Lactobacillus mucosae with anti-feline pestivirus pathogenic effect and application thereof

Abstract

The application provides a mucosal lactobacillus with anti-feline pestivirus pathogenic effect and application thereof, and relates to the technical field of probiotics, veterinary medicine and pet health care. The mucosal lactobacillus with anti-feline pestivirus pathogenic effect is named mucosal lactobacillus WH-3221, and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2026683. The mucosal lactobacillus WH-3221 can realize protection of intestinal barrier by regulating intestinal metabolic product spectrum, and has an anti-feline pestivirus effect, thereby significantly reducing the mortality of cats infected with feline pestivirus and relieving clinical symptoms. The mucosal lactobacillus WH-3221 or a probiotic preparation containing the mucosal lactobacillus WH-3221 can be used for preparing products for preventing and / or treating diseases or symptoms caused by feline pestivirus infection, and for preparing products for regulating the intestinal metabolic product spectrum of cats, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of probiotics, veterinary pharmacology and pet health care technology, specifically to a mucosal lactobacillus with antiviral activity against feline panleukopenia virus and its application. Background Technology

[0002] Feline panleukopenia (FPV) is a highly contagious and fatal infectious disease caused by feline parvovirus (FPV). This virus primarily infects felines, but is extremely dangerous to kittens, clinically manifesting as high fever, severe vomiting, hemorrhagic diarrhea, a sharp decrease in white blood cells, and immunosuppression, with a mortality rate as high as 50%-90%. Currently, there are no specific antiviral drugs available clinically; treatment mainly involves symptomatic supportive care (fluid replacement, antiemetics, and treatment of secondary infections), as well as biological agents such as feline panleukopenia inhibitors or interferon. However, these treatments have limitations, including high cost, unstable efficacy, and inability to effectively reverse intestinal pathological damage. Therefore, developing safe, effective, and easily marketable novel anti-feline panleukopenia agents has significant clinical importance and broad market prospects.

[0003] Probiotics are defined as "live microorganisms that, when ingested in sufficient quantities, produce beneficial effects on the health of the host." Currently reported probiotics with antiviral potential are mostly concentrated in species such as *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, and *Bifidobacterium*. There are currently no reports on the application of *Lactobacillus mucosa* in antiviral activity. Furthermore, existing research largely focuses on the direct effects of probiotics on viral load or immune indicators, neglecting the role of host metabolic regulation in antiviral protection. Currently, there is still a lack of probiotic formulations on the market specifically targeting feline panleukopenia virus that exert their effects through metabolic regulation. Summary of the Invention

[0004] In view of the technical problems existing in the background art, this application provides a mucosal lactobacillus with anti-feline panleukopenia virus pathogenicity and its application. The mucosal lactobacillus can effectively alleviate clinical symptoms such as diarrhea and enteritis caused by feline panleukopenia virus infection by regulating metabolic processes and reduce mortality.

[0005] In a first aspect, embodiments of this application provide a mucosal lactobacillus with antiviral activity against feline panleukopenia virus, named Limosilactobacillus mucosae WH-3221, which was deposited on April 15, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2026683.

[0006] The 16S rDNA sequence of the *Lactobacillus mucosa* WH-3221 is shown in SEQ ID NO:1.

[0007] The *Lactobacillus mucosa* WH-3221 was isolated from cat feces.

[0008] Secondly, embodiments of this application provide a probiotic preparation, wherein the probiotic preparation includes the aforementioned *Lactobacillus mucosa*.

[0009] Thirdly, embodiments of this application provide the application of the above-mentioned *Lactobacillus mucosa* or probiotic preparation in the preparation of a product, the function of which includes: Used for the prevention and / or treatment of diseases or symptoms caused by feline panleukopenia virus infection; Used to regulate the profile of intestinal metabolites in cats.

[0010] Furthermore, in some embodiments, the diseases or symptoms caused by the feline panleukopenia virus infection include at least one of diarrhea, enteritis, intestinal bleeding, weight loss, leukopenia, and death.

