Comparative evaluation of antibody response in rabbits vaccinated with toxoid, alum precipitated and alum precipitated oil adjuvant enterotoxaemia vaccines

Aim: To compare the newly formulated enterotoxaemia vaccine having oil and alum adjuvants, with presently available toxoid and alum precipitated vaccines. Materials and Methods: Three types of enterotoxaemia vaccines, namely toxoid (TV), alum precipitated (APV) and alum precipitated oil adjuvant vaccine (AOV) were prepared using a highly toxigenic strain of Clostridium perfringens type D procured from Division of Biological Standardization, IVRI, Izatnagar. Humoral immunity generated in rabbits with these vaccines was then quantified using indirect enzyme-linked immunosorbent assay (ELISA) and mice neutralization test (MNT). Results: Out of three enterotoxaemia vaccines tested, alum precipitated oil adjuvant vaccine produced higher and persistent antibody titre for more than 45 days without any booster dose and did not produce any untoward reactions at the injection site. Alum precipitated vaccine elicited better and persistent immune response than toxoid vaccine though it was less than alum precipitated oil adjuvant vaccine. In MNT, alum precipitated and alum precipitated oil adjuvant vaccines showed protection at th th 45 day of post vaccination while toxoid vaccine showed only up to 28 day. Conclusion: Results of the study unfolded the synergistic role of adjuvants in the induction of better and persistent immune response and also indicated the superiority of alum precipitated oil adjuvant vaccine over the currently available toxoid and alum precipitated enterotoxaemia vaccines.


Introduction
with antigenic mass and duration of immunity, these drawbacks can be very well addressed by the alum Enterotoxaemia caused by Clostridium perfringens precipitated oil adjuvant formulations. Realizing these is a devastating disease of small ruminants with very needs, a new alum precipitated oil adjuvant enteroshort clinical course [1] and therefore therapeutic intertoxaemia vaccine was prepared in the present study and ventions often do not work. Among the five toxinotypes then compared with commercially available toxoid and of this organism, type D producing epsilon toxin is alum precipitated vaccines in rabbit model. principally associated with caprine and ovine enterotoxaemia [2,3]. Though this disease can be

Materials and Methods
prevented by epsilon toxoid vaccination [3][4][5] double Bacterial cultures: A highly toxigenic strain of C. initial dose is currently recommended for both sheep perfringens type D procured from Division of and goats, followed by a booster every year in sheep [6] Biological Standardization, IVRI, Izatnagar, was used and every 3-4 months in goats [7]. Hence, the present for vaccine preparation. The organisms were vaccination strategy requires heavy input of resources characterized morphologically, culturally and by along with the difficulty in tracing animal for booster toxigenicity studies. Molecular characterization was dose. Therefore there is need for developing a new done by PCR using genomic DNA isolated from the enterotoxaemia vaccine which can induce a sustained strain and etx specific primers (F-5'AAG GAT CCA immunity [8]. AGT TTA GCA ATC GCA TCA GC3'; R -5'TAC CTC Aluminium salts, the widely used adjuvants in GAG TTA TTT TAT TCC TGG TGC C3'). many human and veterinary vaccines [9] can provide only a short duration of immunity. Therefore oil has Experimental animals: Clinically healthy Swiss albino been used along with these to form depot at vaccination mice of either sex weighing not less than 18-20g and site. The duration of immunity provided by these albino rabbits (Lupus cuniculus) of either sex weighing vaccine formulations are longer and amount of not less than 1 kg were procured from the Laboratory antigenic mass required is also less [10]. Since the Animal Resources (LAR) Section, I.V.R.I, and major limitations of the enterotoxaemia vaccine lies Izatnagar and maintained under standard conditions of nutrition and management. The animal ethics committee commercial blender and mixed for 20minutes at room of the Indian Veterinary Research Institute, Deemed temperature. After adding 100ml of alum precipitated University approved the study. toxoid vaccine to the blender emulsification was carried out at room temperature giving 4 runs of 5min Preparation of toxoid vaccine (TV): The production each, with about 5min gap between each run. The medium [11] was inoculated with the seed culture and 0 0 vaccine was stored at 4 C overnight and re-emulsified incubated at 37 C overnight. An active growth was next day. evident by vigorous gas production within 3 hours of Sterility, safety and stability testing of vaccines: incubation. After 24 hours, growth was tested for purity Sterility tests of all three vaccines were done by and aerobic sterility. Trypsin was then added to a final 0 inoculating in nutrient agar and nutrient broth. The concentration of 0.25% and incubated at 37 C for one safety of the vaccines was tested in six swiss albino and half hours. Two ml of the product removed from mice and made a continuous observation for any each flask was pooled and centrifuged. 0.1 ml of serial dilutions of this supernatant was injected i/v into two untoward effects up to 10 days. For testing the stability 0 healthy adult mice (18-20g weight) and the minimum all the prepared vaccines were kept at 4 C as well as at lethal dose per ml was determined. After trypsinization room temperature for 14days before using them for rest of the culture was pooled, formalized with 0.5 % immunization. 0 formalin and incubated at 37 C for 15 days. It was Immunization: The antigenic mass was kept constant tested for aerobic and anaerobic sterility and then in all three vaccines and then each rabbit having not 0 stored at 4 C. Atoxicity test was carried out in mice by less than 1kg weight (4 animals in each group with total i/v inoculation of 0.2 ml of prepared TV. 4 groups including control) was immunized with a Preparation of alum precipitated vaccine (APV): pH of single dose of 2ml TV, 2.5ml of APV and 4ml of AOV 100 ml TV was adjusted to 6.5 by using sterile 1N by deep i/m route. Serum collection was done on 0, 7, sodium hydroxide solution. 20% solution of hot sterile 14, 21, 30, 45 days DPV. alum in distilled water was then added to a final   a, b and c represents significant difference level 1, 2 and 3 a, b and c represents significant difference level 1, 2 and 3 a and b represents significant difference level 1 and 2.
The precipitate was separated by centrifugation and any growth denoting their sterility. All the vaccines dialyzed against 0.01M Phosphate buffer (pH 7.5) and were found safe in mice. AOV did not show separation 0 then concentrated using polyethylene glycol-6000. of different phases (oil and water) when kept at 4 C and Further purification was done by DEAE cellulose room temperature indicating its stability. anion exchange chromatography. The peak fractions Preparation of antigen: On analysis of purified toxin were pooled, concentrated and activated to check by SDS-PAGE, a single band of 32KDa was obtained toxicity then formalized and the protein concentration (Fig. 2). Hyper immune sera raised against TV and was estimated. The confirmation of purified epsilon APV could detect this toxin in western blot analysis toxin was done by SDS-PAGE and western blot (Fig. 3). The concentration of the pooled fractions of analysis.
epsilon toxin was 4.51 mg/ml. Production of hyper immune serum: Two ml of APV .07) to 28 day (2.49±0.14) with a temperature for half an hour. 0.2ml of this mixture was th sudden fall on 45 day. Although it produced less response injected intravenously into two mice. Two mice were th on 45 day compared to AOV, it was significantly greater given 0.1ml of the diluted toxin containing 300 than TV. MLD/ml to make control group.
MNT results obtained with different vaccines are

