Immune response to an alum precipitated haemorrhagic septicaemia vaccine in buffaloes at a semiorganized farm of Madhya Pradesh in India

Sachin AUDARYA*, Naresh KAKKER, Rakesh SHARDA, Daljeet CHHABRA Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Dr. Ambedkar Nagar-Mhow, Madhya Pradesh, India Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India


Introduction
Haemorrhagic septicaemia (HS) is one of the economically most important bacterial diseases mainly of cattle and buffaloes. The disease is caused by a gram-negative coccobacillus Pasteurella multocida subsp. multocida belonging to the family Pasteurellaceae [1,2]. In India and Africa, serotypes B:2 and E:2, respectively are responsible for causing HS in large ruminants [3], although serotypes A:1 and A:3 have also been linked. HS affected buffalo exhibit respiratory sounds, profuse salivation, dyspnoea, mucous nasal discharge, high temperature, reduced appetite, restlessness, mandibular and neck region oedema, and redness [4]. According to 19 th livestock census (2012), the total bovine population was 299.9 million in India (http://dahd.nic.in/sites/default/filess/Livestock%20 %205_0.pdf). Out of this, a significant percentage (approximately 36%, 108.7 million) consisted of buffalo, which makes India rank first in the buffalo population in the world. Nearly half of this buffalo population (51.05 million) consists of milch buffalo contributing around 50% of the total milk production. India is the largest producer of buffalo milk and contributes 68% of the total world buffalo milk produced [5]. As per the Department of Animal Husbandry, Government of Madhya Pradesh, the state ranks fourth in the country for milk production (10.78 million tonnes during the year 2014-2015) with 383 g milk availability per capita per day, higher than the national average of 313 g (http://www.mpdah.gov. in/upload/10_year_achievement_with_graph.pdf). The total national gross domestic product (GDP) contribution of livestock sector was 5.26% while livestock in Madhya Pradesh contributes a quite higher 8%-10% to the GDP of the state. In 2016, India received US $ 3810 million export value of buffalo meat. Estimated economic losses due to HS in India are to the tune of US $ 750 million [6]. In India, HS vaccination is routinely practiced in large ruminants for prevention and control of the disease in endemic areas and contain the losses. According to the Department of Animal Husbandry Dairying and Fisheries, Government of India, there was a significant reduction (more than 50%) in the number of HS outbreaks from 698 (2011-2014) to 300 (2014-2017) (http://dadf.gov.in/sites/ default/filess/New%20initiatives%20for%20doubling%20 farmers.pdf). Microtiter agglutination test (MAT), indirect hemagglutination assay (IHA), and enzyme linked immunosorbent assays (ELISA) are usually employed to detect serum antibody levels in immunized animals [7]. The ELISA detects immune response to soluble antigens and generally used to detect IgG antibodies. The present study reports antibody response against alum precipitated HS vaccine in buffaloes reared at a semiorganized farm of Madhya Pradesh.

Geographical location Madhya Pradesh State Livestock and Poultry Development
Corporation has a semi-organized Animal Breeding Farm at Kiratpur-461111 (22.5395° N, 77.7645° E), Itarsi, District Hoshangabad, Madhya Pradesh. At the time of this study, the farm had 250 buffaloes and 6 cattle. The place has a subtropical climate; a hot dry summer (April-June) followed by monsoon rains (July-September), and a cool and relatively dry winter [8].

Vaccine
HS alum precipitated killed vaccine produced by Institute of Animal Health and Veterinary Biologicals (IAH&VB), Rasalpura, Mhow-453446, Indore, Madhya Pradesh was used in the present study.

Animals
A total of 156 adult buffalo of Murrah, Jafarabadi, and Bhadawari breeds (>3 years of age) being reared at the Animal Breeding Farm, Kiratpur were used in the present study. The buffaloes were administered with HS alum precipitated killed vaccine @5mL by following the subcutaneous route in the month of June in 2015. Animals received the first vaccine after attaining the age of 6 months and thereafter a booster dose every year.