[0011] Furthermore, in some embodiments, the modulation of the cat's intestinal metabolite profile includes at least one of the following a~e: a. Reduce the level of toxins in the intestines; b. Activates the tryptophan AhR axis and inhibits the accumulation of pro-inflammatory polyamines and histamine; c. Promotes the synthesis of primary bile acids and inhibits the production of harmful secondary bile acids; d. Reduces the level of pro-inflammatory lipid mediators and enhances fatty acid oxidation; e. Increase the levels of glucocorticoids, vitamin D, and estrogen metabolites.

[0012] Furthermore, in some embodiments, the toxins in the intestine include at least one of fungal toxins, marine toxins, and drug metabolites.

[0013] Furthermore, in some embodiments, the mycotoxin includes at least one of zearalenone, terbufotenol, and isorhizine, the marine toxin includes ciguatoxin, and the drug metabolite includes thioridazine.

[0014] Furthermore, in some embodiments, the product is a veterinary drug.

[0015] The beneficial effects of this application include: The *Lactobacillus mucosa* WH-3221 provided in this application is a feline probiotic with good colonization ability and biosafety in the feline intestine. It significantly reduces the mortality rate of cats infected with feline panleukopenia virus (FPV), alleviates clinical symptoms such as diarrhea and enteritis, protects the integrity of the intestinal mucosal barrier, and reduces viral invasion into tissues. It can be used to prepare products for the prevention and / or treatment of diseases or symptoms caused by FPV infection. Furthermore, unlike traditional probiotics that directly inhibit viral replication or stimulate leukocyte proliferation, the *Lactobacillus mucosa* WH-3221 provided in this application reduces intestinal inflammation and damage and protects the intestinal mucosal barrier by regulating the intestinal metabolite profile, thereby achieving an anti-pathogenic effect against FPV. This mechanism is substantially different from that of traditional probiotics and has higher safety. Based on the mechanism of action described in this application, *Lactobacillus mucosa* WH-3221 can also be used to prepare products that regulate the feline intestinal metabolite profile, showing broad market application prospects.

[0016] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0018] Figure 1 Gram staining microscopic results of Lactobacillus mucosa WH-3221 (100×); Figure 2 Survival curves for each group of experimental animals; Figure 3 A graph showing the dynamic changes in fecal scores of each group of experimental animals; Figure 4 The graph shows the changes in viral load in the swabs of each group of experimental animals; Figure 5 The images show the pathological examination results of animals in the challenge model group and the bacterial feeding experimental group; where A is the HE staining result of the intestine in the bacterial feeding experimental group; B is the HE staining result of the intestine in the challenge model group; C is the HE staining result of the mesenteric lymph nodes in the bacterial feeding experimental group; and D is the HE staining result of the mesenteric lymph nodes in the challenge model group. Figure 6The graph shows the differences in metabolites between the challenge model group and the bacterial feeding experimental group; where A represents the differences in the top 20 metabolites between the challenge model group and the bacterial feeding experimental group in POS mode; and B represents the differences in the top 20 metabolites between the challenge model group and the bacterial feeding experimental group in NEG mode. Figure 7 The results of KEGG analysis of metabolic pathways in the challenge model group and the feeding experimental group are shown below. Among them, A shows the enrichment results of the top 20 KEGG pathways in the challenge model group and the feeding experimental group under POS mode, and B shows the enrichment results of the top 20 KEGG pathways in the challenge model group and the feeding experimental group under NEG mode. Detailed Implementation

[0019] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0023] Currently, there is a lack of probiotic preparations specifically targeting feline panleukopenia virus on the market. To fill this market gap, this application provides a mucosal lactobacillus with anti-feline panleukopenia virus pathogenicity and its application. This mucosal lactobacillus can protect the intestinal barrier by regulating metabolic processes and play an anti-feline panleukopenia virus role, thereby effectively reducing the mortality rate of cats infected with feline panleukopenia virus and alleviating clinical symptoms such as diarrhea and enteritis caused by feline panleukopenia virus infection.

[0024] Specifically, in the first aspect, embodiments of this application provide a mucosal lactobacillus with antiviral activity against feline panleukopenia virus, named Limosilactobacillus mucosae WH-3221, which was deposited on April 15, 2026 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2026683.