Results
shown in Table 5. APV and AOV showed protection at 45th day of post vaccination while TV showed protec-Characterization of C. perfringens type D: The procured tion only up to 28th day. strain of C. perfringens type D showed typical morphological, cultural and toxigenic properties. During Discussion molecular characterization the desired 997 bp product Eradication of enterotoxaemia, an economically was obtained (Fig-1). The toxin titre was 2000 MLD/ important devastating disease of sheep and goats is ml.
only having a remote possibility owing to the ubiqui-Sterility, safety and stability testing of vaccines: Final tous nature of organism [5].   have also reported protection only for 28 days method of control is vaccination. Inactivated bacterial in toxoid immunized animals. Among three vaccines whole-cell vaccines have been the most widely studied tested by indirect ELISA, AOV produced higher and prophylactic mode of treatment for infectious diseases persistent immune response up to 45DPV which may [3]. Though disease can be prevented by these remain for longer periods. Similar observation vaccines, immunity lasts only for a short period. Thus regarding the superiority of AOV over APV against due to the difficulty in tracing each animal for booster hemorrhagic septicaemia was reported earlier [16]. dose, the current vaccination strategy often fails. In a Though the highest immune response was found with th study evaluating the comparative efficacy of alum TV on 14 day, it dropped suddenly on subsequent th th precipitated and aluminium hydroxide gel adsorbed days. The titre on 45 day was less than 7 day enterotoxaemia vaccines in sheep there was no indicating the fast fall in immune response revealing significant difference between these two vaccines [12]. the reason for short term immunity provided by TV But, it has been proved that duration of immunity reported by the earlier workers [11,15]. APV showed provided by alum precipitated oil adjuvant vaccine an intermediate trend between TV and AOV. APV against hemorrhagic septicemia are longer with less elicited better and persistent immune response than antigenic mass [10]. Keeping these facts new toxoid vaccine though it was less than AOV. This was formulation alum precipitated oil adjuvant vaccine was in agreement with the results of [17] and [12] who made against enterotoxaemia and compared with observed the enhancing effect of alum on anti epsilon presently available toxoid and alum precipitated production. In present study none of animals immuvaccines. The antigenic mass per dose was maintained nized showed any untoward reactions at the site of same in all vaccines and used for immunization in injection. On the contrary to this some authors [18, 19] rabbits. The selection of rabbit was based on the observed adverse reactions following enterotoxaemia suitability of this animal as a model for testing the vaccination. efficacy and protective nature of vaccines.

Conclusion
As humoral immune response against epsilon plays an important role in protective immunity against The findings of this study unfolded the synergistic enterotoxaemia [13], only humoral response was role of adjuvants in induction of better and persistent assayed. The protective antibody titres against C.
immune response against enterotoxaemia. The worth perfringens epsilon toxin are usually measured using mentioning feature of the present study was the the toxin neutralization test in mice (MNT) [3,11]. But development of alum precipitated oil adjuvant this test is cumbersome, very slow, and expensive and enterotoxaemia vaccine which gives better and persistent can be relatively imprecise as often only a ranged result immune response. However, better understanding of is obtained. In addition, apart from the ethical consihigh level of protection which has been observed in derations of the use of living animals, this assay may AOV needs further exploration in larger scale suffers from the disadvantages inherent in biological especially in target species as sheep and goats and on tests such as variation in animal sensitivity [14]. Thus, various molecular determinants like isotypes, cytokine in the present study indirect ELISA was also used along assay etc. Such a study can lead to further development with MNT to monitor the humoral immune response.
of these better vaccines so that these can be finally In MNT, the gold standard test to measure transferred to hands of farmers. protective immunity, AOV and APV showed protection