Title of Invention	NOVEL BIOFILM VACCINES AGAINST BACTERIAL DISEASES OF BIRDS AND DOMESTIC ANIMALS
Abstract	The specification describes a procedure of preparing biofilm vaccine against bacteria causing diseases in birds and domestic animals wherein the bacteria are cultured as biofilm cells and harvested by discarding the supernatant media to remove any planktonic cells. Number of viable bacteria biofilm cells were enumerated by colony counting and counts expressed as colony forming units/gram of bentonite clay and was adjusted to the desired optimal concentration. Bacteria were then inactivated using chemicals such as formalin and stored at 4°C until use, after checking the vaccine for sterility and safety by known methods. Final vaccine preparation was packed into appropriate dose delivery packages.

Title of Invention

NOVEL BIOFILM VACCINES AGAINST BACTERIAL DISEASES OF BIRDS AND DOMESTIC ANIMALS

Abstract

The specification describes a procedure of preparing biofilm vaccine against bacteria causing diseases in birds and domestic animals wherein the bacteria are cultured as biofilm cells and harvested by discarding the supernatant media to remove any planktonic cells. Number of viable bacteria biofilm cells were enumerated by colony counting and counts expressed as colony forming units/gram of bentonite clay and was adjusted to the desired optimal concentration. Bacteria were then inactivated using chemicals such as formalin and stored at 4°C until use, after checking the vaccine for sterility and safety by known methods. Final vaccine preparation was packed into appropriate dose delivery packages.