[0025] The *Lactobacillus mucosa* WH-3221 provided in this application can significantly reduce the mortality rate of cats infected with feline panleukopenia virus (FPV), alleviate clinical symptoms such as diarrhea and enteritis, protect the integrity of the intestinal mucosal barrier, and reduce viral invasion into tissues. Furthermore, unlike traditional probiotics that directly inhibit viral replication or stimulate leukocyte proliferation, the *Lactobacillus mucosa* WH-3221 provided in this application reduces intestinal inflammatory damage and protects the intestinal mucosal barrier by regulating the intestinal metabolite profile, thereby achieving its antiviral effect against FPV. This mechanism is substantially different from that of traditional probiotics and has higher safety.

[0026] The 16S rDNA sequence of Lactobacillus mucosa WH-3221 provided in this application is shown in SEQ ID NO:1.

[0027] This *Lactobacillus mucosa* WH-3221 was isolated from cat feces.

[0028] In the technical solution of this application embodiment, the strain of Lactobacillus mucosa WH-3221 is safe and adaptable. This strain is isolated from the feces of healthy cats and is a cat-derived probiotic. It has good colonization ability and biosafety in the cat's intestine.

[0029] Secondly, embodiments of this application provide a probiotic preparation comprising the aforementioned *Lactobacillus mucosa* WH-3221.

[0030] Since the *Lactobacillus mucosa* WH-3221 provided in this application can protect the intestinal barrier by regulating the intestinal metabolite profile and has an anti-feline panleukopenia virus effect, making it into a probiotic preparation is beneficial to protect the intestinal health of cats, reduce their mortality rate after infection with feline panleukopenia virus, and alleviate the clinical symptoms after infection with feline panleukopenia virus.

[0031] Thirdly, embodiments of this application provide the application of the above-mentioned mucosal lactobacillus or probiotic preparation in the preparation of a product, the function of which includes: Used for the prevention and / or treatment of diseases or symptoms caused by feline panleukopenia virus infection; Used to regulate the profile of intestinal metabolites in cats.

[0032] Because the *Lactobacillus mucosa* WH-3221 provided in this application can significantly reduce the mortality rate of cats infected with feline panleukopenia virus (FPV), alleviate clinical symptoms such as diarrhea and enteritis, protect the integrity of the intestinal mucosal barrier, and reduce viral invasion into tissues, *Lactobacillus mucosa* WH-3221 or probiotic preparations containing this strain can be used to prepare products for the prevention and / or treatment of diseases or symptoms caused by FPV infection. Furthermore, since *Lactobacillus mucosa* WH-3221 provided in this application exerts its anti-FPV effect by regulating the intestinal metabolite profile, this strain or probiotic preparations containing this strain can also be used to prepare products that regulate the intestinal metabolite profile of cats, possessing broad market application prospects.

[0033] Furthermore, in some embodiments, the diseases or symptoms caused by feline panleukopenia virus infection include at least one of diarrhea, enteritis, intestinal bleeding, weight loss, leukopenia, and death.

[0034] The *Lactobacillus mucosa* WH-3221 provided in this application can effectively improve the above-mentioned diseases or symptoms caused by feline panleukopenia virus infection, thereby playing a corresponding preventive and / or therapeutic role.

[0035] Furthermore, in some embodiments, modulating the cat's intestinal metabolite profile includes at least one of the following a~e: a. Reduce the level of toxins in the intestines; b. Activates the tryptophan AhR axis and inhibits the accumulation of pro-inflammatory polyamines and amines; c. Promotes the synthesis of primary bile acids and inhibits the production of harmful secondary bile acids; d. Reduces the level of pro-inflammatory lipid mediators and enhances fatty acid oxidation; e. Increase the levels of glucocorticoids, vitamin D, and estrogen metabolites.

