A method for fluorescent labeling of duck riibergeria

Abstract

This invention discloses a method for fluorescently labeling *R. anatidae*. The method includes: constructing an enhanced green fluorescent protein expression plasmid EGFP-pRES2 to obtain a positive donor strain EGFP-pRES2-S17; introducing the plasmid into *R. anatidae* RAf71 via conjugation transfer; and obtaining the fluorescently labeled strain EGFP-pRES2-RAf71 through resistance screening and PCR identification, with a flow cytometry-detected EGFP positivity rate of 76.8%. Administering this labeled strain to Beijing ducks via gavage allows for dynamic monitoring of its distribution changes in different intestinal segments. After co-administration of this labeled strain with bacteriophage CRP2 / M34, the proportion of fluorescently positive bacteria in each intestinal segment significantly decreased. The method of this invention is feasible, the labeling is sensitive, and the obtained fluorescently labeled strain can be used for monitoring the dynamic in vivo distribution of *R. anatidae* and screening for bacteriophages that kill the bacterium.

Description

Technical Field

[0001] This invention belongs to the field of poultry infectious diseases, specifically relating to a method for fluorescently labeling Riedebrionia anatipestifer. Background Technology

[0002] *Riemerella anatipestifer*, also known as duck plague Riemerella or duck plague Riemerella, belongs to the genus *Riemerella* of the family Flavobacteriaceae. It primarily infects waterfowl aged 2-7 weeks, causing acute or chronic septicemic infectious diseases, commonly known as infectious serositis. The morbidity rate can be as high as 50%, and the mortality rate as high as 90%, leading to death and culling. Its pathological features include fibrinous exudative inflammation of the serosal surface, meningitis, and arthritis. In recent years, cases of oviductitis, serositis, and arthritis caused by this bacterium in chickens have increased.

[0003] Artificial inoculation of susceptible Beijing ducks with *Riegeria anatipestifer* via subcutaneous injection, intravenous injection, nasal inhalation, and oral administration can all induce mortality. While the distribution patterns of *Riegeria anatipestifer* in the blood, brain, and internal organs of poultry after infection can be studied through live bacteria culture, its distribution patterns in the digestive tract are impossible due to interference from other bacteria. Therefore, analysis must rely on PCR detection of *Riegeria anatipestifer* nucleic acid, which does not fully reflect the distribution patterns of live bacteria.

[0004] Current methods for the prevention and treatment of infectious serositis rely on biosafety management, antibiotics, and commercial vaccines. Biosafety management requires significant economic investment, which many small and medium-sized farmers cannot fully afford. The frequent emergence of multidrug-resistant *R. anatidae* in clinical practice reduces the effectiveness of antibiotics, and the policy environment of reducing and limiting antibiotic use further restricts drug application. *R. anatidae* has as many as 21 serotypes, and the low cross-protective ability between different serotypes poses a major challenge to vaccine selection. New prevention and control methods are urgently needed in production, and phage therapy is one direction for development, although more research is required. While phages can specifically kill *R. anatidae* in vitro, the unique acid-base environment, digestive enzymes, and microbial ecosystem within the avian digestive tract raises the question of whether phages still have a killing effect on *R. anatidae* in this environment; currently, there is no good method for evaluation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for fluorescently labeling Riedelium anatipestifer, which can be used to track the distribution pattern of Riedelium anatipestifer in the avian digestive tract by flow cytometry, and can also evaluate the killing effect of Riedelium anatipestifer bacteriophage on Riedelium anatipestifer in the avian digestive tract.

[0006] This invention is achieved through the following technical solution:

[0007] A method for fluorescently labeling Riedelium anatipestifer, comprising the following steps:

[0008] (1) Construction of the expression plasmid EGFP-pRES2 for the enhanced green fluorescent protein gene: The enhanced green fluorescent protein gene was ligated between the NheI and XhoI restriction sites of the pMD19-T Simple vector and transformed into E. coli TOP10 competent cells. Positive strains were screened using LB plates containing 1 μg / mL cefoxitin. After PCR identification and sequencing verification, the positive strain was named EGFP-pMD19-T-TOP10. The plasmid of strain EGFP-pMD19-T-TOP10 was extracted to obtain the EGFP-pMD19-T plasmid. The EGFP-pMD19-T plasmid and pRES2 plasmid were double-digested with NheI and XhoI restriction endonucleases. The digestion products were ligated using T4 DNA ligase and transformed into E. coli S17-1. λpir competent cells were screened for positive strains using LB plates containing 1 μg / mL cefoxitin. After double enzyme digestion and sequencing verification, the positive strains were named EGFP-pRES2-S17.

