Hafnia alvei and application

Abstract

The invention discloses a Chinese grass tortoise source Hafnia alvei strain and application thereof, and relates to the technical field of microorganism application. The virulence gene combination is derived from liver tissues of diseased Chinese grass turtles, the whole genome of the virulence gene combination comprises a phenol glycolipid system gene cluster, a multiple iron acquisition system and type VI secretion system gene cluster, an adhesin array and toxin system gene cluster and an alginate biofilm and multiple secretion system gene cluster, the Hafnia alvei strain is named as Hafnia alvei JX2025 strain, and is preserved in the China Center for Type Culture Collection (Wuhan) on November 17, 2025, and the preservation number is CCTCC M 20252572. The JX2025 strain can be used as a pathogen for preparing a hafnia alvei infected animal model, and the unique virulence gene spectrum of the JX2025 strain provides important molecular basis and data support for pathogenic mechanism research of related diseases, vaccine target screening and development of novel antibacterial drugs.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a strain of Haffniella vesicatoria and its applications. Background Technology

[0002] Haffniella vesicatoria ( Hafnia alvei *Havoc. vesicularis* is a Gram-negative, facultative anaerobic, opportunistic pathogen widely distributed in water, soil, and the intestines of healthy animals (including mammals, birds, and aquatic animals), usually existing in a symbiotic or asymptomatic carrier state. However, under conditions of weakened host immunity or environmental stress, this bacterium can transform into an opportunistic pathogen, causing local or systemic infections. In recent years, with the development of intensive aquaculture and increasing environmental pressure, the pathogenicity of *Havoc. vesicularis* in aquaculture has become increasingly prominent. Serious diseases such as hemorrhagic septicemia, necrotizing enteritis, and liquefactive necrosis of muscle tissue have been reported in fish (such as salmon, trout, carp, and tilapia) and crustaceans (such as shrimp and red swamp crayfish), leading to significant economic losses. Simultaneously, reports of *Havoc. vesicularis* infection in reptiles (such as turtles and lizards) are also on the rise, often manifesting as acute death, hepatosplenomegaly, and multi-organ necrosis, posing a potential threat to the growing reptile breeding and conservation industry.

[0003] Although the pathogenicity of *Havniella vesicatoria* has attracted attention, current research suffers from significant limitations and imbalances: First, existing research is highly focused on mammals (especially human clinical isolates) and a few economically important fish strains, with almost no attention paid to reptiles, particularly turtles and tortoises of significant ecological and economic value. Second, our understanding of the pathogenicity of *Havniella vesicatoria* remains largely at the phenotypic level, such as routine biochemical identification, basic drug susceptibility testing, and histopathological observations. A systematic analysis of the core molecular basis determining its host specificity, tissue tropism, and pathogenicity—namely, the genomic characteristics of virulence factors—is lacking. Currently available publicly available databases lack whole-genome data on turtle and tortoise-derived strains and comprehensive virulence factor analyses based on genomics, leaving our understanding of its pathogenic mechanisms in a "black box" stage. Third, due to the lack of standardized pathogenic strains from turtles and tortoises, along with a corresponding database of biological characteristics (including precise morphological and physicochemical features, standardized pathogenicity experimental models, and drug susceptibility benchmark data based on locally prevalent strains), this field faces the dilemma of lacking diagnostic standards, treatment guidelines, and control targets. In production, it is difficult to quickly distinguish between pathogenic and environmental strains; antibiotic selection is often indiscriminate; and targeted vaccine or antiviral strategies cannot be developed.

[0004] Therefore, isolating and preserving a standard strain of *Haferonepheta harfraseri* from turtles and tortoises with a clear origin and defined pathogenicity, elucidating its biological characteristics (from morphology, physiology, biochemistry to histopathology), and systematically analyzing its virulence determinants at the genomic level, establishing its association with clinicopathological phenotypes, is of irreplaceable value for a deeper understanding of its pathogenic mechanism and the development of precise prevention and control technologies. This invention aims to fill the technological gap in this field and provide core strain resources and key scientific data for solving prevention and control challenges in practical production.

[0005] Currently, there are no reports on the pathogenicity of Haffniella vesicatoria in Chinese pond turtles, and farmers lack sufficient understanding of this pathogen, leading to serious misuse of drugs in production. Summary of the Invention

[0006] This invention aims to provide a novel strain of Haffniella auriculata, JX2025, isolated from a diseased Chinese pond turtle, comprehensively describing its biological characteristics, pathogenicity, and drug susceptibility spectrum, and expanding its application in disease model construction, drug screening, and vaccine development. For the first time, it elucidates the unique virulence gene spectrum carried by the bacterium at the genomic level, providing molecular targets for the study of the pathogenic mechanism and targeted prevention and control of this bacterium.