[0036] Traditional probiotic antiviral mechanisms are achieved by directly inhibiting viral replication or stimulating leukocyte proliferation. However, the *Lactobacillus mucosa* WH-3221 in this application exerts its antiviral effect against feline panleukopenia virus through other mechanisms. Specifically, this application conducted non-targeted metabolomics analysis on fecal samples from experimental animals. The results showed that the fecal metabolic profile of experimental animals treated with *Lactobacillus mucosa* WH-3221 underwent systematic remodeling, mainly manifested as broad-spectrum detoxification (reducing toxin levels in the intestine), anti-inflammatory immune regulation (activating the tryptophan AhR axis and inhibiting the accumulation of pro-inflammatory polyamines and amine inhibitors), bile acid metabolism remodeling (promoting primary bile acid synthesis and inhibiting the production of harmful secondary bile acids), lipid metabolism optimization (reducing the level of pro-inflammatory lipid mediators and enhancing fatty acid oxidation), and steroid hormone regulation (increasing the levels of glucocorticoids, vitamin D, and estrogen metabolites). Therefore, products prepared based on *Lactobacillus mucosa* WH-3221 provided in this application can regulate the feline intestinal metabolic profile through the mechanisms described in a~e above.

[0037] Furthermore, in some embodiments, the intestinal toxins include at least one of fungal toxins, marine toxins, and drug metabolites; even further, in some embodiments, the fungal toxins include at least one of zearalenone, terbufotenol, and isorhizine, the marine toxins include ciguatoxin, and the drug metabolites include thioridazine.

[0038] The *Lactobacillus mucosa* WH-3221 provided in this application can effectively reduce the level of the aforementioned toxins in the intestines, thus achieving a broad-spectrum detoxification effect.

[0039] Furthermore, in some embodiments, the product is a veterinary drug with broad market application prospects.

[0040] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0041] Example 1 This embodiment provides a strain of *Lactobacillus mucosa* WH-3221 with antiviral activity against feline panleukopenia virus. The isolation and identification process of this strain is as follows: 1. Strains Isolation (1) Preparation of culture medium: Weigh 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 2.0 g of ammonium citrate, 0.1 g of magnesium sulfate, 2.0 g of dipotassium hydrogen phosphate, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 1.0 mL of Tween 80, and 15.0 g of agar. Mix them and add distilled water to make up to 1 L. Adjust the pH of the culture medium to 6.2-6.4 and autoclave at 121℃ for 15 min to obtain MRS solid culture medium.

[0042] (2) Strain isolation: Take 0.5g of fresh fecal sample from a healthy cat that has recovered from feline panleukopenia, add 5 mL of sterile saline, vortex to mix and prepare a suspension. Dilute 10-fold with sterile saline to 10 ... -5 10 -6 10 -7 10 -8 100 μL of each dilution of suspension was plated onto MRS solid medium and anaerobically incubated at 37°C for 48 h. Single colonies were picked and streaked onto MRS plates for purification, repeated twice. The purified single colonies were then inoculated into 10 mL of MRS liquid medium and incubated statically at 37°C for 18 h to obtain a pure culture, designated WH-3221.

[0043] 2. Strain identification (1) Morphological identification: On MRS plates, the colonies are round, milky white, smooth, with regular edges, and approximately 1-2 mm in diameter. The Gram staining microscopic examination results of this strain are as follows: Figure 1 As shown, by Figure 1 It can be seen that it is Gram-positive, appears as rod-shaped under a microscope, and is arranged singly or in pairs, without spores.

[0044] (2) Molecular biological identification: Using the purified bacterial culture as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA.

[0045] The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are as follows: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3; 1492R:5'-TACGGYTACCTTGTTACGACTT-3' The PCR reaction system (50 μL) consisted of: 2×Taq PCR Master Mix 25 μL, ddH2O 19 μL, forward primer (25 μmol / L) 2 μL, reverse primer (25 μmol / L) 2 μL, and DNA template 2 μL.

[0046] PCR amplification conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 30 cycles; 72℃ final extension for 7 min; store at 4℃.

[0047] After PCR product detection by 1% agarose gel electrophoresis, the samples were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequence alignment with the NCBI database confirmed that the isolated bacteria were *Lactobacillus mucosa*, specifically *Lactobacillus mucosa* WH-3221, for which protection is sought in this application. This strain is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2026683.

[0048] The 16S rDNA sequence of this *Lactobacillus mucosa* WH-3221 is shown in SEQ ID NO:1.