[0009] (2) Obtaining fluorescently labeled Riesella anatipestifer via conjugation transfer: After treating strains EGFP-pRES2-S17 and Riesella anatipestifer RAf71 separately, the bacterial concentration was adjusted. The two bacterial solutions were mixed, centrifuged, resuspended, and then dropped onto the surface of a sterile NC membrane on a TSA plate for culture. The bacterial sludge was washed off with PBS buffer and spread on a double-antibiotic TSA plate containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin for culture. After single colonies were picked and purified, PCR identification was performed using Riesella anatipestifer detection primers and EGFP detection primers. The strain that was positive for both PCR tests was the fluorescently labeled Riesella anatipestifer EGFP-pRES2-RAf71.

[0010] The nucleotide sequence of the enhanced green fluorescent protein gene described in step (1) is shown in SEQ ID NO.1.

[0011] The treatment method for strain EGFP-pRES2-S17 in step (2) is as follows: strain EGFP-pRES2-S17 is streaked onto resistant LB agar plates containing 1 μg / mL cefoxitin and incubated at 37℃ for 8 h; the bacterial sludge on the plate is washed with 10 mM MgSO4 solution and the OD of the bacterial solution is adjusted. 600 The value was 1.0, and EGFP-pRES2-S17 bacterial culture was obtained.

[0012] The treatment method for *Riebelella anatipestifer* RAf71 in step (2) is as follows: *Riebelella anatipestifer* RAf71 is inoculated onto a TSA plate and cultured at 37°C for 12 hours; the bacterial sludge on the plate is washed with 10 mM MgSO4 solution, and the OD of the bacterial solution is adjusted. 525 The value is 1.0, and RAf71 bacterial culture is obtained.

[0013] The primers used for PCR identification in step (2) for detecting *Riegeria duckweed* are gyrBF: 5'-GGAGACGTAGGAGTAAGAGG-3', gyrBR: 5'-CATGAAACCCCTACACCGTG-3'; and the primers for detecting EGFP are GFPF: 5'-AGTGTGAGCGGTGAAGGTGA-3', GFPR: 5'-GGTGCGTTCCTGAACATAGC-3'.

[0014] The PCR identification reaction procedure described in step (2) is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; and finally 72℃ extension for 10 min.

[0015] A fluorescently labeled *Riegeria anatipestifer* EGFP-pRES2-RAf71 was prepared by the method described above.

[0016] The above-mentioned fluorescently labeled Rhizobium anatipestifer EGFP-pRES2-RAf71 was used in the preparation of a detection reagent for detecting the dynamic distribution of Rhizobium anatipestifer in the digestive tract of ducks.

[0017] The above-mentioned fluorescently labeled *Riegeria anatipestifer* EGFP-pRES2-RAf71 was used in the preparation of a screening model for screening bacteriophages that kill *Riegeria anatipestifer*.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] (1) This invention uses flow cytometer to detect the number of live duck disease Riegeria in the digestive tract, providing accurate data for understanding the distribution pattern of duck disease Riegeria in the digestive tract of poultry.

[0020] (2) By combining the specific bacteriophage of Rhizobium anatipestifer, this invention can explore the killing effect of bacteriophage on Rhizobium anatipestifer in the digestive tract of poultry, and provide accurate data for evaluating the application potential of bacteriophage. Attached Figure Description

[0021] Figure 1 Schematic diagram of EGFP-pRES2 plasmid.

[0022] Figure 2PCR identification of EGFP-pRES2-RAf71.

[0023] Figure 3 Dynamic distribution of EGFP-pRES2-RAf71 in the digestive tract of ducks.

[0024] Figure 4 The killing effect of CRP2 / M34 bacteriophage on EGFP-pRES2-RAf71 in the digestive tract. Detailed Implementation

[0025] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments, but this invention is not limited thereto.

[0026] The pRES2 plasmid involved in the embodiments of the present invention is a circular plasmid, and its nucleotide sequence is shown in SEQ ID NO.2.

[0027] Example 1:

[0028] (1) Constructing the expression plasmid EGFP-pRES2 for the enhanced green fluorescent protein gene, the specific steps are as follows: The enhanced green fluorescent protein (EGFP) gene was optimized to obtain the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence shown in SEQ ID NO.1 was artificially synthesized and ligated between the NheI and XhoI restriction sites of the pMD19-T Simple vector, and then transformed into E. coli TOP10 competent cells. Positive strains were screened using LB plates containing 1 μg / mL cefoxitin. After PCR identification and sequencing verification, the positive strain was named EGFP-pMD19-T-TOP10. The plasmid of strain EGFP-pMD19-T-TOP10 was extracted to obtain the EGFP-pMD19-T plasmid. The EGFP-pMD19-T and pRES2 plasmids were double-digested using NheI and XhoI restriction endonucleases. The digestion products were ligated using T4 DNA ligase, and the ligation products were transformed into *E. coli* S17-1 λpir competent cells. Positive strains were screened using LB agar plates containing 1 μg / mL cefoxitin. After double digestion identification and sequencing verification, the positive strain was named EGFP-pRES2-S17. The plasmid EGFP-pRES2-S17 was extracted to obtain the EGFP-pRES2 plasmid (…). Figure 1 ).