[0007] The objective of this invention is achieved through the following technical solutions: A strain of Haffniella honeycomb ( Hafnia alvei The strain, derived from the liver tissue of a diseased Chinese pond turtle, contains a virulence gene combination in its whole genome. This virulence gene combination includes: phenolic glycolipid (PDIM) system gene cluster, multiple iron acquisition system and type VI secretion system (T6SS) gene cluster, adhesin array and toxin system gene cluster, and alginate biofilm and multiple secretion system gene cluster. It was named *Haffniella vesicae* strain JX2025 and was deposited at the China Center for Type Culture Collection (Wuhan) on November 17, 2025, with accession number CCTCC M 20252572.

[0008] Preferably, the phenolic glycolipid (PDIM) system gene cluster includes: ppsB , fadD22 , Rv2952 The multiple iron acquisition system and type VI secretion system (T6SS) gene cluster includes: ent / ybt and impA / vgrG / hcp The adhesin array and toxin system gene cluster includes: pap , fim , csg , hlyB The alginate biofilm and multiple secretion system gene cluster includes: algZ , algR , algU .

[0009] This invention also provides a method for isolating and culturing Haffniella vesicatoria, comprising the following steps: S1. Liver tissue from diseased Chinese pond turtles was collected, homogenized with sterile physiological saline, and then streaked onto LB solid medium. S2. Incubate at 28.5℃ for 24 hours, then pick single colonies for purification culture; S3. Bacterial species were identified by colony morphology observation, Gram staining, biochemical identification, and 16S rRNA gene sequence analysis.

[0010] The present invention also provides an application of the above-mentioned Haffniella vesicae, wherein the Haffniella vesicae JX2025 strain is used as a pathogen to prepare an animal model of Haffniella vesicae infection.

[0011] Preferably, the animal used in the animal model is the Chinese pond turtle.

[0012] The present invention also provides an application of the above-mentioned Haffniella vesicae, wherein the Haffniella vesicae JX2025 strain is used to screen or evaluate antimicrobial drugs or vaccines against Haffniella vesicae infection.

[0013] Preferred antibacterial drugs include doxycycline, florfenicol, trimethoprim, minocycline, tetracycline, sulfamethoxine, norfloxacin, and ciprofloxacin.

[0014] The present invention also provides an application of the above-mentioned Haffniella vesicae, based on the virulence gene combination of Haffniella vesicae JX2025 strain, to develop inhibitors, monoclonal antibodies or genetically engineered attenuated vaccines targeting key virulence factors such as adhesins and secretion systems.

[0015] The present invention also provides an application of the above-mentioned Haffniella vesicae, based on the virulence gene combination of Haffniella vesicae JX2025 strain, to develop a specific molecular diagnostic method to achieve rapid identification of the pathogenic strain or highly virulent clone.

[0016] Preferably, molecular diagnostic methods include PCR and CRISPR-Cas detection.

[0017] This invention isolated and purified a dominant bacterial strain from the liver of a diseased Chinese pond turtle exhibiting typical symptoms (such as lethargy, loss of appetite, limb weakness, and hepatosplenomegaly), named *Haffniella vesicae* strain JX2025. Colony morphology showed irregularly shaped, small, round, grayish-white, smooth, opaque, glossy surface with a slightly raised center and a soft texture. Gram staining revealed it to be a Gram-negative short rod. Scanning electron microscopy showed the bacteria to be straight or slightly curved rods with blunt ends, each approximately 1.8-2.2 μm in length and 0.5-0.6 μm in width. The most prominent feature was a dense layer of short, straight pili, evenly distributed and approximately 0.2 μm in length, consistent with the typical morphology of type I pili. The bacterial surface was relatively smooth, and no obvious capsule structure was observed. No flagella were observed in the current field of view. The bacteria were dispersed and morphologically intact. Molecular biological identification: Bacterial genomic DNA was extracted, and the 16S rRNA gene sequence was amplified using universal primers 27F / 1492R. The sequencing results were submitted to the NCBI database to obtain a BioProject accession number (e.g., PRJNA1405306). BLAST alignment and phylogenetic tree construction confirmed that this strain clustered with *Haffniella vesicae*, with homology greater than 99%. Physiological and biochemical identification: Microbiological identification tubes showed that this strain could ferment glucose (producing acid and gas) and xylose, but not sorbitol, raffinose, or calendula alcohol; methyl red (MR) and Volta-Pyr (VP) tests were both positive; Simon's citrate utilization was positive; lysine decarboxylase and ornithine decarboxylase were positive; phenylalanine deaminase, urease, and hydrogen sulfide tests were all negative; it exhibited motility. It was identified as *Haffniella vesicae*. Hafnia alvei This strain was deposited at the China Center for Type Culture Collection (Wuhan) on November 17, 2025, with accession number CCTCC M 20252572, and is classified as follows: Hafnia alvei strain .