[0049] Example 2 This embodiment aims to test the protective effect of Lactobacillus mucosa WH-3221 provided in Example 1 against feline panleukopenia virus infection. The specific process is as follows: 1. Laboratory animals and grouping Healthy susceptible cats aged 8-12 weeks were selected, and those that were negative for FPV antibodies and fecal FPV nucleic acid were confirmed by ELISA and PCR testing. The specific grouping method is shown in Table 1.

[0050] Table 1 Grouping of experimental animals 2. Experimental Methods Lactobacillus mucosa WH-3221 was inoculated into MRS liquid medium and incubated statically at 37°C for 18 h. The bacterial cells were collected by centrifugation at 6000 rpm for 10 min, washed twice with sterile physiological saline, and resuspended to a concentration of 1×10⁻⁶. 9 CFU / mL, prepare fresh before use. Oral challenge with the FPV prototype strain, dose 1×10⁻⁶. 7.5 TCID 50 / mL, 1 mL per animal. Daily observation and recording of mental status, appetite, fecal characteristics (scoring criteria: 0 = normal formed stool; 1 = soft stool; 2 = loose stool; 3 = watery / bloody stool), vomiting, and body temperature; daily weight; daily mortality; and plotting survival curves. Rectal swabs were collected daily, and FPV load was measured using the TCID50 method. Peripheral blood was collected every three days, and the total white blood cell count was measured using a hematology analyzer. Necropsy was performed on dead animals and animals euthanized at the end of the experiment; intestines and mesenteric lymph nodes were collected for HE staining and immunohistochemical detection of FPV antigen.

[0051] 3. Experimental Results (1) Survival rate In the viral challenge model group, all four cats died within 11 days after the challenge, a 4 / 4 mortality rate. In the bacterial feeding experimental group, one of the four cats died, and three survived, a 1 / 4 mortality rate and a 3 / 4 survival rate. The survival curves are as follows: Figure 2 As shown in the figure. The Log-rank test showed that the difference between the two groups of survival curves was statistically significant (P<0.05).

[0052] (2) Clinical symptoms and stool score The challenge model group (D1-D4): From day 2 to day 8 after the challenge, the cats began to show signs of depression, loss of appetite, and vomiting. Three-quarters of the cats developed watery or bloody diarrhea (fecal score ≥3) and eventually died.

[0053] In the bacterial feeding experimental group (CJ1-CJ4), only 1 / 4 of the cats experienced intermittent soft stools (fecal score 1-2), which gradually returned to normal in the middle and late stages of the challenge, while the feces of the remaining 3 cats were normal.

[0054] Specifically, the dynamic changes in stool scores are as follows: Figure 3 As shown.

[0055] (3) Viral load (anal swab) The changes in viral load in the swabs of each group of experimental animals are as follows: Figure 4 As shown. By Figure 4 It can be seen that both the challenge model group and the feeding experiment group showed fecal shedding after challenge. The viral load of the three surviving cats in the feeding experiment group gradually decreased to below the detection limit in the later stage of challenge (after day 8-9), while the animals in the model group maintained a high viral load before death.

[0056] (4) White blood cell count Both the challenge model group and the bacterial feeding experimental group showed a sharp decline in white blood cell count after challenge. In the challenge model group, the white blood cell count continued to decrease until death. In the bacterial feeding experimental group, 1 / 4 of the cats experienced a decrease in white blood cell count followed by a return to normal (starting to rise again on day 10 after challenge), 2 / 4 of the cats did not show a significant decrease in white blood cell count, and 1 / 4 of the deceased cats experienced a continuous decrease in white blood cell count. The results are shown in Table 2. This result may suggest that some animals in the bacterial feeding experimental group had enhanced immune recovery capabilities.

