Vaccines for fowl colibacillosis
This invention relates to a vaccine useful for the protection of poultry from colibacillosis infections. More specifically, this invention relates to a vaccine which comprises as an active ingredient ultrasonically disintegrated cells of Escherichia coli which have been pre-inactivated. This invention also relates to a method for protecting poultry from colibacillosis infections which comprises inoculating the vaccine of the invention into the poultry through cloaca. 
     &#34;Fowl colibacillosis&#34; includes various diseases in poultry caused by pathogenic E. coli strains (colibacillus), such as colisepsis, arrthritis, hemorrhagic enterisis, and the like. Mass outbreak of colisepsis in broilers, among others, brings serious economical damage to farmers. Accordingly, development of any efficient method for the treatment and prophylaxis of the fowl colibacillosis has long been a major problem to poultry farmers. 
     Chemotherapeutics and antibiotics have been hitherto used therefor. However, the value of these medicines is now decreasing because of the appearance of resistant strains. Therefore, in place of these medicines, a vaccine useful for the protection of poultry from colibacillosis infections has been developed. Thus, vaccine for colibacillosis which comprises whole cells of inactivated E. coli has already been known. However, this inactivated vaccine is not only highly toxic, but also less effective in protecting poultry from colibacillosis infections. 
     On the other hand, it has already been suggested that toxicity of the inactivated vaccine and appearance of resistant strains can be reduced when the vaccine is inoculated into poultry through cloaca (Japanese Patent Publication (unexamined) No. 131920/1983). 
     It has now been found that prophylactic activity of inactivated E. coli cells is significantly increased when the inactivated cells are ultrasonically disrupted, and that such pre-inactivated and ultrasonicated E. coli cells have desirable properties for use in vaccines to be employed for the treatment and prophylaxis of colibacillosis infections in poultry. It has also been found that the vaccine prepared as such shows less toxicity when administered through cloaca as previously suggested. 
     Accordingly, in one aspect, this invention provides a vaccine useful for the protection of poultry from colibacillosis infections which comprises E. coli cells which have been pre-inactivated and ultrasonicated. 
     This invention also provides a method for protecting poultry from colibacillosis infections by inoculating the vaccine into poultry through cloaca. 
     As use herein, &#34;pre-inactivated E. coli cells&#34;  means E. coli cells which have been inactivated in usual ways. 
     &#34;Pre-inactivated and ultrasonicated E. coli cells&#34; refers to E. coli cells which have been subjected to an inactivating process and then to an ultrasonic disruption process. 
     &#34;Poultry&#34; refers to domestic fowls such as chickens, ducks, quails, pigeons, turkeys, and the like. 
     DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENT 
     Preparation of pre-inactivated E. coli cells 
     Pathogenic E. coli cells are isolated from organs (e.g. air sac) or droppings of poultry suffering from colibacillosis and grown in a culture medium by conventional methods. The resulting cell culture is treated by an inactivating agent such as formalin (a 40% aqueous formaldehyde solution), phenol, acetone, alcohols, and the like. As will be understood to those skilled in the art, any other known inactivating methods, for example, lyophilisation and heat treatment can also be used. 
     Preparation of pre-inactivated and ultrasonicated E. coli cells 
     The inactivated cell culture prepared above is subjected to an ultrasonic disruption treatment using any commercially available apparatus (e.g. Cell Disruper 200, BRANSON) under appropriate conditions. Suitable conditions for the disruption treatment may vary depending on the particular apparatus and other factors. When Cell Disruper 200 is employed, the ultrasonic disruption may be conducted under the following conditions: maximum output, 50% oscillation frequency, for 5 minutes, three times, with cooling on ice. Any other ultrasonicating apparatus can be used for preparing the vaccine of the invention, provided that such apparatus can successfully injure and destroy E. coli cells. 
     Alternatively, the inactivated cell culture may be first centrifuged to collect the cells, which are then suspended in PBS to prepare a bacterial solution. The resulting bacterial solution is then subjected to the ultrasonic disruption treatment as mentioned above. 
     Vaccine preparation 
     A process for the preparation of vaccines which contain inactivated bacteria cells as an active ingredient is well known to the art. The vaccine of the present invention which contains pre-inactivated and ultrasonicated E. coli cells can be prepared in the same manner as the known vaccines. 
     The vaccine of the invention may contain an adjuvant, for example, an aluminum compound such as aluminium hydroxide gel, or Freund&#39;s complete adjuvant. The vaccine of the invention may also contain a preservative for prevention of the growth of undesirable microorganisms. Preferred preservatives are, for example, marzonine, tyrosine, benzoic acid, formaldehyde solution, salicylic acid, crystal violet, surfactants such as benzetonium chloride, polymyxin, gramicidine, and the like. 
     The vaccine of the invention can be conventionally administered, for example, intramuscularly, intravenously, or subcutaneously. However, such administration routes may sometimes cause weight loss of poultry as in the case of known vaccines. However, such disadvantageous effect can be reduced or diminished by inoculating the vaccine through cloaca as stated before. Typically, 0.002-0.2mil (about 2×10 5  -2×10 10  CFU/ml) of vaccine of the invention is inoculated through cloaca into each poultry such as chicken, duck, or turkey. Such doses are sufficient to protect the poultry from colibacillosis and to prevent mass infections of said disease. 
    