[0018] Compared with the prior art, the present invention has the following advantages: Innovative strain resources: For the first time, a strain of *Havococcus vesicatoria* JX2025 derived from the Chinese pond turtle was provided, with clear pathogenicity and complete biological characteristics data, enriching the resource bank of this strain.

[0019] Pathogenicity confirmed: Experiments have confirmed the pathogenicity of strain JX2025 to Chinese soft-shelled turtle and the characteristic histopathological changes it causes, providing a reference for the pathological diagnosis of related diseases.

[0020] Its application value is outstanding: the provided drug susceptibility data can directly guide clinical treatment. This strain can be used as a standard challenge strain to construct stable disease models, serving the research of pathogenic mechanisms, screening of novel antimicrobial drugs, and evaluation of vaccine efficacy.

[0021] Diagnostic basis: The virulence genes unique to the Hafnium vesicantii strain JX2025 provide target sequences for the development of specific molecular diagnostic methods (such as PCR and fluorescent probe detection) against this bacterium or strain.

[0022] The molecular mechanism is clear: for the first time, the potential virulence factors of the *Havococcus harzianum* genome derived from turtles have been provided. This not only explains its pathogenicity at the molecular level, but also provides a clear target for the development of inhibitors, monoclonal antibodies, or genetically engineered attenuated vaccines against key virulence factors (such as adhesins and the secretion system).

[0023] High diagnostic specificity: Based on the unique virulence gene sequence of strain JX2025, more specific molecular diagnostic methods (such as PCR and CRISPR-Cas detection) can be developed to achieve rapid identification of this pathogenic strain or highly virulent clone, distinguishing it from non-pathogenic Haffniella vesicae in the environment. Attached Figure Description

[0024] Figure 1 The colony morphology of the *Haffniella vesicae* strain JX2025 proposed in this invention on LB medium; Figure 2 Gram staining morphology of the Haffniella vesicatoria JX2025 strain proposed in this invention; Figure 3 The scanning electron microscope morphology of the Haffniella avesicae JX2025 strain proposed in this invention; Figure 4 The phylogenetic tree diagram of the *Haffniella vesicae* strain JX2025 proposed in this invention, constructed based on the 16S rRNA gene sequence; Figure 5 The following are histopathological images of the liver tissue of the pathogenic Chinese soft-shelled turtle caused by the *Havococcus pyogenes* strain JX2025 proposed in this invention; where A is the histopathological image of the liver tissue of the control group and B is the histopathological image of the liver tissue of the infected group. Figure 6 The present invention presents histopathological observations of the spleen tissue of the pathogenic Chinese soft-shelled turtle caused by the Hafnium vesicularis strain JX2025. Among them, A is the histopathological observation of the spleen tissue of the control group, and B is the histopathological observation of the spleen tissue of the infected group. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0026] Example 1 A strain of Haffniella honeycomb ( Hafnia alveiThe strain, derived from the liver tissue of a diseased Chinese pond turtle, contains a virulence gene combination in its whole genome. This virulence gene combination includes: phenolic glycolipid (PDIM) system gene cluster, multiple iron acquisition system and type VI secretion system (T6SS) gene cluster, adhesin array and toxin system gene cluster, and alginate biofilm and multiple secretion system gene cluster. It was named *Haffniella vesicae* strain JX2025 and was deposited at the China Center for Type Culture Collection (Wuhan) on November 17, 2025, with accession number CCTCC M 20252572.