[0057] Table 2 White blood cell count results (5) Pathological examination Pathological examinations were performed on the animals in the challenge model group and the surviving animals in the feeding experiment group. The results were as follows: Figure 5 As shown. Figure 5In the figures, A and B show the HE staining results of the intestines of the bacterial feeding experimental group and the virus challenge model group, respectively. The results show that in the virus challenge model group, there is extensive disintegration and ablation of intestinal villi, destruction of crypts, and infiltration of a large number of inflammatory cells. In the bacterial feeding experimental group, the intestinal villi of the surviving animals are neatly arranged and structurally intact, with only mild inflammatory cell infiltration. Figure 5 C and D in the figure show the HE staining results of the mesenteric lymph nodes in the bacterial feeding experimental group and the virus challenge model group, respectively. The results show that the lymph node structure was destroyed, red blood cells were extravasated, and there was obvious bleeding in the virus challenge model group; while the lymph nodes of the surviving animals in the bacterial feeding experimental group were intact and no obvious abnormalities were observed.

[0058] (6) Organ viral load Viral load was detected in tissues of dead animals and animals euthanized at the end of the experiment. The results are shown in Table 3.

[0059] Table 3 Viral load in animal tissues As shown in Table 3, the viral load in the intestines and mesenteric lymph nodes of some of the dead cats in the challenge model group was as high as 10. 7 TCID 50 / mL or higher. In the experimental group, one cat that died after being fed the bacteria had a viral load of only 10 in its intestines and mesenteric lymph nodes. 3.8 TCID 50 / mL and 10 3.67 TCID 50 / mL, lower than that of the challenge model group; and no live virus was detected in the intestines and lymph nodes of the three surviving cats in the feeding experiment group.

[0060] 4. Experimental Conclusions The above results indicate that the *Lactobacillus mucosa* WH-3221 provided in this application can significantly reduce the mortality rate of cats infected with feline panleukopenia virus, alleviate clinical symptoms such as diarrhea, protect the integrity of the intestinal mucosal barrier, and reduce viral invasion into tissues. Notably, this strain does not directly inhibit fecal viral shedding, suggesting that its mechanism of action differs from traditional antiviral drugs.

[0061] Example 3 This embodiment aims to detect the regulatory effect of *Lactobacillus mucosa* WH-3221 provided in Example 1 on the intestinal metabolome of cats. The specific process is as follows: 1. Fecal sample collection Fecal samples were collected from the blank control group, the challenge model group, and the feeding experimental group (cats that survived until the end of the experiment) in Example 2 at three time periods before, during and after the challenge. At least three biological replicates were made for each group. The samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0062] 2. Non-targeted metabolomics detection The above-mentioned fecal samples were sent to Beijing Novogene Technology Co., Ltd. for non-targeted metabolite analysis.

[0063] Specifically, 50 mg of fecal sample was taken and added to 500 μL of extraction buffer (methanol:acetonitrile:water = 2:2:1, containing internal standard), vortexed for 30 s, sonicated in an ice-water bath for 10 min, incubated at -20℃ for 1 h, centrifuged at 13000 rpm for 15 min at 4℃, the supernatant was collected, dried under nitrogen, reconstituted with 100 μL of acetonitrile:water (1:1), centrifuged again, and the supernatant was injected. A Waters ACQUITY UPLC BEHC18 column (2.1 × 100 mm, 1.7 μm) was used at a column temperature of 40℃ and a flow rate of 0.3 mL / min. Mobile phase A was water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid). Gradient elution program: 0–2 min, 5% B; 2–15 min, 5%–95% B; 15–17 min, 95% B; 17–17.1 min, 95%–5% B; 17.1–20 min, 5% B. A Thermo Q-Exactive HF mass spectrometer with electrospray ionization (ESI) was used, acquiring data in both positive and negative ion modes. The scan range was m / z 70–1050, with resolutions of 70,000 (MS1) and 17,500 (MS2). The spray voltage was 3.5 kV (positive ions) or 2.8 kV (negative ions), the sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 10 arb, and the capillary temperature was 320 °C. Raw data were converted to mzXML format using ProteoWizard, and peak detection, alignment, and integration were performed using XCMS software. Isotope and adduct annotations were performed using CAMERA. Metabolite identification was performed using CompoundDiscoverer 3.0 software in conjunction with the HMDB, METLIN, and KEGG databases. Differential metabolite screening criteria: VIP>1, P<0.05, |log2FC|>1.