    
     The following examples are provided to further illustrate and exemplify the present invention. 
     Example 1 
     (1) Vaccine (1) 
     Pathogenic E. coli strains (O 2 ) isolated from air sacs of chickens suffering from colibacillosis were incubated in a broth medium at 37° C. for about 24 hours. To the cell culture was added formalin to a final concentration of 0.2%. The inactivated cell culture thus obtained contained more than 1.5×10 9  cells per ml (CFU/ml). A portion of the cell culture was ultrasonicated using Cell Disruper 200 (BRANSON) at 50% oscillation frequency under maximum output (5 minutes, 3 times) with cooling on ice. This gave vaccine (1) of the invention. 
     (2) Vaccine (2) 
     Inactivated cells were collected by centrifugation (14000×G for 20 minutes at 4° C.), and then suspended into PBS to prepare a bacterial solution which contained inactivated E. coli cells at a density of 2×10 10  CFU/ml. The bacterial solution was then applied to the aforementioned Cell Disruper to yield vaccine (2) of the invention. 
     The PBS solution was prepared as follows: 11.9g of NaH 2  P 4  12H 2  O, 3.6g of NaH 2  PO 4  2H 2  O, and 0.6g of NaC1 were throughly admixed. To the mixture was added distilled water to a final volume of 1 liter, and the resultant solution was sterilized before use. 
     The vaccines prepared above were inoculated into poultries, and the immunization activity thereof was evaluated as descried below. 
     Experiment 
     Pathogenic E. coli solution and its inactivated solution prepared in Examples (1,1) and (1,2) were used as positive control vaccine (1) and positive control vaccine (2) respectively. Eleven chicks (Chankey, broiler species) within 30 hours after hatching were used for tests. Each chick was inoculated with 0.03ml of vaccine (1), vaccine (2), positive control vaccine (1) or positive control vaccine (2) through cloaca by intravenous drip method (see Japanese Patent Publication (unexamined) No. 131920/1983). When the chick become 21-days-old, 6×10 7  CFU of pathogenic E. coli cells, the same strain as used in the preparation of the vaccines, were intravenously injected into each chick. Another group of 12 chicks received only the pathogenic E. coli cells, which served as a negative control. Prophylactic action of the vaccines was evaluated on the basis of the number of survivals on the seventh day after infection, and survival rates were statistically analyzed using χ 2  test. The results are shown below in Table 1. 
     
                                           TABLE 1                                 
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Immunization Effect of Vaccine (1) and Vaccine (2)                        
              Number of Dead Ani-                                         
        Number of                                                         
              mals After Infection                                        
                            Number of                                     
        Chicks                                                            
              1 2 3 4 5 6 7 Survivals                                     
        Tested                                                            
              (Days After Infection)                                      
                            (Survival Rate)                               
                                    χ.sup.2 Test                      
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Negative control                                                          
        12    7 2 l 0 0 0 0 2 (16.7)                                      
                                    --                                    
Vaccine (1)                                                               
        11    2 1 l 0 0 0 0 7 (63.6)                                      
                                    *                                     
Positive control                                                          
        11    4 1 1 0 0 0 0 5 (45.5)                                      
vaccine (1)                                                               
Vaccine (2)                                                               
        11    0 0 0 1 0 0 0 10 (90.9)                                     
                                    **                                    
Positive control                                                          
        11    0 3 2 1 0 0 0 5 (45.5)                                      
vaccine (2)                                                               
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 *P &lt; 0.05 (statistically significant)                                    
 **P &lt; 0.01 (statistically significant)                                   
 
    
     The table shows that the vaccines of the invention exhibit remarkable prophylactic action when compared with the conventional vaccines.