Abstract
Objective
To compared seed culture of hemorrhagic septicemia (HS) bacteria which was used to produce vaccine for its antibody induction efficiency before and after passaging in natural host (calf) using laboratory animals.
Methods
Serial dilution of virulent bacteria was injected in to mice which were immunized with HS vaccine which was obtained from seed bacteria before and after back passaged in calf. Ratio of survived and dead was calculated by Reed-Meunch hypothesis and the LD50 value for each vaccine trial groups were calculated.
Results
The immunological study revealed that vaccine prepared from back passaged seed culture showed greater improvement in its immunopotency than seed vaccine (before back passage). Around 200 mice were used to study the immuno efficiency of vaccine. Each mouse was from the same source, which were free from the Pastuerella infection previous to expose to trial infection. The same broth culture of HS was used to induce infection in mice in both trials (vaccine before back passage and vaccine after back passage). The 0.2 mL of broth dilution from 10−1 to 10−10 was used, as dilution increases, death rate decreases. It indicates the minimum load of bacterium is required to induced infection.
Conclusions
Obtained results revealed that back passaged vaccine seed HS bacteria in its natural host had provided better immune efficiency to the culture than laboratory stock culture, and this findings recommended that regular annual back passage was mandatory for the vaccine seed culture of Pastuerella multocida bacteria for better establishment of immune potent vaccines.
Keywords: Hemorrhagic septicemia, Laboratory animals, Natural host, LD50 values, Annual vaccination
1. Introduction
Heamorrhagic septicemia (HS) is an acute infectious disease of cattle, buffaloes, sheep and goats, caused by Pasteurella multocida serotype B (P. multocida). The disease occurs mainly during rainy season particularly in early monsoon. It spreads rapidly among herds of animals, causing morbidity and mortality between 50% to 100%. Immunization against infectious agent is the only available tool in prevention and control of the disease. Multivalent vaccines were used annually in the endemic area, in spite of annual vaccination the outbreaks of disease were recorded every year. This may be due to improper vaccination and low potent vaccine usage. Keeping potency of vaccine as target this experiment was designed to check the potency of vaccine seed culture of HS before and after back passaged in calf. Enhancement in potency of back passaged vaccine seed culture was determined in detail in this experimental study.
2. Materials and methods
2.1. The preperation of vaccine strain of P. multocida (strain-P52)
The seed vaccine strain of P. multocida (strain-P52) was received from Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, the Drug/Vaccine Control Division of India for the purpose of vaccine production in Biological Production Division, Institute of Animal Health and Veterinary Biologicals, KVAFSU, Hebbal, Bangaluru. In this study, back passaging was done by injecting 1 mL of neat bacterial culture (vaccine strain) into calf (natural host). At 30 h post-injection blood was collected aseptically from jugular vein for preparation of seed bacterial culture for vaccine production. The vaccine prepared from before and after passage seed culture were checked for immune potency using laboratory mice.
2.2. Studies on the potency test of HS vaccine
2.2.1. Potency test of HS batch No: 5/08-09 vaccine (seed culture before back passage)
Potency test of HS batch No: 5/08-09 vaccine (seed culture before back passage) was inoculation to 50 mice, each with 0.2 mL dose by I/M on right thigh. After two weeks booster dose was administered. Tweenty one days after the vaccination, mice were divided into 10 groups of 5 mice each. Simultaneously unvaccinated 50 mice were divided into 10 groups of 5 mice each to serve as control. P. multocida P-52 strain bacteriological stock culture was tested for pathogenicity and virulence in mice. The stock culture was sub cultured in nutrient broth and inoculated for 8 h at 37 °C. The incubated culture was again checked for purity by smearing with grown strain smear and was found to be pure growth of P-52. The broth culture was serially diluted in log dilution in nutrient broth to get 10−1 to 10−10. Theses culture dilutions were injected to group of 5 mice each of vaccinated and unvaccinated control mice. The groups were designated as A1 to A10 for vaccinated and B1 to B10 for unvaccinated control. These grouped mice were housed in different cages labeled appropriately. The inoculated mice were kept on observation for 5 d and the mortality ratio was recorded daily in both the groups in the morning and evening. Results were tabulated and interpreted as for standard procedure (Reed and Muench hypothesis).
2.2.2. Potency test of HS batch No: 8/08-09 vaccine (seed culture after back passage)
The same experimental procedure was repeated for the vaccine which was prepared from the back passaged culture. In this vaccine potency test the same number of mice were used and the mice are from the same source. Other experimental conditions were maintained the same as above. The inoculated mice were kept on observation for 5 d and the mortality ratio was recorded daily in both the groups in the morning and evening. Results were tabulated and interpreted as for standard procedure (Reed and Muench hypothesis).