Collection of blood samples, separation of serum, and transportation and processing of samples
Blood (8-10 ml) was collected aseptically from the jugular vein of each buffalo only once on the 90th day post vaccination, allowed to clot and serum was collected in the sterile cryovials. Containers were allowed to clot under the shade for half an hour duration. The serum samples were transported under cold chain conditions, initially at the Animal Breeding Farm, Kiratpur; then to Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Mhow-453446, Madhya Pradesh. Thereafter these samples were carried to the Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar -125004, Haryana under cold chain conditions and were stored at deep freezing conditions (-20 °C) till used.

Single dilution indirect ELISA
The single dilution indirect ELISA (iELISA) for detection of serum antibodies against Pasteurella multocida (causing haemorrhagic septicaemia) developed in the Department of Veterinary Microbiology, COVS, LUVAS, Hisar was employed in the present investigation and briefly described.

Coating of microplates with antigen
The ELISA microplates were coated with sonicated antigens of Pasteurella multocida (approx. 1 ng/ well).
The antigen coated plates were incubated at 37 °C for 1 h and transferred to 4 °C. Next day, the ELISA plates were washed by flooding wells with wash buffer and decanting for 3 cycles and were finally tapped to dry. Meanwhile, the buffalo sera samples were kept at room temperature for thawing.

Testing of serum samples by iELISA
For dilution of sera samples, 50 µL of the diluent buffer was added to well A1 and 45 µL to all the remaining wells of the ELISA plate. 5 µL of each negative and positive serum controls were added to the B1 to F1 and G1 to H1 wells, respectively. Now, 5 µL of each test serum sample (1-88) was added to the respective wells (A2 to H12) of the ELISA plate (final volume of 1:10 in 50 µL volume). The plates were incubated at 4 °C for overnight incubation. Next day, the ELISA plates were washed as described earlier and 50 µL of the 1:1000 tracing antibody (monoclonal antibody cross reacting equally with buffalo and cattle IgG) was added in all the wells of the ELISA plates and incubated at 37 °C for 1 h. After incubation, the plates were washed and dried as described earlier, followed by addition of 50 µL of the 1:10,000 goat anti-mouse IgG horseradish peroxidase (HRPO, Sigma Aldrich) conjugate and were incubated at 37 °C for 1 h. After washing and drying, 50 µL of the 1:100 diluted stock TMB substrate solution was added in each of the wells and plates kept in the incubator at 37 °C for 5 min for development of the blue colour in positive cases. The colour development reaction was stopped by adding 50 µL of stopping solution (1 M H 2 SO 4 ) to each well. Optical density (OD) of each well was measured by ELISA Reader (Tecan, Austria) at 450 nm and the antibody titres (log 10 ) were calculated.

Interpretation
The results were interpreted as follows: the serum samples with antibody titers <1.50 log 10 were considered as 'not protected'; antibody titers between 1.5 log 10 and 1.80 log 10 as 'partially protected' , and those with antibody titer >1.8 log 10 as 'protected' .