Full Text	"Novel Biofilm Vaccines against bacterial diseases of birds and Domestic animals". Field of Innovation: This innovation describes novel vaccine in the field of poultry vaccines Prior art: Among the bacterial diseases, colibacillosis is the leading cause of morbidity and mortality in poultry and responsible for significant economic loss in all the phases of poultry Industry from production to marketing. The disease is caused by Escherichia coli, consisting of a large number of serotypes. Besides colisepticemia, Escherichia coli is also responsible for various other disease conditions with mortality of five to ten per cent, and some times may be as high as 50 per cent. Immunization of chicken against colibacillosis has been attempted by numerous workers without much success. Escherichia coli antigens used for active immunization of chicken include bacterial pili {Gyimah and Panigrahy, 1985; Gyimah et al., 1986 and Zigterman et a/., 1993); live bacterial cells (Arp, et al., 1979; Arp, 1980; and Frommer ef a/., 1994), and killed bacteria with adjuvants (Deb and Harry, 1976 and 1978; Rosenberger et al., 1985; Lietner et al., 1990; Tengerdy et at., 1990; Franchini et al., 1991 and MeJamed ef al., 1991). ten all these trials, immunity has been found to be seen only against homologous but not to heterologous challenge; that is protection tends to be serotype limited. Thus several attempts of vaccination to achieve protection against avian colibacillosis both in older {active immunization) and younger (passive immunization) birds have been reported. Subsequent attempts to develop effective vaccine by including only the most predominant serigraphs that are frequently associated with colisepticemia, 01 :K1, 02:K1 and O78:K80 were also not successful. It is reported that a vaccine capable of conferring protection must be a broad based one to be most effective against colibacillosis caused by varied serotypes, in view of the serological and chemical differences between different serotypes. {Ewing et ai, 1956 and Orskov et al., 1967 and 1971). There are also reports by Deb and Harry, (1976); Arp, (1980) and Kapur et al, (1992) that cross protection is usually not observed with Escherichia coll serotypes from poultry and that a suitable vaccine should contain at least the most common serotypes, but also opined that efficacy of such a vaccine gets adversely affected by antigenic competition between different serotypes. Further studies were aimed at exploring the common antigens among Escherichia coli in general and APEC in particular. Culturing bacteria under iron-deprived media was one such attempt made to explore common virulence factors of Escherichia coil (Griffiths et al., 1983). Evidently, organisms grown in the host will have their changed characters both structurally and genetically with selective expression of new antigens. These antigens in the form of vaccine may be responsible for providing broad based protection. The above considerations suggest that any vaccine to be successful against Escherichia coli infections should be able to give cross protection against a plethora of serotypes and development of such a safe and effective vaccine has eluded the researchers despite prompt attempts. For a vaccine to be broad based, the part the bacteria (the antigen) with disease producing ability and which is commonly found in other serotypes of Escherichia coli needs to be explored. Bacterial biofilm is a structured community of bacterial cells enclosed in a self-produced glycocalyx matrix and attached to living or non living surface, which constitutes a protected mode of growth that allows survival in hostile environment (Costerton et ai, 1999). Vaccines developed on the basis of the biofilm production represent the growing of organisms in a natural pathogenic ecosystem {Brown and Williams, 1985). Studies on gene expression pattem has indicated that approximately 40 per cent of the genes in in vitro grown biofilm bacteria may be different from those of their planktonic counterparts (OToole et ai, 2000) and there by express some Outer Membrane Proteins (OMPs), which are similar to ones expressed during natural infection (Brown and Williams, 1985). Whether providing a nutrient restricted medium with an appropriate surface for attachment, E. coli would form biofilm, and if so, whether this "simulated in vivo" environment would lead to the expression of some of the antigens of a natural infection has still not been investigated. Therefore, there is a need to test if E. coli can be grown in a laboratory media on a biofilm matrix and test the vaccine potential of protein antigens obtained from such a E. co//culture Summary of invention The biofilm form of E. coli grown under defined in vitro condition, has been exploited to evolve an effective, safe, cross protective vaccine against colibacillosis caused by different serotypes in poultry. The maximum production of E. coli biofilms was found at 0.16 per cent tryptone soya broth with 0.3 per cent w/v bentonite clay as inert surface in seven days. The viability of E. coli in biofilms was significantly higher even at 100 days, on the other hand; nutrients restricted and free cells with bentonite clay, declined very rapidly after 60"" day post inoculation. Analysis of the outer membrane protein profiles of the biofilm, nutrient restricted and free cells revealed repression of the 58 and 42 kDa and over-expression of 61, 35.5, 34 and -28.8 kDa OMPs in biofilm cells and nutrient restricted cells when compared to the free cells. In addition, three unique OMPs of 93.2, 61.0 and 28.8 kDa molecular masses were detected in biofilm cells, which were completely absent in the free cells. Killed biofilm and free cell vaccines were used at the rate of 1X10^ cfu per bird and the same dose was used for challenge studies. Subcutaneous route of vaccination with biofilm vaccine offered 100 per cent protection compared to 58 per cent with free cell vaccine followed by severe live challenge infection by intramuscular route. Vaccination through oral route with biofilm vaccine was efficient in significantly reducing the colonization / elimination of Escherichia co//compared to free cell vaccine, upon challenge by intranasal route, which is considered as natural mode of infection. Field vaccination trials at farm level with vaccine prepared from biofilm grown bacteria conferred cross protection to the extent of 100 and 80 per cent in subcutaneous and oral vaccinated birds respectively. The biofilm vaccine given through subcutaneous route resulted in low feed conversion ratio compared to unvaccinated controls, to the extent 1.68 to 2.06. Challenge studies in field vaccination trials established the ability of biofilm vaccine to give cross protection. Thus it is the first time that cross protection was established among various serotypes of Escherichia coli. The molecular basis for cross protection was established by identifying cross protective OMPs of 93.2, 61, 35.5, 34, 28.8 and 17.6 kDa proteins by western blott. Detailed description of invention Example 1. Method of growing Escherichia coU"in biofilm: c^ For production of biofilm cells, bentonite clay {0.3 % w/v) was suspended in 0.08, 0.16, 0.32 and 3.0 per cent tryptone Soya Broth (TSB). Identical concentrations of TSB media viz., 0.08, 0.16, 0.32 (nutrient restricted) 3.0 per cent (free cell) were used without incorporating bentonite clay to serve as controls. For each concentration, ten different conical flasks of 250 ml capacity with 100 ml each of the above-defined media were used and all the media were sterilized by autoclavlng at 15 lbs pressure at 121° C for 15 min. After checking for sterility, these flasks were ^ Inoculated with the prepared inoculum and incubated at 37°C. The culture flasks with bentonite clay were agitated six times daily in a mechanical shaker at 50 rpm for ^ one hourto keep ttie bentonite clay uniformly suspended in the media. The biofilm cells, nutrients restricted (NR) cells, and free cells with and without bentonite clay were harvested on days one, three, five, seven, ten, twenty, thirty, sixty and one hundred, post-Inoculation. One conical flask of each concentration was used for each of the days, thus making use of nine out of ten flasks inoculated and remaining one flask In each of the concentrations served as reserve. The biofilm cells were quantified by concentrating the cells colonized on bentonite clay surface at 1000 rpm for five min. The supernatant was discarded and the pelleted biofilm colonized on bentonite clay was washed thrice with PBS {pH 7.4) to remove non adherent bacteria, made up the volume to 10 ml with sterile PBS in a test tube and vortexed vigorously for three min to dislodge the biofilm cells from bentonite clay. Biofilm cells thus obtained in supernatant were enumerated. Similarly, viable counts for NR cells and free cells grown with and without bentonite ■, clay were determined up to 100"" day post-Inoculation at different intervals in all the concentrations of TSB. The spread plate method described by Postgate (1969) was employed to enumerate the surface viable counts. The average number of colonies per plate was multiplied by the dilution factor to obtain the viable count in the original suspension and expressed as cfu/ml for the NR and free cells and cfu/g of bentonite clay for biofilm cells. The highest diiution of TSB at which maximum viable counts of bacterial cells were obtained from among the different days of intervals tested was taken as optimum conditions required for good biofilm / NR cell growth. The optimum concentration of TSB required for maximum biofilm formation of Escherichia coli was found to be 0.16 per cent with 0.3 per cent (w/v) bentonite clay for a period of seven days. Biofilm cells showed significantly {P Example 2. Expression of novel proteins In Escherichia cofi when grown in biofilm Escherichia coli used for the extraction of outer membrane proteins (OMPs) was grown under six different conditions with and without bentonite clay and also by incorporating iron chelator, at 37°C and 42°C separately, and for two different A periods viz, one and seven days thus accounting for a total of 24 different antigen preparations. Outer membrane proteins were extracted as per the method described by Bolin and Jensen (1987) and analysed by SDS - PAGE (Laemli 1970) and western blotting (Sambrook etal, 1989). Example 3 Extraction of Outer membrane proteins The culture pellet of Escherichia cofi grown in defined culture conditions for varied temperature and period is suspended in 10 mM HEPES buffer and subjected to sonication at the rate of 80-S pulses in an ice bath for ten cycles of thirty seconds each. Following this, whole cells and debris were pelleted out by centrifugation at 4000 g for 20 min at 5°C and the supernatant containing the cell membranes was collected. 1. The cell membranes in the supernatant was pelleted out by ultra centrifugation (Beckman K-80 ultra centrifuge, Rotor IE58) at 1,05,000 g for 60 min at 5°C. 2. The pellet (membranes) was suspended in lOmM HEPES buffer (pH 7.4) containing two per cent sodium-n-lauryl sarcosinate and incubated for one hour at 22°C. The detergent-insoluble outer membrane protein-enriched fraction was pelleted out by ultra centrifugation at 1,05,000 g for 60 min at 5°C; the pellet was again resuspended in 10mM HEPES buffer (pH 7.4) and stored at -70°C until use. The protein content in the OMP extract was estimated using a protein-dye-binding method according to Bradford (1976). Example 4 SDS-PAGE analysis of outer membrane proteins A 24 hr old culture of Escherichia coli Tryptone Soya Broth was harvested by pelleting at 4000 rpm for 10 min. The pellet was washed thrice in sterile PBS of pH 7.4 and finally resuspended in PBS (pH 7.4). The number of viable cells was enumerated by spread plate method described by Postgate (1969) and the concentration of the inoculum was fixed at 1.5X1 O^cells/ml.) Protein {20 \XQ) was mixed with sample buffer comprising of SDS, Beta mercapto ethanol, glycerol and bromophenolblue and heated to 100°C in a dry bath for three minutes and loaded into the gel comprising of 12% separating gel and 4.5% stacking gel prepared according to the methods of Laemmli (1970). Electrophoresis was carried out at a constant current of 10mA, 50V for the stacking gel and 15mA, 100V for the resolving gel, with pre-stained standard protein molecular weight marker (Phosphorylase b 97,400Da, Bovine serum albumin 68,000Da, Ovalbumin 43,000Da, Carbonic anhydrase 29,000Da, Soyabean trypsin inhibitor 20,000 Da, Lysozyme 14,300Da). After the completion of electrophoresis, gels were stained with Coomassie brilliant blue according to Sambrook etal. (1989). A pennanent record of the stained gel was made by either photographing or documenting in gel documentation system {Alphalmager™ 2200 from Alpha Innotech Corporation). The molecular weight of the protein bands was determined in comparison with the standard protein molecular weight markers using software provided along with the gel documentation system Example 5.0 Western blotting The proteins were transferred to nitrocellulose sheets and membrane was incubated with blocking buffer (5 per cent skimmed milk powder in PBS -Tween - 20). The blots were incubated for one hr at room temperature with individual antisera described in example 6 diluted in PBS -Tween -- 20 in five per cent skim milk powder and then washed. The membrane blot was incubated with rabbit antichicken IgY Horse Radish Peroxidase conjugate at a dilution of 1:2000 in blocking buffer for one hr and washed with washing buffer, thrice at 5 min intervals. Following the final wash, the blot was incubated at room temperature with a freshly prepared substrate - chromogen (Ortho Dianisidin Dihydrochloride) solution, until the desired band intensity was achieved and the blot was transferred