3. Results
The experimental induction of HS disease was done with the broth culture of P. multocida bacteria and its purity and specificity were checked by growing it on specific selective agar medium. The overnight culture gave clear turbidity with smearing from it. And Giemsa stain indicated the presence of bipolar bacilli.
In each trials proportional distance and LD50 values were calculated by using below formula:
 |
-Log of LD50 for vaccinated mice=-Log dilution above 50%+Proportionate distance
3.1. Studies of potency test of HS vaccine(P-52), before passage in calf (natural host)
Potency test result of HS seed vaccine strain (batch No: 5/08-09) (vaccinated group) (Table 1). The Proportional distance for vaccinated mice was 0.3, -Log of LD50 for vaccinated mice was 3.3 and LD50 value was 10−3.3.
Table 1. Studies of potency test of HS vaccine (P-52), before passage in calf (natural host) from June 12 to June 18, 2008.
| Vaccinated group | Culture dilute | Mortality | Dead | Survive | Accumulated value |
Mortality ratio | Percentage of mortality | |
| Dead | Survive | |||||||
| A1 | 10−1 | 3/5 | 3 | 2 | 16 | 2 | 16/18 | 88.88 |
| A2 | 10−2 | 4/5 | 4 | 1 | 13 | 3 | 13/16 | 81.25 |
| A3 | 10−3 | 3/5 | 3 | 2 | 9 | 5 | 9/14 | 64.24 |
| A4 | 10−4 | 4/5 | 4 | 1 | 6 | 6 | 6/12 | 50.00 |
| A5 | 10−5 | 2/5 | 2 | 3 | 2 | 9 | 2/11 | 18.18 |
| A6 | 10−6 | 0/5 | 0 | 5 | 0 | 14 | 0/14 | 00 |
| A7 | 10−7 | 0/5 | 0 | 5 | 0 | 19 | 0/19 | 00 |
| A8 | 10−8 | 0/5 | 0 | 5 | 0 | 24 | 0/24 | 00 |
| A9 | 10−9 | 0/5 | 0 | 5 | 0 | 29 | 0/29 | 00 |
| A10 | 10−10 | 0/5 | 0 | 5 | 0 | 34 | 0/34 | 00 |
Potency test result of HS seed vaccine strain (batch No: 5/08-09) in the unvaccinated control group was shown in Table 2. And the calculated proportional distance for vaccinated mice was 0.77, -Log of LD50 for vaccinated mice was 9.77 and LD50 value was 10−9.77.
Table 2. Potency test result of HS seed vaccine strain (batch No: 5/08) (Unvaccinated control group) from June 12 to June 18, 2008.
| Vaccinated group | Culture dilute | Mortality | Dead | Survive | Accumulated value |
Mortality ratio | Percentage of mortality | |
| Dead | Survive | |||||||
| B1 | 10−1 | 5/5 | 5 | 0 | 42 | 0 | 42/42 | 100 |
| B2 | 10−2 | 5/5 | 5 | 0 | 37 | 0 | 37/37 | 100 |
| B3 | 10−3 | 5/5 | 5 | 0 | 32 | 0 | 32/32 | 100 |
| B4 | 10−4 | 5/5 | 5 | 0 | 27 | 0 | 27/27 | 100 |
| B5 | 10−5 | 5/5 | 5 | 0 | 22 | 0 | 22/22 | 100 |
| B6 | 10−6 | 5/5 | 5 | 0 | 17 | 0 | 17/17 | 100 |
| B7 | 10−7 | 5/5 | 5 | 0 | 16 | 0 | 16/16 | 100 |
| B8 | 10−8 | 5/5 | 5 | 0 | 11 | 0 | 11/11 | 100 |
| B9 | 10−9 | 3/5 | 3 | 2 | 6 | 2 | 6/8 | 75 |
| B10 | 10−10 | 3/5 | 3 | 2 | 3 | 4 | 3/7 | 42.85 |
The difference in LD50 between vaccinated and control was 10−6.47 (10−9.77-10−3.3) and the minimun is Log 4. The LD50 value indicated that HS vaccine was potent.
3.2. Potency test result of HS seed vaccine strain after back passaged in calf (natural host)
In the potency test result of HS back passaged vaccine seed strain (batch No: 8/08-09) (vaccinated group). The proportional distance for vaccinated mice was 0.2, -Log of LD50 for vaccinated mice was 3.2, LD50 value was 10−3.2.