[0064] 3. Experimental Results Metabolite differences and KEGG pathway analysis results between the challenge model group and the feeding experimental group are shown below. Figure 6 , Figure 7 As shown. Figure 6 In the table, A shows the differences in the top 20 metabolites between the challenge model group and the feeding experimental group under POS mode; B shows the differences in the top 20 metabolites between the challenge model group and the feeding experimental group under NEG mode. Figure 6The color of the dot represents the up- or down-regulation result of the feeding experimental group compared to the challenge model group. Blue indicates that the feeding experimental group has down-regulation compared to the challenge model group, and red indicates that the feeding experimental group has down-regulation compared to the challenge model group. The length of the rod connected to the dot represents the size of log2 (Fold Change), and the specific value corresponding to the dot represents the size of the VIP value. Figure 7 In the table, A represents the enrichment results of the top 20 KEGG pathways in the challenge model group and the feeding experimental group under POS mode, and B represents the enrichment results of the top 20 KEGG pathways in the challenge model group and the feeding experimental group under NEG mode. Figure 7 The horizontal axis represents the ratio of the number of differentially expressed metabolites in the corresponding metabolic pathway to the total number of metabolites identified in that pathway. The color of the dot represents the p-value of the hypergeometric test, and the size of the dot represents the number of differentially expressed metabolites in the corresponding flux. A detailed analysis follows: (1) Changes in metabolites related to detoxification function like Figure 6 As shown, the levels of multiple exogenous toxins in the feces of the bacterial feeding experimental group were significantly downregulated. Specifically, β-zearalenol (fungal toxin): log2FC = -21.0, VIP>10; ciguatoxin fragment (marine toxin): log2FC = -20.0, VIP>10; (-)-terpendole D (fungal toxin): log2FC = -19.0; thioridazine (antipsychotic drug metabolite): log2FC = -13.0; and isororidin E (fungal toxin): were also significantly downregulated in the independent validation experiments.

[0065] The above results indicate that Lactobacillus mucosa WH-3221 has a broad-spectrum detoxification ability, which can effectively remove a variety of toxins in the intestine and reduce the damage of toxins to the intestinal mucosa.

[0066] (2) Changes in anti-inflammatory and immune-modulating metabolites like Figure 6As shown, key metabolites in the tryptophan metabolic pathway in the bacterial feeding experimental group underwent significant changes: indole-3-acetic acid (IAA, aryl hydrocarbon receptor AhR agonist): upregulated, log2FC = +2.48; 5-hydroxytryptophan (5-HTP, serotonin precursor): upregulated, log2FC = +1.77; N-acetylputrescine (polyamine metabolite, associated with inflammation): downregulated, log2FC = -19.5; histamine (pro-inflammatory mediator): downregulated, log2FC = -2.06; arachidonic acid ethanolamine (endocannabinoid, anti-inflammatory): upregulated, log2FC = +1.05.

[0067] Figure 7 The KEGG enrichment analysis shown indicates that the tryptophan metabolic pathway (map00380) was significantly enriched in the experimental group (P=0.053).

[0068] The results suggest that *Lactobacillus mucosa* WH-3221 promotes the conversion of tryptophan to anti-inflammatory indole derivatives while inhibiting the accumulation of pro-inflammatory polyamines and histamine.

[0069] (3) Bile acid metabolic remodeling like Figure 6 As shown, the levels of primary bile acid synthesis intermediates in the feces of the bacterial feeding experimental group were significantly upregulated: 27-hydroxycholesterol: log2FC = +3.52; 3β-hydroxy-5-cholestyric acid: log2FC = +3.29; 3α,7α,12α-trihydroxy-5β-cholestane-26-aldehyde: log2FC = +1.48; while the levels of harmful secondary bile acid derivative phenylalanine deoxycholic acid were significantly downregulated: log2FC = -11.5.

[0070] Figure 7 The KEGG enrichment analysis shown indicates that the primary bile acid biosynthesis pathway (map00120) was most significantly enriched in the positive ion mode (P=0.0097).

[0071] The above results indicate that *Lactobacillus mucosa* WH-3221 remodels the bile acid pool through bile salt hydrolase activity, promoting the synthesis of primary bile acids and inhibiting the production of harmful secondary bile acids.