to a tray containing distilled water to stop further development of colour. A permanent record of the western blot was made by either photographing or scanning. Analysis of OMPs is being generally employed to study the strain variation and relatedness among numerous bacterial species. In the current study, analysis of the OMPs of biofilm, nutrient restricted (NR), and free cells of Escherichia coli was carried out by SDS PAGE to detect any variation and / or relatedness among the cells grown under different nutritional and temperature conditions with regard to their expression / repression. It was revealed that several distinct polypeptide bands at 61, 35.5 and 34 kDa and many other faint bands in the range of 28.8 - 95.2 kDa region were expressed, whereas 58 and 42 kDa proteins were repressed. The over-expression of the 61, 35.5 and 34 kDa proteins occurred in the biofilm and nutrient restricted cells when compared to the free cells. Three unique OMPs of 93.2, 61 and 28.8 kDa were detected in the biofilm and nutrient restricted cells that were absent in the free cells. These findings indicate that the bacteria under biofilm mode are demonstrably and profoundly different from their free cell counteiparts in composition of their outer membrane proteins. These observations are in consensus with the findings of Costerton et al. (1995) that the adhesion of bacteria to a surface triggers the expression of a number of genes, making the biofilm cells clearly phenotypically different from the free cells of the same species. Example 6 Antigenicity of novel proteins bv western blott analysis. Antigencity of novel proteins was assessed by western blot using different sera samples, i. Antiserum from vaccinated birds b)Sera from birds orally vaccinated with Escherichia coli 076 c)Sera from birds s/c vaccinated with Escherichia coli 078 d)Sera from birds orally vaccinated with Escherichia coli 02 e)Sera from birds s/c vaccinated with Escherichia coli 02 ti. Antiserum from field outbreaks (convalescent sera) a) Escherichia coli 076 affected colisepticemic birds b) Escherichia coll 02 affected colisepticemic birds c) Escherichia coli06 affected colisepticemic birds d) Escherichia coli 025 affected colisepticemic birds Probing with homologous sera: Probing of Escherichia coll antigens with homologous sera revealed good antibody response to polypeptides at 61, 35.5 and 34 kDa region in biofilm and nutrient restricted antigens besides numerous faint bands detected in 93.2, 32.8, 19.3 and 17.6 kDa regions, compared to strong antibody response to only 58 and 42 kDa proteins antigens grown in conventional media, which are not involved in cross protection, besides faint recognition of 35.5, 34 and 19.3 kDa proteins That is proteins expressed in enriched media lack the properties of cross protection. Probing with heterologous sera: a). Probing E. cod 078 OMP"s with sera obtained from Escheiichia coli 02 serogroup affected colisepticemic birds in a field outbreak and from birds vaccinated with Escherichia coli 02 biofilm vaccine by s/c route revealed a strong antibody response against 61 and 34 kDa proteins and less response to 93.2, 35.5, 19.3 and 17.6 kDa proteins in biofilm antigen compared to moderate response for 35.5 and 34 kDa proteins conventionally grown antigens. b). Probing with sera obtained from a culled chick procured from a hatchery, affected with Escherichia coli 08 serogroup, revealed a moderate to strong antibody response to 61.0, 35.5, 34 and 17.6 kDa proteins in biofilm antigen only. c). Sera obtained from unvaccinated (control) birds with stunted growth from a field vaccination trial affected with Escherichia coli 025 serogroup revealed a strong antibody response to 34 kDa protein and faint response in the region of 17.6, 42, 61 and 93.2 kDa region protein in biofilm antigen only. Probing of 078 OMPs with 02 (heterologous) convalescent sera from a field out break and s/c vaccinated birds revealed identical band profiles. There was recognition of most of the polypeptide bands In biofilm antigen namely 93.2 kDa, an iron regulated OMP and more Importantly 61, 34 and 17.6 kDa proteins, the major cross protective antigens. To conclude, it is very interesting to note that only biofilm grown antigen is able to cross-react with any antisera of heterologous origin, i.e., cross protecting OMPs are expressed only when organisms are confronted with stress situations of nutrient restriction and / or provided with inert surface like bentonite day in vitro or chronic disease condition / pathogenic situation in vivo. Thus it is the first time that cross protection was established among various serotypes of Escherichia coti. The molecular basis for cross protection was established by identifying cross protective OMPs at the regions; 93.2, 61, 35.5, 34, 28.8 and 17.6 kDa by western blott. It is worth to note that heterologous antisera used for probing Escherichia coll OiyPs on two occasions is altogether from a different source, i.e., growth / cultural condition under which the antigen was obtained or antisera raised, are two totally different situations, per se one expressed during disease condition in j/iyo and the other was grown in vitro by simulating natural pathogenic situation through nutrient limitation and / or providing inert surface. So it can be concluded that the biofilm technique that we designed to grow the antigen to express cross-reactive OMPs, which were subsequently incorporated in the vaccine; and the situation under which OMPs expressed during a disease procesgf/"n vivo fe very much simitar. Further more, the OMPs expressed only under such situations are able to give cross protection, which is clearly demonstrated by subsequent challenge studies and western blotting involving antisera of different serotypes. This argument is further supported by the results of subsequent blotting studies, wherein antigens grown under similar conditions are probed with heterologous sera obtained from colisepticemic conditions of two different situations, both yielding Escherichia coll in pure culture upon postmortem examination with pathognomonic colisepticemic lesions. In both the situations. It is possible that the birds have suffered a chronic disease process for quite a long time, thus providing the organism an ideal biofilm situation to grow, leading to an immune response. When such antiserum was used for probing, there is recognition of antigen grown under biofilm and nutrients restricted conditions only. Antibody response during disease process i.e., in vivo processed antigen and against in vitro biofilm grown antigen reveal identical band profile with the same set of antigens, thus, further bolsters the argument that Escherichia coli infections are biofilm associated and vaccines prepared by incorporating antigens expressed only under such situations are cross protective. Example 7. Process of producing E. co/i vaccine using biofilm Biofilm cells grown for seven days in 0.16 per cent TSB with 0.3 per cent bentonite clay were harvested by discarding the supernatant media to remove any planktonic cells. Bentonite clay with biofilm grown E. coli was suspended in PBS. After counting number of viable bacterial cells it was further diluted with PBS to contain a final concentration of 4X10^ cfu/ml. Formalin was added to a final ,^ concentration of 0.1 per cent and stored in PBS at 4^0 until use. Vaccine was subjected to routine sterility and safety tests. Example 8. Use of biofilm grown E. coli as vaccine Two types of Escherichia coli vaccines, (1) vaccine prepared out of E. coli grown in biofilm mode (biofilm vaccine} and (ii) vaccine prepared out of E co//grown in conventional mode (free cell vaccine) were used in experimental vaccination trials. Preparation of free cell vaccine: The culture was grown in 3.0 per cent TSB for 16 hr at 37°C and pelleted at 4000 rpm for 10 min at 4°C. The pellet was washed thrice with PBS. The pellet containing bacterial cells was finally re-suspended in PBS. The pellet was further diluted to contain 4X10^ cfu/ml after counting number of viable cells. Formalin was added to a final concentration of 0.1 per cent and stored in PBS at 4°C until use. Vaccination schedule and experimental protocol. i.Free cell vaccine was given orally for three consecutive days to group T\ on days three to five and boosted on days 21 to 23, whereas parenteral vaccine was given to group T3 by subcutaneous (s/c) route on day five and boosted on day 23 with a dose of 0.25 ml containing 1X10^cfu/bird/day. ii.Biofilm vaccine was given orally for three consecutive days to group Tg on days three to five and boosted on days 21 to 23, whereas parenteral vaccine was given to group T4 by s/c route on day five and boosted on day 23 with a dose of 0.25 ml containing IXIOVu/bird/day. iii.Controls in group T5 were given 0.25ml of sterile PBS by s/c route. First challenge was carried out 10 days post vaccination i.e. on 15"^ day, by separating twelve birds from each group Ti to T5 from main flock and were again sub divided into six each (totally ten sub groups) and were challenged by intranasal (i/n) and intramuscular (i/m) routes using live homologous culture with 1X10^cfu/bird. Similarly second challenge was carried out on day 28*^ i.e., five days after booster and 23 days after primary vaccination and third challenge on day 36 i.e., 31 days after primary vaccination and 13 days after booster dose, 24 birds from each treatment groups were again challenged, by i/n and i/m routes (12 each). Birds were observed for clinical signs, morbidity and mortality for six days. If no death was recorded, then birds were slaughtered at the end of the sixth day post challenge. Samples of liver, spleen, heart and intestine were collected aseptically to estimate the colonization/elimination of Escherichia co//from these organs. Experimental trials (tables 2 and 3) First challenge Upon i/m challenge with homologous strain of both oral and subcutaneous free cell vaccinated birds, at 10 days post vaccination resulted in death of all the six birds In each of the groups compared to one survival out of six each In biofilm orally and s/c vaccinated groups (Table 2). Birds did not show protective level of immunity to withstand the severe fomri of challenge. This could be due to fact that, challenge was very severe (IxlO^cfu/bird) and / or primary vaccination alone may not be sufficient to withstand live severe challenge. Upon i/n challenge, birds that received vaccine had significantly (P£0.05) reduced E coli load from intestine compared to unvaccinated controls. And within the vaccinated group, biofilm vaccinated group were significantly (P£0.05) better than free cell vaccinated birds (table 3) in preventing the invasion of liver by live challenge organisms. No difference in response to parenteral challenge was observed in any of the vaccinated groups and it is concluded that, the challenge 10 days after primary vaccination did not give protection against parenteral challenge. However, It is worth to note that performance of biofilm vaccine was relatively better than free cell vaccine either in eliminating or limiting the colonization of challenge organism or in reducing the mortality due to challenge infection, above all 10 days is too early to expect complete expression of specific immune response. Second challenge Live challenge by intra muscular route of both orally and s/c biofilm vaccinated birds, 23 days after primary and five days after booster, conferred 17 and 83 per cent protection compared to 50 per cent in free cell parenteral vaccinated groups and no protection with free cell oral vaccine group i.e., biofilm vaccine by s/c route, was able to provide effective protection than biofilm vaccine by oral route or free cell vaccine by either of the routes. Upon intra nasal challenge, elimination of challenge organisms from intestine was numerically better in biofilm-vaccinated groups, than free cell vaccinated counterparts and unvaccinated controls. However no growth or non-colonization of challenge organism in liver samples among parenteral biofilm vaccinated birds is noteworthy. And colonization of challenge organism among biofilm orally vaccinated (1.25X10") and free cell s/c vaccinated groups (1.45X10"^) are comparable to each other, but significantly less than free cell oral vaccinated group {5.43X10"") and unvaccinated control group (6.40X10^) (table 3). i.e., biofilm parenterally vaccinated birds provide no opportunity for challenge strain to invade, that othenwise would have lead to septicemic condition, which is evident by presence or absence of challenge organism from liver samples. Third challenge Live intramuscular challenge of both orally and parenterally free cell vaccinated birds, after 31 days primary vaccination and 13 days booster, resulted in vaccine efficacy of 33 and 58 per cent respectively compared to 42 and 100 per cent in biofilm vaccine, So it is obvious that biofilm vaccine by parenteral route alone is capable of providing complete and solid protection against severe form of challenge. Upon i/n challenge, elimination of challenge organism from intestine was significantly (P£0.05) better in biofilm vaccinated groups compared to free cell vaccinated group and unvaccinated controls. Colonization of challenge organism In other visceral organs was quite low in liver and spleen with no colonization in heart sample among biofilm oral vaccinated birds; with absolutely no colonization In any of the visceral organ samples among biofilm parenteral vaccinated birds. Thus there is a significant {PE0.05) reduction in recovery of challenge organism from intestine and liver from biofilm oral vaccinated birds compared to free cell and unvaccinated counterparts. The results of preliminary vaccination trial suggest that vaccine prepared from biofilm grown bacteria is effective In decreasing the mortality and very