For the Potency test result of HS seed vaccine strain (batch No: 8/08-09) (unvaccinated control group), the proportional distance for vaccinated mice was 0.77, -Log of LD50 for vaccinated mice was 9.77, LD50 value was 10−9.77.
The difference in LD50 between vaccinated and control was 10−6.57 (10−9.77-10−3.2) and the minimum is Log 4. The LD50 value indicateed that HS vaccine was potent.
4. Discussion
The immunological study was conducted using laboratory mice to assess the immunopotency of HS vaccine (seed bacteria). Around 200 mice used in the study were from single source belonging to the same colony and were free from the Pasteurella infection by blood smear examination for bipolar organisms, previous to exposure to trial infection. The culture of hemorrhagic septicemia was used to immunized the mice in both trials (vaccine before back passage and vaccine after back passage). The virulent bacterium which was used to induce the infection was available in the form of broth culture. A volume of 0.2 mL of broth dilution from 10−1 to 10−10 was injected into each mice, as dilution increased death rate decreased which indicated the minimum load of bacterium was required to induce infection[1],[2].
Results obtained indicated that potency test conducted for HS vaccine (strain-P52) has shown the increased immunity in the vaccinated animals with passaged vaccine (vaccine produced by the back passaged bacterial strain) than immunity imparted by unpassaged seed vaccine. Passaged strain had better efficiency because of the change in the membrane proteins. When bacteria is in the natural host it is under the iron restricted condition, P. multocida often expresses high molecular weight. Outer membrane proteins called iron regulated outer membrane proteins (IROMP) may be involved in induction of immunity and used as immunogen to produced subunit vaccines[3]. This indicated that back passaging in the natural host may help the bacterial to regain its potential to boost immune stimulation. This finding also supports that bacteria had changed its virulence and immunogenicity when it was grown out of its natural host system and it may become less immune competent in induction of antibody[2]. Two groups of seven animals were vaccinated with haemorrhagic septicaemia vaccine and black quarter vaccine. Pre and post vaccinated sera samples of cattle were collected and tested using Fourier transform infra red spectrometer. The variation observed in peaks indicating the change in protein and lipids levels in the animals was due to introduce of antigens[4]. Our findings also suggested that immune efficiency of vaccine strain may decrease when it was passaged repeatedly in laboratory condition (in vitro).
Immunological studies of HS vaccine (potency test) indicated that all mice were showing the immune response according to the level of antibody titre in them. Propotional distance value for the vaccine (before back passage) in experimental group is 0.30 and 0.20 for back passaged vaccine and proportional distance value for control group in both the trials is 0.77. The difference between proportional distance value for before and after passaged vaccine was only 0.1 but in terms of log values of LD50 is 10−3.3 for before back passaged vaccine and 10−6.57 for back passaged vaccine. This LD50 log values had provided the huge difference in the immune potency of two vaccines used. After challenge studies using virulent HS strain, the obtained logarithmic (LD50) values clearly indicated that back passaged vaccine was more potent than earlier seed vaccine which could be due to the changes in the antigen structures of bacteria. As a principle of attenuation when you grow any bacteria outside its host range for multiple generations it may reduce its virulence and may also altered in its immune efficiency. Such bacteria when you put back into its natural host, it suddenly regains all its virulence and immune efficiency.
The protective efficiency of purified protein (outer membrane proteins) fractions of P. multocida serotype B:2 (P-52) strain were studied in mice[5]. Highest ELISA Log10 titre was observed in mice injected whole outer membrane proteins where as mice injected with purified protein fractions showed low levels of ELISA Log10 titres. It revealed that whole organism was required in its pure form to induce high level of antibody titre in the infected host. This study also indicated that under iron restricted conditions, P. multocida often expresses high molecular weight outer membrane proteins called IROMP which may be involved in inducing the cross protective immunity[3],[6] and when grown in the natural host P. multocida also expresses 18 Kda fimbrial subunit protein which processes potential property and could be potential immunogen in inducing protective immunity[7],[8]. These fimbrial protein which are expressing in the natural host well than in other unnatural host could be the reason to change in the immunostimulation by the vaccine seed before and after back passage into its natural host system. These fimbrial proteins and IROMP may be changed when seed bacterial culture was put in to natural host from the test tube. These altered protein might be the reason for increased immune potency of back passaged vaccine. Biofilm HS vaccine which was used in claves gave satisfactory antibody titre after the third booster dose[9]. It might be due to lack of immune potency inducible dosage in a second dose of vaccine. Although several vaccine formulations including alum precipitated, oil adjuvant and multiple emulsion vaccines are commercially available, the quest for suitable broadly protective HS vaccines with long-lasting immunity is on the upsurge[10].
