio disease model in shrimp glass seedlings.

[0009] This invention also provides a method for constructing a vibrio disease model in shrimp glass seedlings, which involves infecting shrimp with the aforementioned Vibrio parahaemolyticus and then constructing the model.

[0010] The present invention also provides another use of the aforementioned Vibrio parahaemolyticus as a standard or positive control in molecular diagnostic products (such as PCR, LAMP) or immunological detection products (such as ELISA, colloidal gold test strips) for pathogenic bacteria of shrimp glass seedlings.

[0011] The present invention also provides a third use of the aforementioned Vibrio parahaemolyticus, namely, its use as an antigen in the preparation of inactivated vaccines.

[0012] The present invention also provides a method for screening drugs, probiotics, probiotic fermentation products or breeding methods for the prevention and treatment of vibrio parahaemolyticus disease, wherein the method uses the screened Vibrio parahaemolyticus as the pathogen for screening.

[0013] The *Vibrio parahaemolyticus* provided by this invention has a high pathogenicity and strong lethality, and its pathogenicity is higher than that of *Vibrio parahaemolyticus* causing acute hepatopancreatic necrosis listed by the World Organisation for Animal Health (WOAH). Vp AHPND The strain is about 1,000 times stronger than the shrimp strain and is prevalent in major shrimp farming areas, posing a serious threat to my country's shrimp seedling and breeding industry.

[0014] The Vibrio parahaemolyticus provided by this invention can be used as a positive control to prepare diagnostic products for vibrio infection in shrimp vacciniae; or it can be inactivated to prepare an immunization vaccine. Attached Figure Description

[0015] Figure 1 This is a diagram showing the symptoms of "glass shrimp larvae" disease. Black arrows indicate healthy (or early-stage) shrimp larvae, while white arrows indicate severely infected shrimp larvae. Figure 1 a and Figure 1 The scale in the middle b is 10 mm. Figure 1 a and Figure 1 The scale bar in the enlarged image of b is 2 mm.

[0016] Figure 2 The figures show the morphological characteristics of the strain. In the figures, a1 shows the growth status of the strain after culturing in TSA medium at 28℃ for 24 h, and a2 shows the growth status of the strain after culturing in TCBS medium at 28℃ for 24 h. Figure 3 strain Vp TPV Image showing the results of an infection experiment.

[0017] Figure 4 A graph comparing the pathogenicity of two strains of Vibrio parahaemolyticus. Detailed Implementation

[0018] This invention, through systematic epidemiological investigation, histopathological analysis of diseased samples, pathogen isolation and identification, and Koch's postulate verification experiments, screened and obtained a novel highly virulent Vibrio parahaemolyticus strain that causes "glass seedling" disease in Litopenaeus vannamei. This pathogen was named strain Vp-JS20200430001. Vp TPV This study lays the foundation for further research on its pathogenic mechanism, the occurrence pattern of "glass seedling" disease, and effective prevention and control measures, so as to prevent it from causing wider damage to the shrimp farming industry.

[0019] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0020] Example 1: Isolation and purification of pathogens In April 2020, a disease called "glass larvae" broke out in Litopenaeus vannamei at a shrimp farm in Ganyu, Jiangsu Province. Samples were collected from the shrimp, and dominant bacteria were isolated and purified from the hepatopancreas of diseased and dying shrimp. The bacteria were numbered Vp-JS20200430001. Vp TPV The specific steps are as follows: 1) Surface disinfection Place the diseased and dying shrimp in a clean, disposable sterile culture dish and spray it gently with 75% ethanol to disinfect it and eliminate contamination from airborne and shrimp surface bacteria. Then wash the shrimp 2-3 times with sterile 1×PBS to remove the attached ethanol. 2) Isolation and purification of bacterial strains The shrimp were quickly dissected, and the hepatopancreas tissue was ground with a sterile grinding rod. The contents were then streaked onto TSA medium. The culture medium with the bacteria streaked was incubated at 28°C for 24 hours. Single colonies of the dominant bacteria were selected and recultured on TSA medium to purify and propagate the strain. 3) Observation of colony morphology and growth status Colonies on TSA agar are milky white with neat edges and a raised surface. After 24 hours of incubation at 28°C, they are approximately 1.79 mm in diameter. Meanwhile, colonies on TCBS medium are pale yellow after 24 hours of incubation at 28°C, and are approximately 2.04 mm in diameter.