effective in elimination of challenge organism followed by intranasal challenge, compared to that of free cell vaccinated counterparts. Biofilm vaccine by subcutaneous route provides solid immunity not only to resist against very severe form of challenge but also, nullifies any chance for the challenge organism to Invade the visceral organs unlike in birds which received vaccine prepared out of conventionally grown bacteria. Also recovery of challenge organism from liver and spleen was significantly (P Field vaccination trials (table 4) The results of experimental vaccination trials are indeed worth noting, wherein protective ability of the biofilm vaccine was compared with that of free cell vaccine in three different challenges, which were considered to be sufficiently consistent to establish the superiority of the biofilm vaccine over free cell vaccine. Besides conferring 100 per cent protection against any form of challenge, there was a significant (P Mortality per cent in unvaccinated control birds in farm "A" revealed that approximately one out of four birds which died, were due to colibacillosis. constituting 26.12 per cent in a total mortality of 111. Death due to Escherichia coli was not seen in Ti where birds were boosted through s/c route after oral priming. In T2 where birds were both primed and boosted through oral route, mortality due to Escherichia cod was eight per cent in a total mortality of 75 birds. This could partly be due to failure of the orally immunized birds to withstand heterologous severe challenge as this was obsen/ed during challenge studies of field vaccinated birds or probably due to the fact that oral route will not ensure all the birds to consume vaccine uniformly, or it could also be due to degradation of antigen in gastro intestinal tract. At the end of 39 days, the vaccinated groups consumed relatively less feed with a FCR of 1.68 compared to 2.06 in un vaccinated control group, to put on the same body weight. The second field trial was carried out in farm B" to assess the reproducibility of the efficacy of biofilm vaccine at field level under different managemental conditions. In both the trials the biofilm vaccine, was able to reduce the mortality in orally vaccinated and orally primed group to eight per cent and nine per cent in Farm A and Farm B, respectively compared to mortality to the extent of 26.12 and 12.3 per cent in un vaccinated controls, besides totally obviating the mortality in s/c boosted group. At the end of 37 days, the vaccinated groups consumed relatively less feed with a FCR of 1.74 compared 1.96 in un vaccinated control group, to put on the same body weight. The third field trial was carried out in form "C which was consistently affected by collbacillosis in successive batches causing severe economic loss. In this trial mortality due to colibacillosis was, though, not recorded, the scored gross lesions were minimal in vaccinated birds compared to unvaccinated controls. Moreover, total flock mortality was also low at 3.9 per cent in vaccinated birds compared to 10.56% in controls. Vaccinated group consumed relatively less feed with a FCR of 1.91 compared to 2.17 In the un vaccinated control group. 6. Efficacy of biofilm vaccine Challenge studies (table 5) were carried out to assess the efficacy of the biofilm vaccine using birds from three different groups of first field trial (Farm A) and challenged on day 42 with 1X10^ live cells of Escherichia coii intramuscularly using both homologous (078) and heterologous (02) live cultures, to determine the extent to which the biofilm vaccine provides protection and cross protection respectively. Birds boosted through s/c route conferred 100 per cent protection against severe form of challenge compared to 100 per cent protection against homologous and 80 per cent protection against heterologous challenge among orally boosted birds, i.e., both forms of vaccination have provided effective protection under field conditions, but s/c boosting has given absolute protection in terms of ensuring not only 100 per cent cross protection against very severe form of challenge but also Infection free status of flock which Is reflected by better FCR. The ability of vaccine prepared out of biofilm grown bacteria to afford protection against very severe challenge with a homologous vaccine strain is in agreement with the findings of Gross (1957) wherein formolised, heat and p propiolactone inactivated vaccine conferred effective protection against homologous live challenge. However, no details are given about the relative efficacy of this vaccine. Deb and Harry (1978) reported maximum degree of protection (89 to 100%) using inactivated alum precipitated vaccine against an l/m challenge and Gyimah et al. (1986) reported 88 to 100 per cent protection followed by live challenge of birds followed vaccination using polyvalent vaccine. However our findings differ from that of the above researchers as for as cross protection by heterologous challenge is concerned. On the other hand, these workers did not see cross protection against other Escherichia coli serotypes. However, in the present study, the trials showed that, biofilm vaccine was the most effective than conventional vaccine both in terms of effective elimination of challenge organism, Escherichia coli from intestine and cross protection. Our conclusion is, protection conferred by vaccine prepared out of Escherichia coli grown in biofilm mode is quite broad based and complete. Vaccination by s/c route was found to be highly effective when given once in the age of three to five days and boosted once in between 11 and 13 days, keeping in view the fact that primary vaccination alone has practically no protective effect against very severe form of challenge as seen in experimental trials. However single dose is very effective in elimination of Escherichia coli from intestine and preventing the colonization In liver that would be sufficient to protect the birds against intra nasal challenge, one of the most natural ways of field infection. Contrary to parenteral vaccination, the oral vaccination via drinking water, for three consecutive days during first week and again at third week in experimental trials and for two consecutive days at first and second week in field trials consistently eliminated Escherichia coli more efficiently than free cell vaccinated and unvacclnated counterparts. Conclusion It seems pertinent to conclude that Escherichia coli does form biofilms during natural infections. The biofilm form of Escherichia coli was found to alter its outer membrane proteins either by repressing or over expressing the Outer Membrane Proteins, of which some are responsible not only for homologous but also heterologous cross protection among various serotypes. These novel proteins are not expressed in free cell conventional vaccine. In the light of these findings, it becomes clear that the bacteria living in biofilm mode are structurally and functionally very different from the free-living bacterial cells. Hence, it is unwise to extrapolate the results obtained from free cell cultures to bacteria growing in biofilms. The biofilm grown bacterial cells can sustain for very long periods in vitro when compared to their free cell counterparts. The stressful environment such as nutrients restriction in presence of bentonite clay as inert surface for attachment, colonization and trapping of nutrients would simulate the natural in vivo environment during infections with the expression of protective cell surface antigens. References: Arp, L. H., Graham, G. L G. and Cheville, N. F., 1979. Comparision of clearance rates of virulent and avirulent Escherichia coli in turkeys after aerosol exposure. Avian Dis., 23:386-391. Arp, L. H., 1980. Consequences of active or passive immunization of turkeys against Escherichia coiiOlB. Avian Dis., 24:808-815. Bolin, C. A. and Jensen, A. E., 1987. Passive Immunization with antibodies against Iron Regulated Outer Membrane Proteins protects Turkeys from Escherichia CO//septicemia. Infect. Immun., 55:1239-1242. Bradford, M. M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochetn., 72:248-252. Brown, M. R. W. and Williams, P., 1985. The influence of environment on envelope properties affecting survival of bacteria in infections. Annu. Rev. Microbiol., 39:527-56. Costerton, J. W., Levandowski, Z., Caldwell, D. E., Kort)er, D. R. and Lappin-Scott, H. M., 1995. Microbial biofilms. Annu. Rev. Microbioi., 49:711-745. Costerton, J. W., Stewart, P. S. and Greenberg, E. P., 1999. Bacterial biofilms: A common cause of persistent infections. Science, 284:130 -133. Deb, J. R. and Harry, E. G., 1976. Laboratory trials with inactivated vaccines against Escherichia co//(O78:K80) infection in fowls. Res. Vet. Sci, 20:131-138. Deb, J. R. and Harry, E. G., 1978. Laboratory trials with inactivated vaccines against Escherichia co//(02:K1) infection in fowls. Res. Vet. Sci., 24:308-313. Ewing, W. H., Tatum, H. W., Davis, B. R. and Reavis, R. W., Studies on the serology of E. coli group. CDC publication, communicable disease center, Atlanta, Georgia. 1956. Franchini, A., Canti, M., Manfreda, G. and Bertuzzi, S., 1991. Vitamin E as adjuvant in emulsified vaccine for chicks. Poult. Sc/., 70:1709-1715. Frommer, A., and Freilin, P. J., Bock, R. R., Leitner, G., Chaffer, M. and Heller, E. D., 1994. Experimental vaccination of young chickens with a live, non -pathogenic strain of Escherichia coll. Avian Pathol., 23:425-433. Gordon, R. P., 1961. Bull. Off. Int. Epizoot., 56, 507. Griffiths, E., Stevenson, P. and Joyce, P., 1983. Pathogenic Escherichia co//express new outer membrane proteins when growing in vivo. FEMS Microbiol. Lett., 16:95-99. Gross, W. B.,1957. Vaccine against Escherichia coli infection in chickens. Avian Dis., 1:347. Gyimah, J. E. and Panigrahy, B., 1985. Immunogenicity of an Escherichia coli (Serotype 01) Pili vaccine in chickens. Avian Dis., 29:1078-1083. Gyimah, J. E., Panigrahy, B., Hall, C. F. and Williams, J. D., 1985. Immunogenicity of an oil-emulsified Escherichia coil bacterin against heterologous challenge. Avian Dis., 29:540-545. Gyimah, J. E., Panigrahy, B. and Williams, J. D., 1986. Immunogenicity of an Escherichia co//multivalent pilus vaccine in chickens. Avian Dis., 30:687-689. Kapur, v.. White, D. G., Wilson, R. A. and Whittam. T. S., 1992. Outer membrane protein patterns mark clones of Escherichia coil 02 and 078 strains that cause avian septicaemia. Infec. immun. 60:1687-1691. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227:680-685. Lietner, G., Melamed, D., Drabkin, N. and Heller, E. D., 1990. An Enzyme linked immunosorbent assay for detection of antibodies against Escherichia coll: association between indirect hemaglutination test and survival. Avian Dis., 34:58-62. Mckay, K. A., Ruhnke, H. I. and Barnum, D. A., 1965. The results of sensitivity tests on animal pathogens conducted over the period 1956-1963. Can. Vet. J., 16:103-111. Melamed, D., Leitner, G. and Heller, E. D., 1991. A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli. Avian Dis., 35:17-23. Orskov, F., Orskov, I., Jann, B., Jann, K., Muller-settz, E. and Westphal, O., 1967. Acta pathalogica et microblologica scandinavia, 71:339. Orskov, F., Orskov, I., Jann, B. and Jann, K., 1971. Acta pathalogica et microblologica scandinavia, 79:142. O"Toole, G. A., Kaplan, H. B. and Kolter, R., 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol., 54:49-79. Panigrahy, B., Gyimah, J. E., Hall, C. F. and Williams, J. D., 1984. Immunogenic potency of an oil-emulsified Escherichia co//bacterin. Avian Dis., 28(2): 475-481. Postgate, J. R., Viable counts and viability. In: Methods in Microbiology, Vol. I. Norris, Jr. and Ribbons, D. W., Eds. Academic Press, p. 611-628,1969. Rosenberger, J. K., Fries, P. A., Cloud, S. S. and Wilson, R. A., 1985b. In vitro and in vivo characterization of avian Escherichia coli, II. Factors associated with pathogenicity. Avian Dis., 29{4):1094-1107. Sojka, W. J. and Carnaghan, R. B. A., 1961. Escherichia coli infection in poultry. Res. Vet. Sci., 2:340-352. Tengerdy, R. P., Lacetera, N. G. and Nockels, C. F., 1990. Effect of beta carotene on disease protection and humoral immunity in chickens. Avian Dis., 34:848-854. Zigterman, G. J., Van de Ven, W., Van Geffen, C, Loeffen, A. H., Panhuijzen, J. H., Rijke, E. O. and Vermeculen. A. N., 1993. Detection of mucosal immune responses in chickens after immunization or infection. Vet. Immunol. Imunopathol., 36:281-291. We Claim: 1.0: A process of producing vaccine/against colibacillosis of birds and animals) wherein the E. coil bacteria are cultured as a biotin using inert substances such as bentonite clay, harvested using known methods inactivated using chemicals such as formalin and the inactivated bacteria are packaged into convenient doses 2.0: A process of producing vaccine for colibacillosis comprising of the following steps: clay (0.3 % w/v) was suspended In 0.16 per cent tryptone Soya Broth (TSB). -freight media {as prepared in step a) was sterilized by autoclaving at 15 lbs pressure at 121°Cfor15min. c. Check for sterility by known methods, and Inoculate with the prepared Escherichia co/j inoculum’s and incubate at 37°C. d. Shake the culture flasks six times daily in a mechanical shaker at 50 rpm for one hour to keep the bentonite clay uniformly suspended in the media. e. Harvest seven-day-old biopic cells by discarding the supernatant media to remove any plank tonic cells. f. Count the number of viable Escherichia coil biotin cells by colony counting of ten fold serial dilution in sterile phosphate buffered saline pH 7.4 after vigorous overtaxing of bentonite clay In test tube and counts were expressed as colony forming units/g of bentonite clay. g Adjust bentonite clay with biotin growth to a final concentration of h. Add Formalin to a final concentration of 0.1 per cent