[0029] (2) Constructing fluorescently labeled *Riebelella anatipestifer* EGFP-pRES2-RAf71, the specific steps are as follows: streak strain EGFP-pRES2-S17 on LB solid medium plates containing 1 μg / mL cefoxitin and incubate at 37℃ for 8 h; wash the bacterial sludge on the plate with 10 mM MgSO4 solution and adjust the OD of the bacterial solution. 600 The value was 1.0, and EGFP-pRES2-S17 bacterial suspension was obtained. *Riegeria anatipestifer* RAf71 (this strain has been disclosed in existing technology: Cheng Longfei, Zhong Min, Zheng Teng, et al. Isolation and identification of susceptible virulent phages of *Riegeria anatipestifer* [J]. Chinese Journal of Veterinary Science, 2009, 39(2): 106-109.) was inoculated onto TSA solid medium plates and cultured at 37℃ for 12 h; the bacterial sludge on the plates was washed with 10 mM MgSO4 solution, and the OD of the bacterial suspension was adjusted. 525 The value was 1.0, yielding a RAf71 bacterial suspension. 200 μL of EGFP-pRES2-S17 bacterial suspension and 100 μL of RAf71 bacterial suspension were thoroughly mixed, centrifuged, and the bacterial pellet was resuspended in 100 μL of 10 mM MgSO4 solution. The resuspended pellet was added dropwise to the surface of a sterile NC membrane on a TSA solid medium plate and incubated at 30°C for 12 h. The bacterial sludge on the NC membrane was washed off with PBS buffer, and the sludge was spread onto a TSA solid medium plate containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and incubated at 37°C for 48 h. Single colonies were picked from the plate and streaked onto a TSA solid medium plate containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and incubated at 37°C for 48 h for purification. Purified single colonies were picked and resuspended in double-distilled water as PCR templates. Standard PCR reaction systems were prepared using primers for *Riegeria duckweed* detection (gyrBF (85U): 5'-GGAGACGTAGGAGTAAGAGG-3', gyrBR (346L): 5'-CATGAAACCCCTACACCGTG-3') and primers for EGFP detection (GFPF: 5'-AGTGTGAGCGGTGAAGGTGA-3', GFPR: 5'-GGTGCGTTCCTGAACATAGC-3'). The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; and a final extension at 72℃ for 10 min. The amplified products were separated by 10 g / L agarose gel electrophoresis, and the results were observed using a gel imaging system. The expected amplified band sizes were 280 bp (*Riegeria duckweed*) and 210 bp (EGFP). The strain that tested positive in both PCR tests was labeled EGFP-pRES2-RAf71 ( Figure 2 ).

[0030] (3) Flow cytometry detection of EGFP-pRES2-RAf71: Rhizobium anatipestifer RAf71 was inoculated onto TSA solid medium plates and cultured at 37°C for 16 h. Then, a single colony was picked and inoculated onto TSB liquid medium and cultured at 37°C with shaking until the concentration reached 5 × 10⁻⁶. 9 CFU / mL was used to obtain RAf71 bacterial culture. The strain EGFP-pRES2-RAf71 was inoculated onto TSA solid medium plates containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and incubated at 37°C for 36 h. Then, single colonies were picked and inoculated onto TSB liquid medium containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and cultured at 37°C with shaking until the concentration reached 5 × 10⁻⁶ CFU / mL. 9 CFU / mL was used to obtain EGFP-pRES2-RAf71 bacterial suspension. Using RAf71 bacterial suspension as a blank control, flow cytometry was performed on the EGFP-pRES2-RAf71 bacterial suspension, and the results showed that its EGFP positivity rate was 76.8%.