[0021] Example 2: Identification of the strain Bacterial DNA was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using primers for bacterial identification of the 16S rRNA gene (primer sequences are shown in Table 1). The DNA was then electrophoresed, recovered, cloned, and sequenced. The sequencing results were analyzed. The PCR amplification products were sent to a sequencing company for sequencing. After sequence alignment with NCBI, the 16S rRNA gene sequence was found to be consistent with that of Vibrio parahaemolyticus (Vibrio parahaemolyticus). Vibrio parahaemolyticus It has the highest homology with ) and is named Vibrio parahaemolyticus ( Vibrio parahaemolyticus Vp-JS20200430001 was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33883.

[0022] Further bacterial classification was then determined through multiple site sequence analysis (MLSA). The ligation sequence of the rpoD-rctB-toxR gene (primer sequences are shown in Table 1) also clearly identified strain Vp-JS20200430001 as being most closely related to Vibrio parahaemolyticus.

[0023] The strain (Vp-JS20200430001) was then tested for the thermolabile hemolysin gene (tlh) and the pirAvp and pirBvp genes (primer sequences shown in Table 1) associated with acute hepatopancreatic necrosis disease (AHPND) in shrimp. The results showed that this strain carried the tlh gene, which showed 100% homology with Vibrio parahaemolyticus (GenBank accession number: MT646121). The pirABVp gene test was negative, indicating that this strain did not carry the pirAvp and pirBvp virulence protein genes that can cause AHPND in shrimp.

[0024] Table 1: Primer sequence information for each gene

[0025] * The pirAvp, pirBvp, and pirABvp primer sets listed in the table are recommended by the World Organisation for Animal Health (WOAH) for the identification and detection of Vibrio parahaemolyticus, an important pathogen in shrimp that causes acute hepatopancreatic necrosis. Vp AHPND ) Specific primers for the strain.

[0026] The above results confirm the effectiveness of the strain (Vp-JS20200430001) screened in this invention. Vp TPV Unlike previously reported cases of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis in shrimp ( ) Vp AHPND ), which is a novel, highly virulent Vibrio parahaemolyticus.

[0027] Example 3: Study on the pathogenicity of the strain According to Koch's postulates, the above-mentioned Vibrio parahaemolyticus isolates were used Vp TPV A re-infection experiment was conducted on PL7 shrimp larvae, and the results were as follows: Figure 2 The results showed that the 24-hour median lethal concentration (LC50) of this pathogen in shrimp larvae (PL7) was 1.07 × 10⁻⁶. 6 CFU / mL. Its concentration is 1.83 × 10⁻⁶. 7 CFU / mL, 1.83×10 6 CFU / mL and 1.83×10 5 The times to reach 100% cumulative mortality for shrimp larvae (PL7) at CFU / mL immersion doses were 28, 40, and 56 hours, respectively, and 1.83 × 10⁻⁶. 4Shrimp larvae (PL7) immersed at CFU / mL also achieved a cumulative mortality rate of 75% after 48 hours. The median lethal time (LT50) for the four doses of shrimp larvae (PL7) were 38.6, 28.9, 24.6, and 15.7 hours, respectively. No mortality was observed in the control group during the experiment. The identification results of the 16S rRNA and toxR genes of the dominant strain isolated from the dying shrimp in the infection experiment also showed... Vp TPV The strains were completely identical. Meanwhile, most individual shrimp in the infected group exhibited typical clinical symptoms of TPV, including decreased vitality, empty intestines and stomachs, and pale hepatopancreas. Figure 3 ).