Full Text

"Novel Biofilm Vaccines against bacterial diseases of birds and
Domestic animals".
Field of Innovation: This innovation describes novel vaccine in the field of poultry
vaccines Prior art:
Among the bacterial diseases, colibacillosis is the leading cause of morbidity and mortality in poultry and responsible for significant economic loss in all the phases of poultry Industry from production to marketing. The disease is caused by Escherichia coli, consisting of a large number of serotypes. Besides colisepticemia, Escherichia coli is also responsible for various other disease conditions with mortality of five to ten per cent, and some times may be as high as 50 per cent.
Immunization of chicken against colibacillosis has been attempted by numerous workers without much success. Escherichia coli antigens used for active immunization of chicken include bacterial pili {Gyimah and Panigrahy, 1985; Gyimah et al., 1986 and Zigterman et a/., 1993); live bacterial cells (Arp, et al., 1979; Arp, 1980; and Frommer ef a/., 1994), and killed bacteria with adjuvants (Deb and Harry, 1976 and 1978; Rosenberger et al., 1985; Lietner et al., 1990; Tengerdy et at., 1990; Franchini et al., 1991 and MeJamed ef al., 1991). ten all these trials, immunity has been found to be seen only against homologous but not to heterologous challenge; that is protection tends to be serotype limited. Thus several attempts of vaccination to achieve protection against avian colibacillosis both in older {active immunization) and younger (passive immunization) birds have been reported. Subsequent attempts to develop effective vaccine by including only the most predominant serigraphs that are frequently associated with colisepticemia, 01 :K1, 02:K1 and O78:K80 were also not successful.

It is reported that a vaccine capable of conferring protection must be a broad based one to be most effective against colibacillosis caused by varied serotypes, in view of the serological and chemical differences between different serotypes. {Ewing et ai, 1956 and Orskov et al., 1967 and 1971). There are also reports by Deb and Harry, (1976); Arp, (1980) and Kapur et al, (1992) that cross protection is usually not observed with Escherichia coll serotypes from poultry and that a suitable vaccine should contain at least the most common serotypes, but also opined that efficacy of such a vaccine gets adversely affected by antigenic competition between different serotypes.
Further studies were aimed at exploring the common antigens among Escherichia coli in general and APEC in particular. Culturing bacteria under iron-deprived media was one such attempt made to explore common virulence factors of Escherichia coil (Griffiths et al., 1983). Evidently, organisms grown in the host will have their changed characters both structurally and genetically with selective expression of new antigens. These antigens in the form of vaccine may be responsible for providing broad based protection.
The above considerations suggest that any vaccine to be successful against Escherichia coli infections should be able to give cross protection against a plethora of serotypes and development of such a safe and effective vaccine has eluded the researchers despite prompt attempts. For a vaccine to be broad based, the part the bacteria (the antigen) with disease producing ability and which is commonly found in other serotypes of Escherichia coli needs to be explored.

Bacterial biofilm is a structured community of bacterial cells enclosed in a self-produced glycocalyx matrix and attached to living or non living surface, which constitutes a protected mode of growth that allows survival in hostile environment (Costerton et ai, 1999). Vaccines developed on the basis of the biofilm production represent the growing of organisms in a natural pathogenic ecosystem {Brown and Williams, 1985). Studies on gene expression pattem has indicated that approximately 40 per cent of the genes in in vitro grown biofilm bacteria may be different from those of their planktonic counterparts (OToole et ai, 2000) and there by express some Outer Membrane Proteins (OMPs), which are similar to ones expressed during natural infection (Brown and Williams, 1985). Whether providing a nutrient restricted medium with an appropriate surface for attachment, E. coli would form biofilm, and if so, whether this "simulated in vivo" environment would lead to the expression of some of the antigens of a natural infection has still not been investigated. Therefore, there is a need to test if E. coli can be grown in a laboratory media on a biofilm matrix and test the vaccine potential of protein antigens obtained from such a E. co//culture
Summary of invention
The biofilm form of E. coli grown under defined in vitro condition, has been exploited to evolve an effective, safe, cross protective vaccine against colibacillosis caused by different serotypes in poultry. The maximum production of E. coli biofilms was found at 0.16 per cent tryptone soya broth with 0.3 per cent w/v bentonite clay as inert surface in seven days. The viability of E. coli in biofilms was significantly higher even at 100 days, on the other hand; nutrients restricted and free cells with bentonite clay, declined very rapidly after 60"" day post inoculation. Analysis of the

outer membrane protein profiles of the biofilm, nutrient restricted and free cells revealed repression of the 58 and 42 kDa and over-expression of 61, 35.5, 34 and -28.8 kDa OMPs in biofilm cells and nutrient restricted cells when compared to the free cells. In addition, three unique OMPs of 93.2, 61.0 and 28.8 kDa molecular masses were detected in biofilm cells, which were completely absent in the free cells. Killed biofilm and free cell vaccines were used at the rate of 1X10^ cfu per bird and the same dose was used for challenge studies. Subcutaneous route of vaccination with biofilm vaccine offered 100 per cent protection compared to 58 per cent with free cell vaccine followed by severe live challenge infection by intramuscular route. Vaccination through oral route with biofilm vaccine was efficient in significantly reducing the colonization / elimination of Escherichia co//compared to free cell vaccine, upon challenge by intranasal route, which is considered as natural mode of infection. Field vaccination trials at farm level with vaccine prepared from biofilm grown bacteria conferred cross protection to the extent of 100 and 80 per cent in subcutaneous and oral vaccinated birds respectively. The biofilm vaccine given through subcutaneous route resulted in low feed conversion ratio compared to unvaccinated controls, to the extent 1.68 to 2.06. Challenge studies in field vaccination trials established the ability of biofilm vaccine to give cross protection. Thus it is the first time that cross protection was established among various serotypes of Escherichia coli. The molecular basis for cross protection was established by identifying cross protective OMPs of 93.2, 61, 35.5, 34, 28.8 and 17.6 kDa proteins by western blott.

Detailed description of invention
Example 1. Method of growing Escherichia coU"in biofilm:
c^
For production of biofilm cells, bentonite clay {0.3 % w/v) was suspended in 0.08, 0.16, 0.32 and 3.0 per cent tryptone Soya Broth (TSB). Identical concentrations of TSB media viz., 0.08, 0.16, 0.32 (nutrient restricted) 3.0 per cent (free cell) were used without incorporating bentonite clay to serve as controls. For each concentration, ten different conical flasks of 250 ml capacity with 100 ml each of the above-defined media were used and all the media were sterilized by autoclavlng at 15 lbs pressure at 121° C for 15 min. After checking for sterility, these flasks were ^ Inoculated with the prepared inoculum and incubated at 37°C. The culture flasks with bentonite clay were agitated six times daily in a mechanical shaker at 50 rpm for ^ one hourto keep ttie bentonite clay uniformly suspended in the media.
The biofilm cells, nutrients restricted (NR) cells, and free cells with and without bentonite clay were harvested on days one, three, five, seven, ten, twenty, thirty, sixty and one hundred, post-Inoculation. One conical flask of each concentration was used for each of the days, thus making use of nine out of ten flasks inoculated and remaining one flask In each of the concentrations served as reserve.
The biofilm cells were quantified by concentrating the cells colonized on bentonite clay surface at 1000 rpm for five min. The supernatant was discarded and the pelleted biofilm colonized on bentonite clay was washed thrice with PBS {pH 7.4) to remove non adherent bacteria, made up the volume to 10 ml with sterile PBS in a test tube and vortexed vigorously for three min to dislodge the biofilm cells from

bentonite clay. Biofilm cells thus obtained in supernatant were enumerated. Similarly, viable counts for NR cells and free cells grown with and without bentonite ■, clay were determined up to 100"" day post-Inoculation at different intervals in all the concentrations of TSB.
The spread plate method described by Postgate (1969) was employed to enumerate the surface viable counts. The average number of colonies per plate was multiplied by the dilution factor to obtain the viable count in the original suspension and expressed as cfu/ml for the NR and free cells and cfu/g of bentonite clay for biofilm cells. The highest diiution of TSB at which maximum viable counts of bacterial cells were obtained from among the different days of intervals tested was taken as optimum conditions required for good biofilm / NR cell growth.
The optimum concentration of TSB required for maximum biofilm formation of Escherichia coli was found to be 0.16 per cent with 0.3 per cent (w/v) bentonite clay for a period of seven days. Biofilm cells showed significantly {P Example 2. Expression of novel proteins In Escherichia cofi when grown in biofilm
Escherichia coli used for the extraction of outer membrane proteins (OMPs) was grown under six different conditions with and without bentonite clay and also by incorporating iron chelator, at 37°C and 42°C separately, and for two different

A

periods viz, one and seven days thus accounting for a total of 24 different antigen preparations. Outer membrane proteins were extracted as per the method described by Bolin and Jensen (1987) and analysed by SDS - PAGE (Laemli 1970) and western blotting (Sambrook etal, 1989).
Example 3 Extraction of Outer membrane proteins
The culture pellet of Escherichia cofi grown in defined culture conditions for varied temperature and period is suspended in 10 mM HEPES buffer and subjected to sonication at the rate of 80-S pulses in an ice bath for ten cycles of thirty seconds each. Following this, whole cells and debris were pelleted out by centrifugation at 4000 g for 20 min at 5°C and the supernatant containing the cell membranes was collected.
1. The cell membranes in the supernatant was pelleted out by ultra centrifugation
(Beckman K-80 ultra centrifuge, Rotor IE58) at 1,05,000 g for 60 min at 5°C.
2. The pellet (membranes) was suspended in lOmM HEPES buffer (pH 7.4)
containing two per cent sodium-n-lauryl sarcosinate and incubated for one hour
at 22°C.
The detergent-insoluble outer membrane protein-enriched fraction was pelleted out by ultra centrifugation at 1,05,000 g for 60 min at 5°C; the pellet was again resuspended in 10mM HEPES buffer (pH 7.4) and stored at -70°C until use. The protein content in the OMP extract was estimated using a protein-dye-binding method according to Bradford (1976).

