Patent Publication Number: US-11045537-B2

Title: Klebsiella pneumoniae from minks and its application

Description:
TECHNOLOGY FIELD 
     The present innovation belongs to the technical field of veterinary biological products, and in particular relates to a  Klebsiella pneumoniae  from mink and its application. 
     TECHNOLOGY BACKGROUND 
     Mink farming is a special farming mainly to produce skin. Mink skin is thin, pliable and strong. It is the main raw material for making clothes, collars, hats, cuffs and ornaments. From the perspective of the quality and value, its utilization rate is higher than that of fox skin, raccoon skin and other large-size skins, so it has great potential in the domestic market. The large-size skins such as fox skin and raccoon skin are thicker and longer than mink skin, and are not as gorgeous as mink skin, so they are not suitable for making coats. They are only suitable for making collars, cuffs, shawls and other accessories. Therefore their utilization rate is lower than that of mink skin. 
     However, in recent years, with the increase in mink prices, the farming volume has become larger and larger, and the farming density has continued to increase, resulting in an increase of diseases, among which, hemorrhagic pneumonia in minks is the most harmful to mink farming. Hemorrhagic pneumonia in minks mainly occurs when the minks&#39; summer hair falls off in autumn. When the minks&#39; summer hair falls off, the hair that has fallen off is flying around in the farm, and the bacteria adhering to the hair can contaminate feed and drinking water at any time and cause infection. The attack of hemorrhagic pneumonia in minks is mainly acute, and infected minks often die without any symptoms. Bloody foam comes out of the mouths and noses of the sick and dead minks. The symptoms of hemorrhagic pneumonia in minks mainly include dyspnea, hemorrhagic pneumonia changes, pulmonary edema, hemorrhagic pathological changes of organs and gastrointestinal mucosal ulcers, and individual mink will also occur increased eye secretions, paw enlargement, etc. Hemorrhagic pneumonia in minks is often endemic, causing greater economic losses to farmers. 
     When hemorrhagic pneumonia in minks occurs, it is often too late to treat. The commonly used drugs tilmicosin, azithromycin, kanamycin are not effective, and the abuse of antibiotics will make the pathogenic bacteria resistant to drugs, so effective vaccines are needed to prevent it. At present, in the prevention of hemorrhagic pneumonia in minks, most countries mainly use inactivated bacterial vaccine products, that is preparing polyvalent inactivated vaccines by inactivating epidemic serotype strains, which can effectively prevent the disease caused by similar serotype strains. 
     SUMMARY OF THE INVENTION 
     The purpose of the present innovation is to provide a  Klebsiella pneumoniae  from mink and its application. The provided  Klebsiella  is a new strain with mutation, and the vaccine prepared by using it can provide more comprehensive protection for minks, thereby making up for the deficiencies of the existing technology. 
     The  Klebsiella pneumoniae  SD-1 strain of the present innovation was deposited in the China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing on Jun. 5, 2019, and the collection number is CGMCC NO: 17901. 
     The  Klebsiella pneumoniae  SD-1 strain of the present innovation is used for preparing vaccine. 
     The preferred form of the said vaccine is the inactivated vaccine. 
     The present innovation also provides an inactivated vaccine, which comprises an inactivated antigen and a vaccine adjuvant, wherein the inactivated antigen is inactivated  Klebsiella pneumoniae  ( Klebsiella  sp.) SD-1 strain. 
     The said vaccine adjuvant is an aqueous vaccine adjuvant, more specifically, aluminum hydroxide gel. 
     The  Klebsiella pneumoniae  present of the present innovation was screened from minks that died after the vaccination. Compared with the current  Klebsiella pneumoniae , the strain has mutated and is a new type of mutated pathogenic strain. The said strain is used to prepare the vaccine to provide better immune protection for minks. 
    