Current finding indicated that back passage might be useful to in vitro adopted bacteria in getting membrane proteins with altered motifs and even chances of mutation such as single nucleotide polymorphism and definitely there is increased fimbrial activity in natural host than in vitro.These hypothetical queries has to be addressed by analyzing molecular level changes in bacteria by genomic study of bacterial seed used for vaccine production[11]. Information available on HS vaccines developed from time to time using whole bacteria or their components. Kinetics and isotype of antibody and cell-mediated immune responses have also been poorly understood so far. Hence information on their role in protection against HS is reviewed[12]. However, detailed laboratory mice experiment indicated that vaccine which was produced by back passaged bacteria strain had better and improved immune potency than unpassaged in vitro culture.
HS is one of the most important bacterial diseases of cattle and buffalo in India. The disease occurs mainly during the rainy season. It spreads rapidly among groups of animals, causing morbidity and mortality between 50 to 100%. Vaccination is the only available preventive measure for the control of disease, so regular annual vaccination is recommended in the endemic areas. The vaccine produced by the seed vaccine strain may lose its potential or may decrease in its immune potential to stimulate high antibody titre in the vaccinated animal because of repeated passaging in the unnatural host and stressful laboratory conditions (growth factors and environmental conditions). When we back passaged the vaccine seed culture in its natural host, it improves the immune potency of bacterial culture. The molecular and genomic level changes are needed to be resolved by proper genomic analysis and analysis of genomic level mutations which could reveal the reason for improvement of immune potency. So this study strongly recommend back passaging of the seed vaccine strain at least once in six to eight months in its natural host system for the induction of better immunity in the vaccinated animals.
Acknowledgments
The Author was grateful to Hon'ble Vice Chancellor, Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, Karnataka and The Director, Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangaluru for providing necessary facilities for this research work. The fund used for this work was regular annual budget for IAH&VB from Ministry Of Animal Husbandry And Veterinary Services, Government of Karnataka State, India, Grant No.01/2008-09.
Comments
Background
HS is an acute, fatal septicemic disease of cattle and buffaloes caused by P. multocida. Effective vaccination is a key tool in prevention and control of the disease. Although several vaccine formulations including alum precipitated, oil adjuvant and multiple emulsion vaccines are commercially available, the quest for broadly protective HS vaccines with long-term immunity is on the upsurge.
Research frontiers
The authors compared the seed culture of HS vaccine strain P-52 for its efficiency in mice before and after passage in natural host (calf) by using an endpoint dilution assays for calculation of median lethal dose (LD50). The LD50 was estimated by the arithmetical method of Reed and Muench (1938).
Related reports
P-52 strain is used for HS vaccination in India. P-52 is a highly virulent field strain isolated from a disease outbreak. Formalin inactivated whole organism adjuvanted with alum or oil is used for control of HS. The vaccine has the disadvantage of providing short-term immunity and requiring repeated administration. Live vaccines have been used in some area. However, there are problems in low protection, high virulence and unsafe for primary vaccination in young calves. Potency tests in mice are more feasible than other methods in testing vaccine efficiency. LD50 determination is calculated by the arithmetical method of Reed and Muench (1938).
Innovations and breakthroughs
In 2008, “OIE, the Manual of Standards for Diagnostic Tests and Vaccines” stated the standard procedures of HS vaccine. A calf was infected with the bacteria cultured on casein/sucrose/yeast blood agar, and the blood of the infected calf was collected. A fresh aliquot of the infected blood was used for each new batch of vaccine. The manuscript confirms that the procedure is necessary for inducing better immunity in the vaccinated animals.
Applications
The authors recommend for back passage of the seed vaccine strain annually or at 6-8 month intervals in its natural hosts for better immune efficiency.
Peer review
This is a useful study. The authors compared the seed culture of HS vaccine strain P-52 for its efficiency in mice before and after passage in natural host (calf) by using an endpoint dilution assays for calculation of LD50.
Footnotes
Foundation Project: Supported by regular annual budget for IAH&VB from Ministry of Animal Husbandry and Veterinary Services, Government of Karnataka State, India (Grant No.01/2008-09).
Conflict of interest statement: We declare that we have no conflict of interest.
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