Estimation of titers of antibody against Pasteurella multocida in cattle vaccinated with haemorrhagic septicemia alum precipitated vaccine

Aim: The present study was carried out in 100 cattle to assess the antibody response to Haemorrhagic Septicaemia alum precipitated vaccine by Microtiter Agglutination Test (MAT), Indirect Haemaaglutination Assay (IHA) and Monoclonal Antibody based Indirect Enzyme Linked Immunosorbent Assay (ELISA). Materials and Methods: One hundred cattle from a local gaushala of Ludhiana were vaccinated with alum precipitated Haemorrhagic Septicaemia vaccine. Serum was collected at 0, 42, 84 and 128 days post immunization and antibody titers at different stages were estimated by MAT, IHA and ELISA. Results: The animals exhibited the classical pattern of humoral immune response with gradual increase and achievement of peak antibody titers plateau by 42DPI and gradual decline by 128 DPI. The IHA titers in cattle were significantly higher (P<0.05) at 42 days post immunization than the corresponding MAT titers on the same day. ELISA titers were significantly higher (P<0.05) than MAT and IHA titers at 42 DPI. IHA was found to be more sensitive than MAT, and the titers were higher by ELISA than by MAT and IHA throughout the observation period. Conclusion: The results indicate that animals vaccinated with commercial alum precipitated HS vaccine could not develop and sustain adequate levels of antibody for long duration.


Introduction
compared to foot and mouth disease, rinderpest, anthrax and black quarter, respectively [4].The estimated Pasteurella multocida, a commensal of digestive annual economic losses in India due to Pasteurella and respiratory tracts of warm-blooded animals, causes multocida infections alone were found to be to the tune disease in animals (bovines, porcines, rabbits, and of Rs. 228 millions [5].poultry) weakened by stresses such as viral infections, Humoral immunity plays an important role in heat, cold or humidity with aerosol transmission of protection against the disease.Vaccination has a infection between animals [1].Haemorrhagic Septigreater effect in controlling mortality in HS than any caemia (HS) is an acute, fatal septicaemic disease of other measure [6].The vaccines in use against HS cattle and buffaloes caused by the bacterium Pasteurella include broth bacterins, alum precipitated, aluminium multocida and occur as a catastrophic epizootic in hydroxide gel and oil adjuvant vaccines [7].The most many Asian and African countries resulting in high widely used vaccines in Asia being the whole cell mortality and morbidity.Serotypes B: 2 and E: 2 of P.
formalin killed P. multocida P bacterin precipitated 52 multocida are associated with hemorrhagic septicaewith alum or emulsified in aluminium hydroxide gel.mia in cattle and buffaloes in Asia and Central Africa The alum precipitated and the aluminium hydroxide respectively [2].The interactions between host factors gel vaccines are reported to confer immunity for four to and certain specific bacterial virulence factors, like six months.Oil adjuvant vaccine that imparts higher lipopolysaccharides (LPS), capsule, outer membrane level of immunity (up to 1 year) poses difficulty in proteins (OMP), fimbrial protein etc are responsible for injecting owing to its viscous nature and it induces the pathogenesis of the disease [3].
inflammation at the site of injection making it In India, HS has been identified as number one unpopular among the field users (w/v), and incubated at 37 ºC for 30 min with occasional

Materials and Methods
shaking.The tanned G-SRBCs (TG-SRBCs) were Ethical approval: The animal experimentation was pelleted and washed three times with PBS to yield a approved by the Institutional Animal Ethics Committee.
10% suspension.The antigen prepared as described o One hundred adult cattle from a local gaushala earlier was heated at 56 C for 30 min in a water bath (cow shelter) of Ludhiana were included in the study.
with frequent shaking.Heat treated suspension was 0 Zebus as well as cross bred of zebu and Jersey/Holstein then centrifuged at 6000xg for 15 min at 4 C.The clear 0 cattle were present in the population studied.The supernatant was separated and stored at -20 C. One animals included both male and female cattle which volume of packed TG-SRBCs and 15 volumes of the antigen, approximately were mixed and incubated for were in production or non-producers.The average age 0 of the animals was 3 years.The animals were 1-2 hrs at 37 C in a water bath with frequent shaking.