    
     DETAILED DESCRIPTION 
     The present innovation uses the conventional techniques and methods in the fields of genetic engineering and molecular biology, such as MOLECULAR CLONING: the method described in A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the field. 
     The present innovation will be described in detail in combination with the following embodiments. 
     Embodiment 1: Analysis of Purification and Physicochemical Properties of Isolation of Strains 
     In 2018, the symptoms of hemorrhagic pneumonia in mink appeared in a mink farm in Qingdao, but the affected minks had previously been injected with an officially launched triple inactivated vaccine against hemorrhagic pneumoniae  P. aeruginosa  in minks,  Pasteurella multocida  and  Klebsiella pneumoniae.    
     1) Take a dead mink and use a scalpel to aseptically open the chest and abdominal cavity of the sick and dead mink. Cut the liver of the sick and dead mink to fully expose its internal tissues. Dip the contents of the liver with a sterile cotton swab and then carry out gradient dilution on it with normal saline, then make a streak on the LB solid agar medium, and culture the culture dish with bacteria at 37° C. for 10-12 h. Select single bacterial colonies. Culture each single colony on the urinary tract flora chromogenic medium. 
     2) Strains showing different colors on the chromogenic medium will be inoculated into LB liquid medium as a group of 3 for expanded culture, and then centrifuge it at 4000 rpm to collect bacterial cells. Resuspend the bacterial cells with 0.5% sterile normal saline, and set the bacterial concentration of the strains as 1×10 7  CFU/mL by turbidimetric method for injection infection. 
     4) Carry out inject infection experiments with healthy minks that have been inoculated with the triple inactivated vaccine against hemorrhagic pneumoniae  P. aeruginosa  in minks,  Pasteurella multocida  and  Klebsiella pneumoniae . Inject 0.2 mL of bacterial suspension of different strains to be screened into the abdominal cavity, and inject 0.5% sterile normal saline, of the same volume into the control group. For the affected minks, the three single colonies in the combined bacterial suspension will be separately tested for injection infection. 
     Finally, a strain that is blue on the urinary tract flora chromogenic medium is determined. Injection infection shows that it will cause the symptoms of hemorrhagic pneumonia in minks who have been injected with the mink hemorrhagic pneumonia vaccine. 
     The strain can form grayish white and round raised colonies on the serum LB agar medium. Grain-negative. 
     Extract the bacterial DNA with a bacterial genomic DNA extraction kit, perform PCR amplification with the 16S rRNA bacterial identification primers, then go through electrophoresis, recovery, cloning and sequencing, and analyze the sequencing results. Send the PCR amplification products to the sequencing company for sequencing. After sequence alignment on NCBI, the 16S rRNA sequence has the highest homology with  Klebsiella , so it is suspected to belong to  Klebsiella.    
     Since the Khe gene can be used as a target gene for identifying  Klebsiella pneumoniae , primer pairs that amplify the Khe gene are used to detect the genomic DNA of the screened strain. The sequence information of the primer pairs is as follows: 
     
       
         
           
               
               
            
               
                   
                 Upstream primer: 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 5′-TGATTGCATTCGCCACTGG-3′ 
               
               
                   
                   
               
               
                   
                 Downstream primer:  
               
               
                   
                 ( SEQ ID NO: 4) 
               
               
                   
                 5′-GGTCAACCCAACGATCCTG-3′ 
               
            
           
         
       
     
     The amplification results show that positive fragments appear in the strain, which is consistent with 16S rRNA, confirming that the screened strain is  Klebsiella pneumoniae  and is named SD-1 strain. 
     Considering that SD-1 strain will cause disease in minks that have been vaccinated, in order to verify whether the pathogenic gene of SD-1 strain has been mutated, the  Klebsiella pneumoniae  SD-1 strain and pathogenesis-related rmpA gene, uge gene, magA gene, wabG gene, capsular polysaccharide K2 gene are amplified and sequenced. Use EasyPure Quick Gel Extraction Kit to recover PCR products, connect to pEASY-Blunt Cloning vector or pMD18-T Vector (Promega) to transform Trans-5α competent cells, and then sequence after identification. Use DNAMAN software to deduce the amino acid sequence. 
     Perform amino acid homology comparison of the pathogenesis-related gene fragments with the  Klebsiella pneumoniae  sequences published on GenBank. The results show that the pathogenesis-related uge gene, magA gene, and wabG gene have multiple mutations at the amino acid level, and the nucleotide sequence of rmpA gene is SEQ ID NO: 1 and the amino acid sequence is SEQ ID NO: 2. Compared with what have been reported, the amino acid homology is only 70%, which may be the reason why the immunity of existing  Klebsiella  genes is not good. 
     