[0028] Comparison of the pathogenicity of two strains of Vibrio parahaemolyticus ( Figure 4 Vibrio parahaemolyticus isolates Vp TPV The cumulative mortality rate of infected shrimp was 100%, not... Vp TPV The cumulative mortality rate of Vibrio parahaemolyticus was 45% over the same time period. Within the same time period, Vibrio parahaemolyticus isolates... Vp TPV The cumulative mortality rate in the infected group was higher than that in the non-infected group. Vp TPV Vibrio parahaemolyticus, indicating Vibrio parahaemolyticus isolate Vp TPV It has a high mortality rate.

[0029] The above results indicate that the Vibrio parahaemolyticus isolates screened in this invention are more pathogenic to shrimp larvae (PL7), and the mortality process of infected shrimp is consistent with the natural disease process of "glass larvae".

[0030] Vp TPV Under strictly controlled laboratory conditions, an effective method can be used to screen for potential treatments for vibriosis. The procedure is as follows: First, a variety of candidate drugs were prepared and made into drug sensitivity test discs with fixed concentrations. Then, different types of drug sensitivity test discs were placed in sections on agar plates uniformly coated with Vibrio hygroscopicus bacterial suspension, and each drug was labeled for subsequent recording and analysis.

[0031] Next, the plates were placed upside down in a 28°C incubator to ensure the stability and consistency of the experimental conditions. After incubation, the diameter of the inhibition zone formed around each drug sensitivity test disc was observed and measured, and the data were recorded in detail. Generally, a larger inhibition zone diameter indicates a stronger antibacterial effect of the drug at the same concentration; at the same time, drugs that produce significant inhibition zones at lower concentrations are considered to have higher antibacterial potential under laboratory conditions.

[0032] Finally, to further verify the reliability of the laboratory results, aquaculture experiments can be conducted to evaluate the effectiveness of the candidate drug in combating host resistance to Vibrio hygroscopic infection in practical applications. This step not only confirms the drug's effectiveness but also provides important reference data for its subsequent development.

[0033] Through the above systematic process, the present invention selects... Vp TPV The strain provides a scientific and reliable basis for screening and evaluating diagnostic agents for vibriosis of glassy seedlings, as well as for the development and testing of therapeutic drugs, which helps to improve the TPD control technology capabilities.

Claims

1. A Vibrio parahaemolyticus (Vp) that causes vibrioiosis in shrimp glass shrimp larvae, characterized in that, Vibrio parahaemolyticus The preservation number of the Vibrio parahaemolyticus is CGMCC No. 33883. ​ 2. The Vibrio parahaemolyticus of claim 1 is used as a pathogen to construct a model of Vibrio penaeicida in glass shrimp.

3. A method of constructing a model of Vibrio penaeicida disease in shrimp glass shrimp, characterized by, The method is to use the Vibrio parahaemolyticus of claim 1 to infect the model of shrimp.

4. The Vibrio parahaemolyticus of claim 1 is used as a standard or positive control in the preparation of a detection product of a pathogen of glass shrimp.

5. The use according to claim 4, wherein the compound is ###0002### The detection product is a molecular diagnostic product or an immunological detection product.

6. The use according to claim 5, wherein the compound is ###0002### The molecular diagnostic product is a PCR or LAMP detection kit; the immunological detection product is an ELISA detection kit or a colloidal gold test strip.

7. A detection product for detecting pathogenic bacteria in shrimp larvae, characterized in that, The detection product uses the Vibrio parahaemolyticus of claim 1 as a standard or positive control.

8. The Vibrio parahaemolyticus of claim 1 is used as an antigen to prepare an inactivated vaccine.

9. An inactivated vaccine, characterized in that, The antigen of the inactivated vaccine is the inactivated Vibrio parahaemolyticus of claim 1.

10. A method for screening a drug or a farming method for preventing treatment of v. philippinavibrio disease, characterized by, The method is to use the Vibrio parahaemolyticus of claim 1 as a pathogen for screening.

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