The sensitized cells thus prepared were washed 3 times apparently healthy and reported to be vaccinated and with NSS and resuspended in chilled NSS to obtain a dewormed annually.The animals were administered 5 1% suspension.To remove the heterophile antibodies, ml of alum precipitated HS vaccine through subcutaall the test serum samples (3 volumes) were adsorbed neous route.All the animals were bled prior to vaccination 0 with packed SRBCs (1 volume) for 2 hr at 37 C with and blood samples were collected at 0, 42, 84 and 128 periodic shaking before the test proper.The RBCs were days post immunization (DPI).Serum samples were removed by centrifugation.The supernatant was subjected to analysis by MAT, IHA and ELISA to collected and used for the test.Normal saline (160 µl) measure antibody titers in response to the vaccine.and adsorbed serum (40 µl) were added to the first well Preparation of whole cell killed antigen: The whole (1:5 dilution), and 100 µl of NSS was added to all the cell killed antigen was prepared from P. multocida other wells of a U bottom 96 well microtitre plate (B:2) vaccine strain P .The organisms were grown 52 (Tarson).Two fold serial dilutions of serum were made.overnight on BHI agar in Roux flasks, and the bacterial An equal volume of the 0.5% sensitized RBC cells were harvested in normal saline solution (NSS).
suspension was added to all the wells.The plates were The purity of the culture was tested by Gram's staining.agitated briefly and left at room temperature for 2 hr.The bacteria were killed by adding formalin at a final Appropriate antigen, RBC and serum controls were concentration of 0.3% and incubated at room temperaalso incorporated in the test.ture overnight.The sterility of the suspension was ELISA: Indirect ELISA procedure was carried out as tested by inoculation of 1 ml suspension on to the blood per method described earlier [12].The antibody titre of agar on which no growth was observed.The cell the serum samples was determined by monoclonal suspension was centrifuged at 1000xg for 15 minutes antibody (Mab) based indirect ELISA Kit prepared at and subjected to three washings with NSS.The the Department of Veterinary Microbiology, College of bacterial sediment was suspended in NSS to match the Veterinary Science, Lala Lajpat Rai University of opacity with tube no. 3 of Mac Farland opacity scale.
Veterinary and Animal Sciences (LLRUVAS), Hisar, MAT: MAT was performed as per the standard method Haryana.The kit is based on indirect ELISA using outer membrane proteins of the P strain of the bacteria [10].Serum samples were serially diluted two fold in a 52 final volume of 100 µl. 100 µl of formalin killed as antigen (dilution1:1000).The binding of the antibodies antigen was added to each well.Negative control in serum samples with the plate bound antigen was contained 100 µl of sterilized normal saline solution detected by a monoclonal antibody against cattle IgG (NSS) and 100 µl of the antigen.The formation of matt conjugated with horse radish peroxidase.Using an signified agglutination whereas button formation was equation of regression, antibody titers were calculated indicative of a negative reaction.Titers (log ) were from the intensity of color developed in a chosen single

Results and Discussion
from OMPs and bacterin supplemented with 10% OMPs, the mean IHA titers 21 days post vaccination The post vaccination antibody titers in cattle were 27.9, 168.9 and 42.4,respectively [15].It was vaccinated with alum precipitated HS vaccine revealed concluded that subunit or whole bacterin based vaccines an increase over the initial prevaccination stage, with had less immunogenic potential in comparison to antipeak antibody response being observed at 42  was observed to be the most practical test among the However, the decline by 128 DPI to a mean log titre of 10 four serological tests in the sero-diagnosis of haemorr-1.349(MAT), 1.355 (IHA) and 1.862 (ELISA) indicated hagic septicaemia [17].that the immune response generated was of low In a study conducted by Sangwan V. P.