Example 4 SDS-PAGE analysis of outer membrane proteins
A 24 hr old culture of Escherichia coli
Tryptone Soya Broth was harvested by pelleting at 4000 rpm for 10 min. The pellet was washed thrice in sterile PBS of pH 7.4 and finally resuspended in PBS (pH 7.4). The number of viable cells was enumerated by spread plate method described by Postgate (1969) and the concentration of the inoculum was fixed at 1.5X1 O^cells/ml.)
Protein {20 \XQ) was mixed with sample buffer comprising of SDS, Beta mercapto ethanol, glycerol and bromophenolblue and heated to 100°C in a dry bath for three minutes and loaded into the gel comprising of 12% separating gel and 4.5% stacking gel prepared according to the methods of Laemmli (1970). Electrophoresis was carried out at a constant current of 10mA, 50V for the stacking gel and 15mA, 100V for the resolving gel, with pre-stained standard protein molecular weight marker (Phosphorylase b 97,400Da, Bovine serum albumin 68,000Da, Ovalbumin 43,000Da, Carbonic anhydrase 29,000Da, Soyabean trypsin inhibitor 20,000 Da, Lysozyme 14,300Da). After the completion of electrophoresis, gels were stained with Coomassie brilliant blue according to Sambrook etal. (1989). A pennanent record of the stained gel was made by either photographing or documenting in gel documentation system {Alphalmager™ 2200 from Alpha Innotech Corporation).
The molecular weight of the protein bands was determined in comparison with the standard protein molecular weight markers using software provided along with the gel documentation system Example 5.0 Western blotting
The proteins were transferred to nitrocellulose sheets and membrane was incubated with blocking buffer (5 per cent skimmed milk powder in PBS -Tween -

20). The blots were incubated for one hr at room temperature with individual antisera described in example 6 diluted in PBS -Tween -- 20 in five per cent skim milk powder and then washed. The membrane blot was incubated with rabbit antichicken IgY Horse Radish Peroxidase conjugate at a dilution of 1:2000 in blocking buffer for one hr and washed with washing buffer, thrice at 5 min intervals. Following the final wash, the blot was incubated at room temperature with a freshly prepared substrate - chromogen (Ortho Dianisidin Dihydrochloride) solution, until the desired band intensity was achieved and the blot was transferred to a tray containing distilled water to stop further development of colour. A permanent record of the western blot was made by either photographing or scanning.
Analysis of OMPs is being generally employed to study the strain variation and relatedness among numerous bacterial species. In the current study, analysis of the OMPs of biofilm, nutrient restricted (NR), and free cells of Escherichia coli was carried out by SDS PAGE to detect any variation and / or relatedness among the cells grown under different nutritional and temperature conditions with regard to their expression / repression. It was revealed that several distinct polypeptide bands at 61, 35.5 and 34 kDa and many other faint bands in the range of 28.8 - 95.2 kDa region were expressed, whereas 58 and 42 kDa proteins were repressed. The over-expression of the 61, 35.5 and 34 kDa proteins occurred in the biofilm and nutrient restricted cells when compared to the free cells. Three unique OMPs of 93.2, 61 and 28.8 kDa were detected in the biofilm and nutrient restricted cells that were absent in the free cells. These findings indicate that the bacteria under biofilm mode are demonstrably and profoundly different from their free cell counteiparts in composition of their outer membrane proteins. These observations are in consensus with the

findings of Costerton et al. (1995) that the adhesion of bacteria to a surface triggers
the expression of a number of genes, making the biofilm cells clearly phenotypically
different from the free cells of the same species.
Example 6 Antigenicity of novel proteins bv western blott analysis.
Antigencity of novel proteins was assessed by western blot using different
sera samples, i. Antiserum from vaccinated birds
b)Sera from birds orally vaccinated with Escherichia coli 076 c)Sera from birds s/c vaccinated with Escherichia coli 078 d)Sera from birds orally vaccinated with Escherichia coli 02 e)Sera from birds s/c vaccinated with Escherichia coli 02
ti. Antiserum from field outbreaks (convalescent sera)
a) Escherichia coli 076 affected colisepticemic birds
b) Escherichia coll 02 affected colisepticemic birds
c) Escherichia coli06 affected colisepticemic birds
d) Escherichia coli 025 affected colisepticemic birds
Probing with homologous sera:
Probing of Escherichia coll antigens with homologous sera revealed good antibody response to polypeptides at 61, 35.5 and 34 kDa region in biofilm and nutrient restricted antigens besides numerous faint bands detected in 93.2, 32.8, 19.3 and 17.6 kDa regions, compared to strong antibody response to only 58 and 42 kDa proteins antigens grown in conventional media, which are not involved in cross protection, besides faint recognition of 35.5, 34 and 19.3 kDa proteins That is proteins expressed in enriched media lack the properties of cross protection.

Probing with heterologous sera:
a). Probing E. cod 078 OMP"s with sera obtained from Escheiichia coli 02 serogroup affected colisepticemic birds in a field outbreak and from birds vaccinated with Escherichia coli 02 biofilm vaccine by s/c route revealed a strong antibody response against 61 and 34 kDa proteins and less response to 93.2, 35.5, 19.3 and 17.6 kDa proteins in biofilm antigen compared to moderate response for 35.5 and 34 kDa proteins conventionally grown antigens.
b). Probing with sera obtained from a culled chick procured from a hatchery, affected with Escherichia coli 08 serogroup, revealed a moderate to strong antibody response to 61.0, 35.5, 34 and 17.6 kDa proteins in biofilm antigen only.
c). Sera obtained from unvaccinated (control) birds with stunted growth from a field vaccination trial affected with Escherichia coli 025 serogroup revealed a strong antibody response to 34 kDa protein and faint response in the region of 17.6, 42, 61 and 93.2 kDa region protein in biofilm antigen only.
Probing of 078 OMPs with 02 (heterologous) convalescent sera from a field out break and s/c vaccinated birds revealed identical band profiles. There was recognition of most of the polypeptide bands In biofilm antigen namely 93.2 kDa, an iron regulated OMP and more Importantly 61, 34 and 17.6 kDa proteins, the major cross protective antigens.
To conclude, it is very interesting to note that only biofilm grown antigen is able to cross-react with any antisera of heterologous origin, i.e., cross protecting OMPs are expressed only when organisms are confronted with stress situations of nutrient restriction and / or provided with inert surface like bentonite day in vitro or

chronic disease condition / pathogenic situation in vivo. Thus it is the first time that cross protection was established among various serotypes of Escherichia coti. The molecular basis for cross protection was established by identifying cross protective OMPs at the regions; 93.2, 61, 35.5, 34, 28.8 and 17.6 kDa by western blott.
It is worth to note that heterologous antisera used for probing Escherichia coll OiyPs on two occasions is altogether from a different source, i.e., growth / cultural condition under which the antigen was obtained or antisera raised, are two totally different situations, per se one expressed during disease condition in j/iyo and the other was grown in vitro by simulating natural pathogenic situation through nutrient limitation and / or providing inert surface. So it can be concluded that the biofilm technique that we designed to grow the antigen to express cross-reactive OMPs, which were subsequently incorporated in the vaccine; and the situation under which OMPs expressed during a disease procesgf/"n vivo fe very much simitar. Further more, the OMPs expressed only under such situations are able to give cross protection, which is clearly demonstrated by subsequent challenge studies and western blotting involving antisera of different serotypes.
This argument is further supported by the results of subsequent blotting studies, wherein antigens grown under similar conditions are probed with heterologous sera obtained from colisepticemic conditions of two different situations, both yielding Escherichia coll in pure culture upon postmortem examination with pathognomonic colisepticemic lesions. In both the situations. It is possible that the birds have suffered a chronic disease process for quite a long time, thus providing the organism an ideal biofilm situation to grow, leading to an immune response. When such antiserum was used for probing, there is recognition of antigen grown

under biofilm and nutrients restricted conditions only. Antibody response during disease process i.e., in vivo processed antigen and against in vitro biofilm grown antigen reveal identical band profile with the same set of antigens, thus, further bolsters the argument that Escherichia coli infections are biofilm associated and vaccines prepared by incorporating antigens expressed only under such situations are cross protective.
Example 7. Process of producing E. co/i vaccine using biofilm
Biofilm cells grown for seven days in 0.16 per cent TSB with 0.3 per cent bentonite clay were harvested by discarding the supernatant media to remove any planktonic cells. Bentonite clay with biofilm grown E. coli was suspended in PBS. After counting number of viable bacterial cells it was further diluted with PBS to contain a final concentration of 4X10^ cfu/ml. Formalin was added to a final ,^ concentration of 0.1 per cent and stored in PBS at 4^0 until use. Vaccine was subjected to routine sterility and safety tests.
Example 8. Use of biofilm grown E. coli as vaccine
Two types of Escherichia coli vaccines, (1) vaccine prepared out of E. coli grown in biofilm mode (biofilm vaccine} and (ii) vaccine prepared out of E co//grown in conventional mode (free cell vaccine) were used in experimental vaccination trials.
Preparation of free cell vaccine: The culture was grown in 3.0 per cent TSB for 16 hr at 37°C and pelleted at 4000 rpm for 10 min at 4°C. The pellet was washed thrice with PBS. The pellet containing bacterial cells was finally re-suspended in PBS. The pellet was further diluted to contain 4X10^ cfu/ml after counting number of viable cells. Formalin was added to a final concentration of 0.1 per cent and stored in PBS at 4°C until use.