[0031] (4) Dynamic distribution of EGFP-pRES2-RAf71 in the duck digestive tract: Duck plague bacillus RAf71 was inoculated onto TSA solid medium plates and cultured at 37℃ for 16 h. Then, single colonies were picked and inoculated onto TSB liquid medium and cultured at 37℃ with shaking until the concentration reached 5 × 10⁻⁶. 9 CFU / mL was used to obtain RAf71 bacterial culture. The strain EGFP-pRES2-RAf71 was inoculated onto TSA solid medium plates containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and incubated at 37°C for 36 h. Then, single colonies were picked and inoculated onto TSB liquid medium containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and cultured at 37°C with shaking until the concentration reached 5 × 10⁻⁶ CFU / mL. 9 CFU / mL was used to obtain EGFP-pRES2-RAf71 bacterial suspension. Ten-day-old Beijing ducks were randomly divided into two groups of nine ducks each, ensuring that the ducks in each group had similar health status and weight. Each duck in group 1 was orally administered 2 mL of RAf71 bacterial suspension, and each duck in group 2 was orally administered 2 mL of EGFP-pRES2-RAf71 bacterial suspension. At 6, 12, and 24 hours post-administration, three ducks from each group were randomly euthanized, and the contents of the anterior intestinal segment (duodenum to yolk stalk), posterior intestinal segment (yolk stalk to rectum), and cecum were collected. Twice the weight of physiological saline was added to each segment, and the mixture was thoroughly shaken and filtered through a 300-mesh sieve. Using the filtrate from group 1 as a control, flow cytometry was performed on the filtrates from each intestinal segment of group 2 to analyze the distribution of fluorescently labeled bacteria. Results are shown below. Figure 3Six hours after administration, the proportions of fluorescently positive bacteria in each intestinal segment were 20.34% (anterior intestinal segment), 23.61% (posterior intestinal segment), and 5.96% (cecum), respectively. This indicates that fluorescently labeled bacteria were mainly distributed in the anterior and posterior intestinal segments, with a relatively low bacterial count in the cecum. Twelve hours after administration, the proportions of fluorescently positive bacteria in each intestinal segment decreased to 7.86%, 13.54%, and 7.13%, respectively. The bacterial counts in the anterior and posterior intestinal segments decreased significantly, while the bacterial count in the cecum increased slightly. Twenty-four hours after administration, the proportions of fluorescently positive bacteria in each intestinal segment were 2.17%, 10.88%, and 8.66%, respectively. The bacterial counts in the anterior and posterior intestinal segments continued to decrease, while the bacterial count in the cecum increased slightly.

[0032] (5) The bacteriophage mutant strain CRP2 / M34 of *Rhizobium anatipestifer* (disclosed in CN120718861A) inactivates EGFP-pRES2-RAf71 in the duck digestive tract: *Rhizobium anatipestifer* RAf71 was inoculated onto TSA solid medium plates and cultured at 37°C for 16 h. Then, single colonies were picked and inoculated onto TSB liquid medium and cultured at 37°C with shaking until the concentration reached 5 × 10⁻⁶. 9 CFU / mL was used to obtain RAf71 bacterial culture. The strain EGFP-pRES2-RAf71 was inoculated onto TSA solid medium plates containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and incubated at 37°C for 36 h. Then, single colonies were picked and inoculated onto TSB liquid medium containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin, and cultured at 37°C with shaking until the concentration reached 5 × 10⁻⁶ CFU / mL. 9 CFU / mL was used to obtain EGFP-pRES2-RAf71 bacterial culture. Wild-type RAf71 was used as the host bacteriophage, and the phage mutant strain CRP2 / M34 was added for co-culture and amplification. The lysate was centrifuged, filtered through a 0.22 μm filter for sterilization, and the titer was adjusted to 5 × 10⁻⁶. 8PFU / mL is the working solution of phage CRP2 / M34. Healthy 10-day-old Beijing ducks were randomly divided into three groups of three. Group 1 received 2 mL of RAf71 bacterial suspension via gavage. Group 2 received 2 mL of EGFP-pRES2-RAf71 bacterial suspension via gavage. Group 3 received 2 mL of both EGFP-pRES2-RAf71 bacterial suspension and 2 mL of phage CRP2 / M34 working solution via gavage. Six hours after administration, the ducks were euthanized, and the contents of the anterior intestinal segment (duodenum to yolk stalk), posterior intestinal segment (yolk stalk to rectum), and cecum were collected. Each sample was weighed, and twice its weight of physiological saline was added, followed by thorough shaking and filtration through a 300-mesh sieve. Using the filtrate from Group 1 as a negative control, flow cytometry was used to gate the samples, and the proportion of fluorescently positive bacteria in the filtrates from each intestinal segment of Groups 2 and 3 was detected. As shown in Figure 4, compared with experimental group 2 which was administered EGFP-pRES2-RAf71 by gavage alone, experimental group 3, which was combined with phage, showed a significant decrease in the proportion of fluorescent bacteria in each intestinal segment: the proportion in the anterior intestinal segment decreased from 20.54% to 11.03%, the proportion in the posterior intestinal segment decreased from 24.06% to 12.88%, and the proportion in the cecum decreased from 5.85% to 2.34%, with all differences reaching a highly significant level (P<0.01).