[18] on magnitude and of short duration and may not confer buffaloes, administered alum precipitated vaccine, adequate immunity.The IHA titers in cattle were antibody levels were found to be high up to 84 days significantly higher (P<0.05) at 42 days post immunipost vaccination after which they declined.In an earlier zation than the corresponding MAT titers on the same study, the mean antibody titers (log ) by ELISA four 10 day.Mean ELISA titers were significantly higher months post vaccination in buffaloes vaccinated with (P<0.05)than MAT and IHA titers at 42 DPI.
aluminium hydroxide gel precipitated HS vaccine were However, MAT and IHA titers at 84 and 128 DPI did found to be 2.22±0.06,2.09±0.02,3.46±0.08and 2.14 not reveal any significant difference.ELISA titers were ±0.03, respectively, and were claimed to be protective, significantly higher than IHA and MAT titers at 84 and whereas at six months post vaccination they were 128 DPI, respectively.The results of our study are in found to be marginally protective vaccine whereas in bivalent FMD-HS vaccine, the In the present study IHA was found to be more peak level of antibody titre was detected on 48 days sensitive than MAT in assessing the antibody levels.The different titers observed in the same animals by post priming which persisted for a longer time than the agglutination assays (MAT/IHA) and ELISA can be monovalent alum precipitated HS vaccine.reconciled with the fact that these two assays target The mean antibody titers of thirty pregnant Sahiwal antigens of different nature i.e. agglutination assays are cows vaccinated with alum precipitated formalin killed directed towards particulate antigen whereas ELISA bacterin 15 days prior to parturition and after parturition detects immune response to soluble antigens.ELISA is were 213 and 182, respectively by IHA [21].generally used to detect IgG antibodies [13], while IHA Humoral immune response in mice against P. is used to detect the major immunoglobulin fraction multocida vaccine grown in presence of serum was IgM induced in response to such vaccination [14].compared using ELISA and IHA .The highest antibody In a comparative study of alum adsorbed HS titers as measured by IHA and ELISA were observed on vaccine, P. multocida anti-idiotypic vaccine prepared 7, 14, 45, and 75, DPI.No significant difference in the Values with different letters in superscript differ significantly at P< 0.05.
10 dilution of the test sera.The optical density (O.D.) was recorded as the reciprocal of the highest dilution of the read on an ELISA Reader (Tecan) at 450nm.serum giving at least 50 per cent agglutination.Antibody titre (Log ) Y = a + bX 10 IHA: IHA was performed as per the method reported = 1.35 + 0.05 X earlier [11] with minor modifications.A 10 % (v/v) Where constant a = 1.35, constant b = 0.05, suspension of sheep RBCs was prepared in NSS and X = O.D. value of a test well/ (Mean + 3S.D. value of 0 stored at 4 C. Equal volumes of glutaraldehyde solution negative controls wells).(1% v/v in NSS) and 10% washed RBC suspension 0 Statistical analysis: SPSS 16 software was used for were mixed and allowed to stand at 4 Cfor 30 min with data analysis.The values were converted to log before intermittent gentle stirring.The sensitized sheep RBCs 10 (SRBCs) were given three washes in NSS to remove analysis.
weeks PI which decreased to 1.52 at 24 MAT, IHA and ELISA are presented in Table-1.The weeks PI in bovines vaccinated with alum precipitated mean value of peak antibody titers (log ) induced by 10 vaccine thus necessitating vaccination at six monthly vaccine in these cattle was 1.948 as measured by MAT, intervals.In a study conducted on American bison, IHA 2.020 by IHA and 2.076 by ELISA, respectively.
DPI, idiotypic vaccine prepared against P. multocida B:2.plateauing up to 84 DPI and gradually declining It has earlier been reported by Venkatesh et al [16] thereafter as determined by MAT, IHA and ELISA that maximum antibody titers (log ) of 2.46 were 10 (Figure-1).The antibody titers (log ) as determined by 10 observed at 12