       
         
           
               
               
            
               
                   
                 ( SEQ ID NO: 1) 
               
               
                   
                 ACTGGGCTACCTCTGCTTAACAAGACATTTGAAGCAGTAAT 
               
               
                   
                   
               
               
                   
                 TAATAAATCAATATTGATGAAGCACAAAAAAAACATAAGAG 
               
               
                   
                   
               
               
                   
                 TATTGGcTGACTGCGGGtTTTTTTATTCATCCACATGGAGA 
               
               
                   
                   
               
               
                   
                 GGGTACAAAATGTTAAGATCCACATTAcATATGATAAGCCA 
               
               
                   
                   
               
               
                   
                 ATGGAcAcGGCTTcATGTTTCGGGGGGGGGGCGGTTTcACC 
               
               
                   
                   
               
               
                   
                 TTTGcAGCAGGTGTGcTTATTACcTtTATGTTAAaATGCAA 
               
               
                   
                   
               
               
                   
                 GATCCACTAAAAAGTATTGGcTcAAAACTATATcTTGCAcT 
               
               
                   
                   
               
               
                   
                 CTcAAAGAAAAATGTACTGGGAATTGTAAAaCATTATCCtC 
               
               
                   
                   
               
               
                   
                 GGCTAACAAAAAAAGAACAATGGATGCTGCAA 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 TGLPLLNKTFEAVINKSILMKHKKNIRVLADCGFFYSSTWR 
               
               
                   
                   
               
               
                   
                 GYKMLRSTLHMISQWTRLHVSGGGRFHLCSRCAYYLYVKMQ 
               
               
                   
                   
               
               
                   
                 DPLKSIGSKLYLALSKKNVLGIVKHYPRLTKKEQWMLQ 
               
            
           
         
       
     
     Embodiment 2: Detection of the Pathogenic Effect of SD-1 Strain 
     First, inoculate  Klebsiella pneumoniae  SD-1 strain on the LB solid plate medium and cultivate it at 37° C. for 24 hours. Then select single colonies to inoculate into the LB liquid medium, stir it at 37° C. in a ventilated environment and then culture it for 24 h, then centrifuge it at 4000 rpm for 10 min to collect the precipitate, then resuspend the precipitate with sterile normal saline, and count the viable bacteria in the suspension. Prepare for animal injection infection experiments. 
     Select twenty heads of 30-40 day-old healthy and susceptible minks and randomly divide them into 4 groups with 5 heads in each group. Dilute the fermentation solution of  Klebsiella pneumoniae  SD-1 strain into three different gradients of 1.0×10 6  CFU, 4.0×10 6  CFU/ml and 6.0×10 6  CFU/ml, and inoculate 2.0 ml into each of the minks of a group and inject 2.0 l of sterile normal saline, into each of the minks of another group as a control. 
     Observe for 7-20 days and record the clinical situation. The results show that most minks in the group injected with the fermentation solution experience clinical symptoms of hemorrhagic pneumonia in minks such as increased body temperature, decreased appetite, abdominal breathing, hemoptysis or red foamy liquid from the nostrils or even death. The body temperature of the minks in the control group does not change significantly, and there are no clinical symptoms or death in the group (Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Infectivity of Klebsiella pneumoniae SD-1 Strain 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Number 
                 Challenged  
                 Number of 
                 Number of 
               
               
                   
                 of 
                 viral 
                 challenged 
                 infected 
               
               
                 Group 
                 injections 
                 dosage 
                 bacteria 
                 minks 
               
               
                   
               
               
                 SD-1 
                 5 
                 2 ml 
                 1.0 × 10 6  CFU 
                 4/5 
               
               
                 strain 
                   
                   
                   
                 incidence 
               
               
                   
                 5 
                 2 ml 
                 4.0 × 10 6  CFU 
                 5/5 
               
               
                   
                   
                   
                   
                 incidence 
               
               
                   
                 5 
                 2 ml 
                 6.0 × 10 6  CFU 
                 5/5 
               
               
                   
                   
                   
                   
                 incidence 
               
               
                 Control 
                 5 
                 2 ml 
                 Normal 
                 0/5 
               
               
                 group 
                   
                   
                 saline 1 
                 incidence 
               
               
                   
               
            
           
         
       
     