[0027] Example 2 The isolation and culture method of a strain of *Havococcus faecium* JX2025 from *Turtleidae* species includes the following steps: S1: Preparation of culture media: LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, dissolved in 1000mL ultrapure water, sterilized at 121℃ for 15min, and stored at 4℃ after cooling to room temperature; LB solid medium: 10g peptone, 5g yeast extract, 10g NaCl, 20g agar powder, dissolved in 1000mL ultrapure water, sterilized at 121℃ for 15min, the culture medium was cooled to 50℃ and poured into disposable bacterial culture dishes, and stored at 4℃ after solidification. S2: Isolation and culture of strains: Liver tissue (2cm tissue block) of diseased and dying Chinese pond turtles in the breeding farm was aseptically collected into a sterile centrifuge tube containing 5ml of physiological saline, and ground using a disposable grinding device to prepare tissue homogenate. The homogenate was then streaked onto LB solid medium with an inoculation loop and incubated at 28.5℃ for 24h to grow single colonies.

[0028] S3: Select dominant single colonies (colon diameter of 2.5mm-4.0mm on LB agar plates, irregular small round shapes, grayish-white, smooth and opaque surface, glossy, slightly raised in the middle, and relatively soft in texture) and inoculate them onto LB solid medium for purification culture (incubate at 28.5℃ for 24h, repeat the operation until the colony morphology is consistent). Select single colonies and inoculate them into LB liquid medium, and incubate them in a shaker at 30℃ and 200rpm for 24h to obtain a bacterial suspension.

[0029] Example 3 Identification of Haffniella vesicatoria JX2025 strain 1) Morphological identification: On LB agar plates, colonies are 2.5mm-4.0mm in diameter, with irregularly shaped small round edges, grayish-white in color, smooth and opaque with a glossy surface, slightly raised in the center, and relatively soft in texture. Figure 1 As shown.

[0030] 2) Gram staining: shows as a Gram-negative short bacillus (see attached image). Figure 2 ).

[0031] 3) Scanning electron microscopy revealed that the bacteria were straight or slightly curved rods with blunt, rounded ends. Individual bacteria were approximately 1.8–2.2 μm long and 0.5–0.6 μm wide. The most prominent feature was the dense, short, straight pili covering the surface. These pili were evenly distributed and approximately 0.2 μm long, consistent with the typical morphology of type I pili. The bacterial surface was relatively smooth, and no obvious capsule structure was observed. No flagella were observed in the current field of view. The bacteria were dispersed and morphologically intact. (See attached image) Figure 3 ).

[0032] 4) Molecular biological identification: Bacterial genomic DNA was extracted, and the 16S rRNA gene sequence was amplified using universal primers 27F / 1492R. The sequencing results were submitted to the NCBI database to obtain the BioProject accession number (PRJNA1405306). A phylogenetic tree was constructed using BLAST alignment (see attached). Figure 4 The strain was found to cluster with the Haffni apigenis strain, showing a homology greater than 99%.

[0033] 5) Physiological and biochemical identification: The results of the bacterial biochemical micro-identification tubes (as shown in Table 1) show that this strain can ferment glucose (producing acid and gas) and xylose, but cannot ferment sorbitol, raffinose, and calendula alcohol; methyl red (MR) and Volta-Pyr (VP) tests are both positive; Simon's citrate utilization is positive; lysine decarboxylase and ornithine decarboxylase are positive; phenylalanine deaminase, urease, and hydrogen sulfide tests are all negative; and it is motile.

[0034] Table 1. Results of Physiological and Biochemical Identification

[0035] 6) Strain preservation: The identified and purified strain was named *Havniella vesicae* JX2025 and was deposited at the China Center for Type Culture Collection (Wuhan) on November 17, 2025, with accession number: CCTCC NO: M20252572.

[0036] Example 4 Pathogenicity test of Haffniella vesicae strain JX2025 1) Experimental animals: 30 healthy Chinese soft-shelled turtles (50±5g) were randomly divided into an infection group (15 turtles) and a control group (15 turtles).

[0037] 2) Infection method: Each Chinese pond turtle (50±5g) in the infection group was intraperitoneally injected with 0.2mL of JX2025 bacterial suspension (1×10⁻⁶). 9 (CFU / mL), while the control group was injected with an equal volume of sterile PBS.

[0038] 3) Results Observation: The infected group developed symptoms (limb weakness, lethargy, and loss of appetite) within 24-96 hours, with a mortality rate of 70%. Bacterial isolation was performed again from infected turtles exhibiting typical symptoms. 16S rDNA identification showed that the isolated bacteria had 100% homology with *Havococcus vesicularis*. Autopsy revealed enlarged and congested liver and spleen. Liver and spleen tissues were collected for pathological sectioning and HE staining.