[0072] (4) Changes in lipid metabolism and oxidative stress-related metabolites like Figure 6As shown, pro-inflammatory lipid mediators in the feces of the bacterial feeding experimental group were significantly downregulated: LysoPC (16:1): log2FC = -15.0; LysoPC (20:4): log2FC = -12.5; Ceramide: significantly downregulated; Cholesterol-5α,6α-epoxide (a marker of oxidative stress): significantly downregulated. Simultaneously, fatty acid β-oxidation-related metabolites were upregulated: Sebacoylcarnitine: log2FC = +4.40; (9Z)-nonadecanoylcarnitine: log2FC = +2.60. Furthermore, antioxidant metabolites were significantly upregulated, including Chrysophanol, caffeic acid-3'-sulfate, and luteolin-4'-methyl ether-3'-sulfate.

[0073] (5) Changes in steroid hormones and vitamin metabolism-related metabolites like Figure 6 As shown, the following metabolites were significantly upregulated in the feces of the bacterial feeding experimental group: hydrocortisone (anti-inflammatory glucocorticoid): log2FC = +2.81; calcidiol (25-hydroxyvitamin D3): log2FC = +2.56; 7-dehydrocholesterol (vitamin D3 precursor): log2FC = +3.40; 4-hydroxyestradiol: log2FC = +1.85; 4-hydroxyestradiol: log2FC = +5.36.

[0074] Figure 7 The KEGG enrichment analysis shown that the steroid biosynthesis (map00100) and steroid hormone biosynthesis (map00140) pathways were significantly enriched in the bacterial feeding experimental group (P<0.05).

[0075] 4. Experimental Conclusions The metabolomics results summarized above indicate that intervention with *Lactobacillus mucosa* WH-3221 resulted in a systemic remodeling of the feline intestinal metabolome, including the clearance of fungal toxins, marine toxins, and drug metabolites; activation of anti-inflammatory immune regulation, including activation of the tryptophan-AhR axis, inhibition of histamine, and upregulation of endocannabinoids; bile acid metabolic remodeling, promoting the synthesis of primary bile acids and inhibiting harmful secondary bile acids; lipid metabolism optimization, reducing pro-inflammatory LysoPC and ceramides, enhancing fatty acid oxidation, and alleviating oxidative stress; and steroid hormone regulation, upregulating glucocorticoids, vitamin D, and estrogen metabolites. These metabolic changes collectively constitute the molecular basis for *Lactobacillus mucosa*'s anti-feline panleukopenia virus activity, intestinal barrier protection, and reduced mortality rate. Furthermore, its mechanism of action does not rely on directly reducing viral load or stimulating leukocyte proliferation, but rather on metabolic reprogramming.

[0076] In summary, this application provides a *Lactobacillus mucosa* with antiviral activity against feline panleukopenia virus (FPV) and its application. This *Lactobacillus mucosa* with antiviral activity is named WH-3221 and is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026683. *Lactobacillus mucosa* WH-3221 can protect the intestinal barrier by regulating the intestinal metabolite profile and exert an antiviral effect against FPV, thereby significantly reducing the mortality rate of cats infected with FPV and alleviating their clinical symptoms. *Lactobacillus mucosa* WH-3221 or probiotic preparations containing *Lactobacillus mucosa* WH-3221 can be used to prepare products for the prevention and / or treatment of diseases or symptoms caused by FPV infection, and can also be used to prepare products that regulate the intestinal metabolite profile of cats, showing broad application prospects.

[0077] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A mucosal lactobacillus with antiviral activity against feline panleukopenia virus, characterized in that, It was named Limosilactobacillus mucosa WH-3221 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026683.

2. A probiotic preparation, characterized in that, Includes the mucosal lactobacillus described in claim 1.

3. The use of the *Lactobacillus mucosa* as described in claim 1 or the probiotic preparation as described in claim 2 in the preparation of veterinary drugs, characterized in that, The functions of the veterinary drug include: Used for the prevention and / or treatment of feline panleukopenia virus infection.

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