Claims

1. A method for fluorescently labeling Riedelella anatipestifer, characterized in that: Includes the following steps: (1) Constructing the expression plasmid EGFP-pRES2 of the enhanced green fluorescent protein gene: The enhanced green fluorescent protein gene was linked between the NheI and XhoI restriction sites of the pMD19-T Simple vector and transformed into Escherichia coli TOP10 competent cells. Positive strains were screened using LB plates containing 1 μg / mL cefoxitin. After PCR identification and sequencing verification, the positive strains were named EGFP-pMD19-T-TOP10. The plasmid EGFP-pMD19-T-TOP10 was extracted to obtain the EGFP-pMD19-T plasmid. The EGFP-pMD19-T plasmid and the pRES2 plasmid were double-digested with NheI and XhoI restriction endonucleases. The digestion products were ligated with T4 DNA ligase. The ligation products were transformed into E. coli S17-1 λpir competent cells. Positive strains were screened by LB plates containing 1 μg / mL cefoxitin. After double digestion identification and sequencing verification, the positive strain was named EGFP-pRES2-S17. (2) Obtaining fluorescently labeled Riesella anatipestifer via conjugation transfer: After treating strains EGFP-pRES2-S17 and Riesella anatipestifer RAf71 separately, the bacterial concentration was adjusted. The two bacterial solutions were mixed, centrifuged, resuspended, and then dropped onto the surface of a sterile NC membrane on a TSA plate for culture. The bacterial sludge was washed off with PBS buffer and spread on a double-antibiotic TSA plate containing 1 μg / mL cefoxitin and 50 μg / mL kanamycin for culture. After single colonies were picked and purified, PCR identification was performed using Riesella anatipestifer detection primers and EGFP detection primers. The strain that was positive for both PCR tests was the fluorescently labeled Riesella anatipestifer EGFP-pRES2-RAf71.

2. The method according to claim 1, characterized in that: The nucleotide sequence of the enhanced green fluorescent protein gene described in step (1) is shown in SEQ ID NO.

1.

3. The method according to claim 1, characterized in that: The treatment method of the strain EGFP-pRES2-S17 in step (2) is that the strain EGFP-pRES2-S17 is streak-inoculated on a LB plate containing 1 μg / mL cefoxitin, and cultured at 37°C for 8 h; the bacterial slurry on the plate is washed with 10 mM MgSO4solution and washed, and the OD value of the bacterial solution is adjusted to 1.0 to obtain the EGFP-pRES2-S17 bacterial solution. 600 value is 1.0 to obtain the EGFP-pRES2-S17 bacterial solution.

4. The method according to claim 1, characterized in that: The treatment method of the duck Riemerella anatipestifer RAf71 in step (2) is as follows: the duck Riemerella anatipestifer RAf71 is inoculated on a TSA plate and cultured at 37℃ for 12 hours; the bacterial slurry on the plate is washed with a 10 mM MgSO4 solution and washed, and the OD value of the bacterial solution is adjusted to 1.0 to obtain the RAf71 bacterial solution. 525 value is 1.0, to obtain the RAf71 bacterial solution.

5. The method according to claim 1, characterized in that: The primers used for PCR identification in step (2) for detecting *Riegeria duckweed* are gyrBF: 5'-GGAGACGTAGGAGTAAGAGG-3', gyrBR: 5'-CATGAAACCCCTACACCGTG-3'; and the primers for detecting EGFP are GFPF: 5'-AGTGTGAGCGGTGAAGGTGA-3', GFPR: 5'-GGTGCGTTCCTGAACATAGC-3'.

6. The method according to claim 1, characterized in that: The PCR identification reaction procedure described in step (2) is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; and finally 72℃ extension for 10 min.

7. A fluorescently labeled *Riebelella anatipestifer* EGFP-pRES2-RAf71, characterized in that: It is prepared by the method described in any one of claims 1 to 6.

8. The use of the fluorescently labeled *Riegeria anatipestifer* EGFP-pRES2-RAf71 as described in claim 7 in the preparation of a detection reagent for detecting the dynamic distribution of *Riegeria anatipestifer* in the digestive tract of ducks.

9. The application of the fluorescently labeled *Riegeria anatipestifer* EGFP-pRES2-RAf71 as described in claim 7 in the preparation of a screening model for screening bacteriophages that kill *Riegeria anatipestifer*.

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