[0039] 4) Pathological observation: Liver (attached) Figure 5 The liver tissue structure is severely disordered, with multifocal to diffuse hepatocellular necrosis. In necrotic areas, hepatocellular structures disintegrate, with nuclei pyknosis, fragmentation, or dissolution; the cytoplasm shows increased eosinophilicity or granular degeneration. Infiltrating inflammatory cells (mainly lymphocytes and neutrophils) are visible in some areas. Hepatic sinusoids are dilated and congested, with hemorrhage observed in some areas. The portal tract structure is blurred, accompanied by mild edema and aggregation of inflammatory cells. Overall, the condition presents the typical pathological features of acute hepatitis with multifocal necrosis.

[0040] Spleen (attached) Figure 6 The tissue structure is disordered, the boundary between red and white pulp is unclear, and focal or diffuse lesions may occur. White pulp atrophy, red pulp congestion and inflammatory infiltration suggest that the bacteria not only directly cause tissue inflammation and damage, but may also inhibit the host's specific immune response, thereby promoting the development of systemic infection.

[0041] No obvious abnormalities were observed in the control group. The results indicate that the Haffniella auriculata strain JX2025 is highly pathogenic to the Chinese soft-shelled turtle.

[0042] This embodiment demonstrates through artificial infection experiments that this strain is highly pathogenic to the Chinese pond turtle, causing death in experimental animals and inducing typical pathological damage to the liver and spleen, showing lesions such as hepatocyte necrosis and unclear demarcation between the red and white pulp of the spleen.

[0043] Example 5 Antimicrobial susceptibility testing of strain JX2025 The disk diffusion method (KB method) was used, and the operation and result interpretation were performed according to CLSI standards. The results are shown in Table 2, providing direct evidence for clinical medication. The *Haffniella vesicae* strain JX2025 was highly sensitive to 8 out of 12 antibiotics (doxycycline, florfenicol, trimethoprim, minocycline, tetracycline, sulfamethoxazole, norfloxacin, ciprofloxacin), moderately sensitive to 2 (enrofloxacin, neomycin), and resistant to 2 (erythromycin, penicillin). Therefore, in actual production, medication should be prescribed based on drug sensitivity results.

[0044] Table 2. Antimicrobial susceptibility test results for strain JX2025

[0045] Note: S: Sensitive; I: Intermediate; R: Drug Resistance Example 6 Whole genome sequencing and virulence factor analysis of Haffniella vesicae JX2025 strain 1) Genome sequencing: Genomic DNA of strain JX2025 was extracted and sequenced using the Illumina HiSeq high-throughput sequencing platform based on sequencing by synthesis (SBS) technology to obtain whole genome framework data.

[0046] 2) Gene prediction and annotation: Gene prediction was performed using Prodigal-2.6.2 software, and the predicted gene sequences were submitted to databases such as VFDB for virulence factor homology comparison.

[0047] 3) Results Analysis: 208 virulence-related genes were predicted in the genome of *Havococcus pyogenes* strain JX2025, belonging to 14 virulence categories (Table 3). This strain exhibits complex virulence characteristics, mainly including the following key virulence systems:  PDIM (phenolic glycolipid) system gene cluster, core genes ppsB , fadD22 , Rv2952 The presence of these features suggests that the system has the ability to evade the recognition and response of the host's innate immune system by modifying cell wall lipid components to mask pathogen-associated molecular patterns (PAMPs).

[0048]  Multiple iron acquisition system and type VI secretion system (T6SS) gene cluster, core genes of the iron acquisition system ent , ybt and T6SS core component genes impA , vgrG , hcp The presence of this suggests a systemic function that involves efficiently competing for host iron resources through secreting siderophores, utilizing T6SS to mediate interbacterial competition, and directly delivering toxic effector factors to host cells.

[0049]  Adhesin array and toxin system gene cluster, key adhesion factors pap (P fimbriae) fim (Type I fimbriae) csg (curli) and toxin genes hlyB The co-existence of (α-hemolysin) suggests that the process involves a multi-stage, multi-mechanism adhesion system that transforms reversible adhesion into irreversible colonization, and that it works in conjunction with toxins such as hemolysin to directly cause host tissue damage and inflammatory responses.

[0050] Alginate biomembranes and multiple secretion systems gene clusters, key genes in biomembrane synthesis algZ , algR , algU The presence of T3SS and T6SS-related gene clusters in the secretory system suggests that alginate-mediated biofilm formation enhances the persistent colonization and resistance of bacteria on the host or environmental surface. Furthermore, the synergistic effect of T3SS / T6SS allows the strain to flexibly switch between acute virulence challenge and chronic persistent infection phenotypes, adapting to different infection stages and host environments.