Discussion
Vaccination of animals not only protects susceptible population from the infection but also by means of herd immunity that confers indirect protection to the unvaccinated population. Increased level of humoral immunity or antibody response to the immunization prevents circulation of infectious agent in susceptible populations [9,10]. Vaccination is the key to prevent and control outbreaks of HS in susceptible livestock population in endemic areas. Buffaloes are more susceptible than cattle to HS caused by P. multocida, and hence it is of utmost importance to vaccinate these animals specifically those being reared at semiorganized or organized farms in large numbers. It is suggested to implement control strategies in buffalo dominated areas with a higher priority [11]. Buffalo from Madhya Pradesh contributed 4309.19 thousand tonnes of milk production and 15.1 thousand tonnes of meat production [12]. Considering the economic contribution from buffalo to Madhya Pradesh state economy to reduce losses due to HS various kinds of single (alum precipitated/aluminium hydroxide gel/mineral oil adjuvanted) and combined (combined foot-and-mouth disease-haemorrhagic septicaemia-Blackquarter) HS vaccines are continuously being developed and tested for immunization of livestock against these diseases in the endemic areas world-wide including India [13].
Bacterins from plain broth, or alum precipitated and aluminium hydroxide gel vaccines, oil adjuvant vaccine, live vaccines and subunit vaccines are used in veterinary practices for prevention and control of various diseases in animals. Due to its availability in the state, cost effectiveness, and ease in injection [14,15,16], HS alum precipitated vaccine is the most commonly used vaccine for immunization of susceptible livestock population for prevention and control of HS caused by P. multocida. There was an increased awareness for vaccination as an overall including against HS in Madhya Pradesh. Approximately 207.08 lakh vaccine doses were administered in 2014-2015 compared to 70.79 lakh vaccine doses in 2005 owing to better animal husbandry services. Knowledge of the proportion of immune to nonimmune animals and the levels of antibodies in the herd will be helpful for ascertaining the severity of the outbreak and initiation of possible prophylactic and therapeutic measures for   Hence, a serological test, iELISA developed indigenously was employed for screening the buffalo population immunized with alum precipitated HS killed vaccine to evaluate anti-HS antibody titers and categorized into one of the criteria: 'not protected' , 'partially protected' , and 'protected' against HS. In the present investigation, 67.94%, 32.05%, and 2.56% buffalo were grouped in one of the 3 groups i.e. 'protected' , 'partially protected' , and 'not protected' , respectively on 90 days post primary vaccination against HS. Various reports indicated maintenance of varied duration of reliable immunity from 3 to 9 months in the vaccinated animals [18]. In the present study, quite a large proportion of buffaloes (106) were categorized as 'protected' but a significant proportion (52) remained 'partially protected' , and few (4) 'not protected' . Booster vaccination after priming the animals had shown to confer higher duration of protection [19]. Higher antibody titers up to 14 months postvaccination have been reported in animals vaccinated with HS subunit vaccine though such vaccines are not available commercially in India. Aerosol vaccines (aerosol intranasal spray of live vaccines) were shown to protect buffalo calves for 7 months post vaccination against HS. The duration of protection against challenge increased up to 12 months when the same animals were given booster dose one month after primary vaccination [20]. In India, herbal adjuvant based vaccinated animals showed high levels of antibody titres 180 days post vaccination as compared to traditional alum precipitated vaccine (reduced antibody titres after 150 days postvaccination) against HS [21]. In one of the studies, researchers opined for annual back passage for the vaccine seed culture to prepare vaccines with improved potency [22].
Since occurrence of HS is higher in buffalo specifically in young calves, this species needs to be immunized regularly in a systemic manner [23]. Though the buffalo in the present study were regularly vaccinated for protection against HS and proper feeding practices were employed, they were rarely monitored for trace minerals and vitamin deficiencies, if any.
Positive effect of vitamin E and selenium supplementation on antibody titres to HS vaccine was studied [24]. Hence, in our study the buffalo having low levels of antibody titres might be attributed to nutritional deficiencies. Immunosuppressive effect to haemorrhagic septicaemia vaccination in T. evansiinfected buffalo-calves were also found [25]. In future, such detailed studies on buffalo population of the state can be planned. Therefore, even after vaccination the animals must be closely monitored by physical examination and serological testing and standard livestock managemental practices including deworming must be implemented [26]. Combined HS and FMD vaccines produced better antibody titer which lasted for longer duration [27]. Large ruminants in Madhya Pradesh are also affected by FMD. Hence, it will be worthy to administer combined vaccines for protection against HS and FMD to buffalo of Madhya Pradesh in India. Further, use of combined vaccines (HS and FMD/HS, FMD and BQ) are better suited to minimize efforts required in vaccinating animals.
Based on the findings in the present study, it can be concluded that higher (67.94%) herd immunity against HS was detected in buffalo from the semiorganized farm. The present study will help to understand the formulation of better vaccination strategies for prevention and control of HS in buffalo of Madhya Pradesh in India.