     The above results show that the minimum amount of  Klebsiella pneumoniae  SD-1 strain that can cause the 5/5 incidence of a healthy and susceptible mink 30 to 40 days old is 4.0×10 6  CFU/ml, which indicates that the strain selected by the present innovation has strong pathogenicity. 
     Embodiment 3: Preparation of Vaccines Using  Klebsiella Pneumoniae  SD-1 Strain 
     1. Preparation of Inactivated Vaccines: 
     First, inoculate  Klebsiella pneumoniae  SD-1 strain on the LB solid plate medium and cultivate it at 37° C. for 24 hours. Then select single colonies to inoculate into the LB liquid medium, stir it at 37° C. in a ventilated environment and then culture it for 24 h, then add 0.1% formaldehyde solution to the total amount of the bacterial solution, inactivate it for 24 hours, and stir 3 times during this period. Add aluminum hydroxide gel with a volume percentage of 30% into the obtained inactivated bacterial solution to prepare vaccines. Prepare three batches of vaccines in this way. 
     Vaccine inactivation test: Take and add 100 μl of formaldehyde-inactivated bacterial solution into 3 ml liquid medium, and take three samples per culture and inoculate each sample in three test tubes and observe for five days for inactivated security test, if the liquid medium in the test tubes remains clear without turbidity after five days, it indicates that the prepared vaccines meet the requirements. 
     The prepared vaccines are tested without bacterial growth according to page 42 of Appendix of  Veterinary Pharmacopoeia of the People&#39;s Republic of China  (2010 edition). 
     Inject the three batches of prepared vaccines into the inner muscles of the hind limbs of healthy minks with three injection volumes of single dose, single dose repeat and overdose. After 10 days of inoculation, the body temperature and appetite of the inoculated minks are normal, there is no swelling and inflammation in the inoculated area, there is no adverse local and systemic immune reactions, and the inoculated minks eat normally, indicating that the vaccines are safe for minks. 
     2. The Immune Effect of the Vaccines 
     Randomly divide the 2-3 month-old healthy minks into six groups, 10 heads in each group. There were 40 heads in the vaccine injection groups and 20 in the control groups. Inject 0.2 ml of the 5×10 7  cfu/mL mink  Klebsiella  inactivated vaccine prepared in Embodiment 2 into the inner muscles of the hind limbs of each of the 20 minks in the first vaccine injection group. Inject the commercially available triple inactivated vaccine for hemorrhagic pneumonia in mink into the other 20 minks in the second vaccine injection group, and the minks in the control groups are injected with normal saline, only. 
     Thirty days after inoculation, conduct infection experiments using CMCC (B) 46117, the standard strain of  Klebsiella pneumoniae , ATCC 13883, the mixed strain of  Klebsiella pneumoniae , and SD-1 strain, respectively. 
     Inoculate infected bacteria into the muscles of minks in the experimental groups and the control groups, feed and observe the minks for 7 days, and record the attack and mortality of the minks in each group. The experimental results are shown in Table 1 and Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Vaccine Immunization Efficacy Data Sheet for Infection 
               
               
                 with a Mixed Strain of CMCC (B) 46117 and  
               
               
                 ATCC 13883  Klebsiella pneumoniae   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Number of 
                 Survival  
                   
               
               
                   
                   
                 challenged minks 
                 number 
                 Survival  
               
               
                   
                 Group 
                 (head) 
                 (head) 
                 rate (%) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Vaccine group 1 
                 10 
                 9 
                 90 
               
               
                   
                 Vaccine group 2 
                 10 
                 10 
                 100 
               
               
                   
                 Control group 
                 10 
                 3 
                 30 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Vaccine Immunization Efficacy Data Sheet for  
               
               
                 Infection with QDNDSD-1 Strain 
               
            
           
           
               
               
               
               
            
               
                   
                 Number of 
                 Survival  
                   
               
               
                   
                 challenged minks 
                 number 
                 Survival  
               
               
                 Group 
                 (head) 
                 (head) 
                 rate (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Vaccine group 1 
                 10 
                 10 
                 100 
               
               
                 Vaccine group 2 
                 10 
                 6 
                 60 
               
               
                 Control group 
                 10 
                 2 
                 20 
               
               
                   
               
            
           
         
       
     
     Results of the above infection experiments show that the inactivated vaccine prepared by the SD-1 strain of the present innovation can provide complete protection against the infection of SD-1 strain. The minks in vaccine group 1 do not show any clinical symptoms and there is no death phenomenon. However, the commercially available vaccine only provides partial protection against infection with the SD-1 strain. Therefore, the vaccine prepared by existing mink  Klebsiella  can not sufficiently cross-protect the mink  Klebsiella  SD-1 strain screened by the present innovation, and combined vaccine is needed to be prepared with the QDNDSD-1 strain to provide more complete protection.