[0051] This complete virulence gene spectrum lays the foundation for subsequent functional verification and application development.

[0052] This invention further constructed a whole-genome framework of strain JX2025 using next-generation sequencing technology and conducted systematic comparative analysis using tools such as VFDB (Virulent Factor Database). The results showed that, in addition to carrying common virulence factors of *Havococcus vesicatoria*, strain JX2025 also possesses a unique combination of virulence genes. This combination includes, but is not limited to: the PDIM (phenolic glycolipid) system gene cluster, which may be related to the recognition and response of escaping the host's innate immune system; the multiple iron acquisition system and type VI secretion system (T6SS) gene cluster, which may be related to efficient competition for host iron resources and direct invasion of host cells; the adhesin array and toxin system gene cluster, which may be related to enhanced colonization ability of host epithelial cells and causing host tissue damage; and the adhesin array and toxin system alginate biofilm and multiple secretion system gene cluster, which are associated with persistent infection. This unique virulence gene spectrum may be an important molecular basis for the strong pathogenicity of strain JX2025 against *Trionyx sinensis*.

[0053] Based on the above characteristics, this invention proposes the application of strain JX2025 in constructing animal models of Haffniella vesicatoria infection, screening and evaluating antimicrobial drugs and vaccines.

[0054] Table 3. Potential virulence genes of Haffniella vesicatoria JX2025

Claims

1. A strain of Haffniella apiacea ( Hafnia alvei ), characterized in that, The liver tissue derived from a diseased Chinese pond turtle contained a virulence gene cluster in its whole genome. The virulence gene cluster included: phenolic glycolipid (PDIM) system gene cluster, multiple iron acquisition system and type VI secretion system (T6SS) gene cluster, adhesin array and toxin system gene cluster, and alginate biofilm and multiple secretion system gene cluster. It was named *Haffniella vesicae* strain JX2025 and deposited at the China Center for Type Culture Collection (Wuhan) on November 17, 2025, with accession number CCTCC M 20252572.

2. The strain of *Haffniella vesicae* according to claim 1, characterized in that, The phenolic glycolipid (PDIM) system gene cluster includes: ppsB , fadD22 , Rv2952 The multiple iron acquisition system and type VI secretion system (T6SS) gene cluster includes: ent / ybt and impA / vgrG / hcp The adhesin array and toxin system gene cluster includes: pap , fim , csg , hlyB The alginate biofilm and multiple secretion system gene cluster includes: algZ , algR , algU .

3. A method for isolating and culturing *Haffniella vesicae* as described in any one of claims 1 or 2, characterized in that, Includes the following steps: S1. Liver tissue from diseased Chinese pond turtles was collected, homogenized with sterile physiological saline, and then streaked onto LB solid medium. S2. Incubate at 28.5℃ for 24 hours, then pick single colonies for purification culture; S3. Bacterial species were identified by colony morphology observation, Gram staining, biochemical identification, and 16S rRNA gene sequence analysis.

4. An application of *Haffniella vesicae* as described in any one of claims 1 or 2, characterized in that, The strain JX2025 of Haffniella vesicae was used as a pathogen to prepare an animal model of Haffniella vesicae infection.

5. The application according to claim 4, characterized in that, The animal used in the animal model is the Chinese pond turtle.

6. An application of *Haffniella vesicae* as described in any one of claims 1 or 2, characterized in that, The JX2025 strain of Haffniella vesicae is used to screen or evaluate antimicrobial agents or vaccines against Haffniella vesicae infection.

7. The application according to claim 6, characterized in that, Antibacterial drugs include doxycycline, florfenicol, trimethoprim, minocycline, tetracycline, sulfamethoxine, norfloxacin, and ciprofloxacin.

8. An application of *Haffniella vesicae* as described in any one of claims 1 or 2, characterized in that, Based on the virulence gene combination of Haffniella auriculata JX2025 strain, a target is provided for the development of inhibitors, monoclonal antibodies or genetically engineered attenuated vaccines against key virulence factors such as adhesins and secretion systems.

9. An application of *Haffniella vesicae* as described in any one of claims 1 or 2, characterized in that, Based on the virulence gene combination of Haffniella auriculata JX2025 strain, a specific molecular diagnostic method was developed to achieve rapid identification of this pathogenic strain or highly virulent clone.

10. The application according to claim 9, characterized in that, Molecular diagnostic methods include PCR and CRISPR-Cas detection.

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