Vaccination schedule and experimental protocol.
i.Free cell vaccine was given orally for three consecutive days to group T\ on days three to five and boosted on days 21 to 23, whereas parenteral vaccine was given to group T3 by subcutaneous (s/c) route on day five and boosted on day 23 with a dose of 0.25 ml containing 1X10^cfu/bird/day.
ii.Biofilm vaccine was given orally for three consecutive days to group Tg on days three to five and boosted on days 21 to 23, whereas parenteral vaccine was given to group T4 by s/c route on day five and boosted on day 23 with a dose of 0.25 ml containing IXIOVu/bird/day.
iii.Controls in group T5 were given 0.25ml of sterile PBS by s/c route.
First challenge was carried out 10 days post vaccination i.e. on 15"^ day, by separating twelve birds from each group Ti to T5 from main flock and were again sub divided into six each (totally ten sub groups) and were challenged by intranasal (i/n) and intramuscular (i/m) routes using live homologous culture with 1X10^cfu/bird. Similarly second challenge was carried out on day 28*^ i.e., five days after booster and 23 days after primary vaccination and third challenge on day 36 i.e., 31 days after primary vaccination and 13 days after booster dose, 24 birds from each treatment groups were again challenged, by i/n and i/m routes (12 each). Birds were observed for clinical signs, morbidity and mortality for six days. If no death was recorded, then birds were slaughtered at the end of the sixth day post challenge. Samples of liver, spleen, heart and intestine were collected aseptically to estimate the colonization/elimination of Escherichia co//from these organs.

Experimental trials (tables 2 and 3) First challenge
Upon i/m challenge with homologous strain of both oral and subcutaneous free cell vaccinated birds, at 10 days post vaccination resulted in death of all the six birds In each of the groups compared to one survival out of six each In biofilm orally and s/c vaccinated groups (Table 2). Birds did not show protective level of immunity to withstand the severe fomri of challenge. This could be due to fact that, challenge was very severe (IxlO^cfu/bird) and / or primary vaccination alone may not be sufficient to withstand live severe challenge.
Upon i/n challenge, birds that received vaccine had significantly (P£0.05) reduced E coli load from intestine compared to unvaccinated controls. And within the vaccinated group, biofilm vaccinated group were significantly (P£0.05) better than free cell vaccinated birds (table 3) in preventing the invasion of liver by live challenge organisms. No difference in response to parenteral challenge was observed in any of the vaccinated groups and it is concluded that, the challenge 10 days after primary vaccination did not give protection against parenteral challenge. However, It is worth to note that performance of biofilm vaccine was relatively better than free cell vaccine either in eliminating or limiting the colonization of challenge organism or in reducing the mortality due to challenge infection, above all 10 days is too early to expect complete expression of specific immune response.
Second challenge
Live challenge by intra muscular route of both orally and s/c biofilm vaccinated birds, 23 days after primary and five days after booster, conferred 17 and 83 per cent

protection compared to 50 per cent in free cell parenteral vaccinated groups and no protection with free cell oral vaccine group i.e., biofilm vaccine by s/c route, was able to provide effective protection than biofilm vaccine by oral route or free cell vaccine by either of the routes.
Upon intra nasal challenge, elimination of challenge organisms from intestine was numerically better in biofilm-vaccinated groups, than free cell vaccinated counterparts and unvaccinated controls. However no growth or non-colonization of challenge organism in liver samples among parenteral biofilm vaccinated birds is noteworthy. And colonization of challenge organism among biofilm orally vaccinated (1.25X10") and free cell s/c vaccinated groups (1.45X10"^) are comparable to each other, but significantly less than free cell oral vaccinated group {5.43X10"") and unvaccinated control group (6.40X10^) (table 3). i.e., biofilm parenterally vaccinated birds provide no opportunity for challenge strain to invade, that othenwise would have lead to septicemic condition, which is evident by presence or absence of challenge organism from liver samples.
Third challenge
Live intramuscular challenge of both orally and parenterally free cell vaccinated birds, after 31 days primary vaccination and 13 days booster, resulted in vaccine efficacy of 33 and 58 per cent respectively compared to 42 and 100 per cent in biofilm vaccine, So it is obvious that biofilm vaccine by parenteral route alone is capable of providing complete and solid protection against severe form of challenge.

Upon i/n challenge, elimination of challenge organism from intestine was significantly (P£0.05) better in biofilm vaccinated groups compared to free cell vaccinated group and unvaccinated controls. Colonization of challenge organism In other visceral organs was quite low in liver and spleen with no colonization in heart sample among biofilm oral vaccinated birds; with absolutely no colonization In any of the visceral organ samples among biofilm parenteral vaccinated birds. Thus there is a significant {PE0.05) reduction in recovery of challenge organism from intestine and liver from biofilm oral vaccinated birds compared to free cell and unvaccinated counterparts.
The results of preliminary vaccination trial suggest that vaccine prepared from biofilm grown bacteria is effective In decreasing the mortality and very effective in elimination of challenge organism followed by intranasal challenge, compared to that of free cell vaccinated counterparts. Biofilm vaccine by subcutaneous route provides solid immunity not only to resist against very severe form of challenge but also, nullifies any chance for the challenge organism to Invade the visceral organs unlike in birds which received vaccine prepared out of conventionally grown bacteria. Also recovery of challenge organism from liver and spleen was significantly (P
Field vaccination trials (table 4)
The results of experimental vaccination trials are indeed worth noting, wherein protective ability of the biofilm vaccine was compared with that of free cell vaccine in three different challenges, which were considered to be sufficiently consistent to establish the superiority of the biofilm vaccine over free cell vaccine. Besides conferring 100 per cent protection against any form of challenge, there was a significant (P Mortality per cent in unvaccinated control birds in farm "A" revealed that approximately one out of four birds which died, were due to colibacillosis.

constituting 26.12 per cent in a total mortality of 111. Death due to Escherichia coli was not seen in Ti where birds were boosted through s/c route after oral priming. In T2 where birds were both primed and boosted through oral route, mortality due to Escherichia cod was eight per cent in a total mortality of 75 birds. This could partly be due to failure of the orally immunized birds to withstand heterologous severe challenge as this was obsen/ed during challenge studies of field vaccinated birds or probably due to the fact that oral route will not ensure all the birds to consume vaccine uniformly, or it could also be due to degradation of antigen in gastro intestinal tract. At the end of 39 days, the vaccinated groups consumed relatively less feed with a FCR of 1.68 compared to 2.06 in un vaccinated control group, to put on the same body weight.
The second field trial was carried out in farm B" to assess the reproducibility of the efficacy of biofilm vaccine at field level under different managemental conditions. In both the trials the biofilm vaccine, was able to reduce the mortality in orally vaccinated and orally primed group to eight per cent and nine per cent in Farm A and Farm B, respectively compared to mortality to the extent of 26.12 and 12.3 per cent in un vaccinated controls, besides totally obviating the mortality in s/c boosted group. At the end of 37 days, the vaccinated groups consumed relatively less feed with a FCR of 1.74 compared 1.96 in un vaccinated control group, to put on the same body weight.
The third field trial was carried out in form "C which was consistently affected by collbacillosis in successive batches causing severe economic loss. In this trial mortality due to colibacillosis was, though, not recorded, the scored gross lesions

were minimal in vaccinated birds compared to unvaccinated controls. Moreover, total flock mortality was also low at 3.9 per cent in vaccinated birds compared to 10.56% in controls. Vaccinated group consumed relatively less feed with a FCR of 1.91 compared to 2.17 In the un vaccinated control group.
6. Efficacy of biofilm vaccine
Challenge studies (table 5) were carried out to assess the efficacy of the biofilm vaccine using birds from three different groups of first field trial (Farm A) and challenged on day 42 with 1X10^ live cells of Escherichia coii intramuscularly using both homologous (078) and heterologous (02) live cultures, to determine the extent to which the biofilm vaccine provides protection and cross protection respectively.
Birds boosted through s/c route conferred 100 per cent protection against severe form of challenge compared to 100 per cent protection against homologous and 80 per cent protection against heterologous challenge among orally boosted birds, i.e., both forms of vaccination have provided effective protection under field conditions, but s/c boosting has given absolute protection in terms of ensuring not only 100 per cent cross protection against very severe form of challenge but also Infection free status of flock which Is reflected by better FCR.
The ability of vaccine prepared out of biofilm grown bacteria to afford protection against very severe challenge with a homologous vaccine strain is in agreement with the findings of Gross (1957) wherein formolised, heat and p propiolactone inactivated vaccine conferred effective protection against homologous live challenge. However, no details are given about the relative efficacy of this

vaccine. Deb and Harry (1978) reported maximum degree of protection (89 to 100%) using inactivated alum precipitated vaccine against an l/m challenge and Gyimah et al. (1986) reported 88 to 100 per cent protection followed by live challenge of birds followed vaccination using polyvalent vaccine. However our findings differ from that of the above researchers as for as cross protection by heterologous challenge is concerned. On the other hand, these workers did not see cross protection against other Escherichia coli serotypes. However, in the present study, the trials showed that, biofilm vaccine was the most effective than conventional vaccine both in terms of effective elimination of challenge organism, Escherichia coli from intestine and cross protection.
Our conclusion is, protection conferred by vaccine prepared out of Escherichia coli grown in biofilm mode is quite broad based and complete. Vaccination by s/c route was found to be highly effective when given once in the age of three to five days and boosted once in between 11 and 13 days, keeping in view the fact that primary vaccination alone has practically no protective effect against very severe form of challenge as seen in experimental trials. However single dose is very effective in elimination of Escherichia coli from intestine and preventing the colonization In liver that would be sufficient to protect the birds against intra nasal challenge, one of the most natural ways of field infection. Contrary to parenteral vaccination, the oral vaccination via drinking water, for three consecutive days during first week and again at third week in experimental trials and for two consecutive days at first and second week in field trials consistently eliminated Escherichia coli more efficiently than free cell vaccinated and unvacclnated counterparts.

Conclusion
It seems pertinent to conclude that Escherichia coli does form biofilms during natural infections. The biofilm form of Escherichia coli was found to alter its outer membrane proteins either by repressing or over expressing the Outer Membrane Proteins, of which some are responsible not only for homologous but also heterologous cross protection among various serotypes. These novel proteins are not expressed in free cell conventional vaccine. In the light of these findings, it becomes clear that the bacteria living in biofilm mode are structurally and functionally very different from the free-living bacterial cells. Hence, it is unwise to extrapolate the results obtained from free cell cultures to bacteria growing in biofilms.
The biofilm grown bacterial cells can sustain for very long periods in vitro when compared to their free cell counterparts. The stressful environment such as nutrients restriction in presence of bentonite clay as inert surface for attachment, colonization and trapping of nutrients would simulate the natural in vivo environment during infections with the expression of protective cell surface antigens.

References:
Arp, L. H., Graham, G. L G. and Cheville, N. F., 1979. Comparision of clearance rates of virulent and avirulent Escherichia coli in turkeys after aerosol exposure. Avian Dis., 23:386-391.
Arp, L. H., 1980. Consequences of active or passive immunization of turkeys against Escherichia coiiOlB. Avian Dis., 24:808-815.
Bolin, C. A. and Jensen, A. E., 1987. Passive Immunization with antibodies against Iron Regulated Outer Membrane Proteins protects Turkeys from Escherichia CO//septicemia. Infect. Immun., 55:1239-1242.
Bradford, M. M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochetn., 72:248-252.
Brown, M. R. W. and Williams, P., 1985. The influence of environment on envelope properties affecting survival of bacteria in infections. Annu. Rev. Microbiol., 39:527-56.
Costerton, J. W., Levandowski, Z., Caldwell, D. E., Kort)er, D. R. and Lappin-Scott, H. M., 1995. Microbial biofilms. Annu. Rev. Microbioi., 49:711-745.
Costerton, J. W., Stewart, P. S. and Greenberg, E. P., 1999. Bacterial biofilms: A common cause of persistent infections. Science, 284:130 -133.
Deb, J. R. and Harry, E. G., 1976. Laboratory trials with inactivated vaccines against Escherichia co//(O78:K80) infection in fowls. Res. Vet. Sci, 20:131-138.
Deb, J. R. and Harry, E. G., 1978. Laboratory trials with inactivated vaccines against Escherichia co//(02:K1) infection in fowls. Res. Vet. Sci., 24:308-313.
Ewing, W. H., Tatum, H. W., Davis, B. R. and Reavis, R. W., Studies on the serology of E. coli group. CDC publication, communicable disease center, Atlanta, Georgia. 1956.
Franchini, A., Canti, M., Manfreda, G. and Bertuzzi, S., 1991. Vitamin E as adjuvant in emulsified vaccine for chicks. Poult. Sc/., 70:1709-1715.
Frommer, A., and Freilin, P. J., Bock, R. R., Leitner, G., Chaffer, M. and Heller, E. D., 1994. Experimental vaccination of young chickens with a live, non -pathogenic strain of Escherichia coll. Avian Pathol., 23:425-433.
Gordon, R. P., 1961. Bull. Off. Int. Epizoot., 56, 507.
Griffiths, E., Stevenson, P. and Joyce, P., 1983. Pathogenic Escherichia co//express new outer membrane proteins when growing in vivo. FEMS Microbiol. Lett., 16:95-99.

Gross, W. B.,1957. Vaccine against Escherichia coli infection in chickens. Avian Dis., 1:347.
Gyimah, J. E. and Panigrahy, B., 1985. Immunogenicity of an Escherichia coli (Serotype 01) Pili vaccine in chickens. Avian Dis., 29:1078-1083.
Gyimah, J. E., Panigrahy, B., Hall, C. F. and Williams, J. D., 1985. Immunogenicity of an oil-emulsified Escherichia coil bacterin against heterologous challenge. Avian Dis., 29:540-545.
Gyimah, J. E., Panigrahy, B. and Williams, J. D., 1986. Immunogenicity of an Escherichia co//multivalent pilus vaccine in chickens. Avian Dis., 30:687-689.
Kapur, v.. White, D. G., Wilson, R. A. and Whittam. T. S., 1992. Outer membrane protein patterns mark clones of Escherichia coil 02 and 078 strains that cause avian septicaemia. Infec. immun. 60:1687-1691.
Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227:680-685.
Lietner, G., Melamed, D., Drabkin, N. and Heller, E. D., 1990. An Enzyme linked immunosorbent assay for detection of antibodies against Escherichia coll: association between indirect hemaglutination test and survival. Avian Dis., 34:58-62.
Mckay, K. A., Ruhnke, H. I. and Barnum, D. A., 1965. The results of sensitivity tests on animal pathogens conducted over the period 1956-1963. Can. Vet. J., 16:103-111.
Melamed, D., Leitner, G. and Heller, E. D., 1991. A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli. Avian Dis., 35:17-23.
Orskov, F., Orskov, I., Jann, B., Jann, K., Muller-settz, E. and Westphal, O., 1967. Acta pathalogica et microblologica scandinavia, 71:339.
Orskov, F., Orskov, I., Jann, B. and Jann, K., 1971. Acta pathalogica et microblologica scandinavia, 79:142.
O"Toole, G. A., Kaplan, H. B. and Kolter, R., 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol., 54:49-79.
Panigrahy, B., Gyimah, J. E., Hall, C. F. and Williams, J. D., 1984. Immunogenic potency of an oil-emulsified Escherichia co//bacterin. Avian Dis., 28(2): 475-481.
Postgate, J. R., Viable counts and viability. In: Methods in Microbiology, Vol. I. Norris, Jr. and Ribbons, D. W., Eds. Academic Press, p. 611-628,1969.

Rosenberger, J. K., Fries, P. A., Cloud, S. S. and Wilson, R. A., 1985b. In vitro and in vivo characterization of avian Escherichia coli, II. Factors associated with pathogenicity. Avian Dis., 29{4):1094-1107.
Sojka, W. J. and Carnaghan, R. B. A., 1961. Escherichia coli infection in poultry. Res. Vet. Sci., 2:340-352.
Tengerdy, R. P., Lacetera, N. G. and Nockels, C. F., 1990. Effect of beta carotene on disease protection and humoral immunity in chickens. Avian Dis., 34:848-854.
Zigterman, G. J., Van de Ven, W., Van Geffen, C, Loeffen, A. H., Panhuijzen, J. H., Rijke, E. O. and Vermeculen. A. N., 1993. Detection of mucosal immune responses in chickens after immunization or infection. Vet. Immunol. Imunopathol., 36:281-291.

We Claim:
1.0: A process of producing vaccine/against colibacillosis of birds and animals) wherein the E. coil bacteria are cultured as a biotin using inert substances such as bentonite clay, harvested using known methods inactivated using chemicals such as formalin and the inactivated bacteria are packaged into convenient doses
2.0: A process of producing vaccine for colibacillosis comprising of the following steps:
clay (0.3 % w/v) was suspended In 0.16 per cent tryptone Soya Broth
(TSB). -freight media {as prepared in step a) was sterilized by autoclaving at 15 lbs pressure at 121°Cfor15min.
c. Check for sterility by known methods, and Inoculate with the prepared Escherichia
co/j inoculum’s and incubate at 37°C.
d. Shake the culture flasks six times daily in a mechanical shaker at 50 rpm for one
hour to keep the bentonite clay uniformly suspended in the media.
e. Harvest seven-day-old biopic cells by discarding the supernatant media to
remove any plank tonic cells.
f. Count the number of viable Escherichia coil biotin cells by colony counting of ten
fold serial dilution in sterile phosphate buffered saline pH 7.4 after vigorous
overtaxing of bentonite clay In test tube and counts were expressed as colony
forming units/g of bentonite clay.
g Adjust bentonite clay with biotin growth to a final concentration of

h. Add Formalin to a final concentration of 0.1 per cent

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Patent Number

216990

Indian Patent Application Number

730/CHE/2003

PG Journal Number

17/2008

Publication Date

25-Apr-2008

Grant Date

24-Mar-2008

Date of Filing

15-Sep-2003

Name of Patentee

Dr. B. M. VEEREGOWDA

Applicant Address

NO. 293, LAKSHMI NIVAS, 1ST MAIN, 4TH CROSS, KERE ANGALA ROAD, KAMALANAGAR, BANGALORE - 560 079,

Inventors:

#	Inventor's Name	Inventor's Address
1	Dr. B. M. VEEREGOWDA	NO. 293, LAKSHMI NIVAS, 1ST MAIN 4TH CROSS, KERE ANGALA ROAD, KAMALANAGAR, BANGALORE - 560 079,
2	Dr. G. KRISHNAPPA	NO. 260, 7TH MAIN ROAD, 3RD STAGE, BASAVESHWARANAGAR, BANGALORE - 560 079,

PCT International Classification Number

A61K 39/108

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA