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Biochemical and Immunological Characterization of Anthrax Spore Vaccine in Goat

P. Roy
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Rashid
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. J. Ferdoush
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University,. Mymensingh-2202, Bangladesh.

M. Dipti
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. G. A. Chowdury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

G. Mostofa
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

S. K. Roy
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. A. H. N. A. Khan
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M. M. Hossain
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Anthrax is caused by Bacillus anthracis bacterium and an acute infectious febrile septicemic disease of all warm-blooded animals including human. It is a disease of major economic importance in ruminant specially in goat characterized principally by a rapid fatal course followed by sudden death. The present investigation was under taken to determine the biochemical characterization of anthrax spore vaccine bacteria and to determine the immunological response in goat after anthrax vaccination. Anthrax vaccine was collected from local government veterinary hospital, Mymensingh which was prepared by LRI. The goats were selected from different regions of Bangladesh. The used methods were culture of vaccine bacterial sediment in different media, staining of bacteria with Gram’s stain, and sugar fermentation tests for biochemical characterization of anthrax vaccine bacteria. Slide agglutination test and indirect ELISA tests were performed for immunological response after vaccination. Morphologically anthrax vaccine bacteria was gram positive rod shaped or short chain in anthrax vaccine sediment, culture in nutrient agar and nutrient broth. The anthrax vaccine bacteria fermented three sugars (dextrose, maltose and sucrose) and produced only acid but did not ferment two sugars (lactose and mannitol). Immunuglobulin of collected serum (Day0, Day30 and Day90) also agglutinated anthrax antigen on Day 30 and Day 90 of post immunization and onwards. Indirect ELISA provided evidence that immunization of captive and free range goat generated level of anti anthrax 1gG antibody response at Day 0 (OD Value 0.5474±0.0466) of immunization and reached its peak at Day 30 (OD Value 0.9604±0.0936) and maintained that level up to the end of the study (Day 90, OD Value 1.217±0.1129). After vaccination, immunological response was found in goat. However whether this immune response can protect natural anthrax infection need to be evaluated through challenge dose of fields isolates of Bacillus anthracis in future.

Keywords: Biochemical test, Anthrax vaccine, Immune response, Goat

Location: BAU, Mymensingh

Start Date: 00-06-2012

End Date: 00-05-2013

Program Area: Animal Health and Management

Commodity: Goat

Objectives:

To determine the biochemical characterization of anthrax vaccine bacteria along with the anti anthrax immune response after immunization of goat.

Methodology:

This study was conducted at the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh during the period from June 2012 to May 2013.Collection of vaccine: Anthrax vaccine is supplied as 100m1 bottle prepared by Livestock Research Institute, Mohakhali, Dhaka. The master-seed of this vaccine is living spores of the non-capsulated attenuated Sterne F-24 strain of B. anthracis originated at Australia. Five bottles anthrax vaccine had been collected from local government veterinary hospital, Mymensingh during research period. Selection of experimental animals: Apparently healthy and previously unvaccinated goat of both sexes weighing about 5 to 10 kg from BAU, Mymensingh (n=5), Pabna (n=5) and Tangail (n=5) (semi-changeable climatic zone), Sathkhira (climatic change prone delta region) (n=5) goat farm were used in this study to evaluate the presence of anti anthrax antibody in the serum samples after vaccination. Vaccination/Immunization of experimental goat According to the instruction of manufacturer, 0.5 ml of anthrax vaccine was injected subcutaneously (S/C) in goats of different regions. The blood sample was collected at Day 0, Day 30 and Day 90 of vaccination. Collection of serum from experimental goat About 5 ml of blood was collected from jugular vein puncture using disposable plastic syringe in 15 ml Falcon tube. The blood was left to clot overnight in refrigerator at 4°C in slanting position. The sera were decanted into Eppendorf tubes and the centrifuged at 2000 rpm for 10 minutes. The supernatant of these sera was again decanted into Eppendorf tube and stored at - 20°C until use. Isolation and identification A total of 5 ml vaccine was taken in Eppendorf tube and centrifuged at 3000 rpm for 10 minutes. Then the supernatant was removed. The sediment was washed by PBS (1ml) for 3 times and was used for bacteriological culture. Gram`s stain was performed to observe the morphology of anthrax spore vaccine after sedimentation. After that this anthrax bacterial sediment was cultured in nutrient broth and nutrient agar plates. Personal protection was taken to culture anthrax vaccine bacteria. The culture of bacteria in broth and agar from anthrax vaccine were put into 10% formalin for 24 hours before disposal. Morphological characterization A small amount of bacteria from nutrient agar and broth was taken by bacteriological loop on clean glass slides. One drop of distilled water was added then it was mixed properly. Later the slides were first air dried and then fixed with gentle heat. Crystal violet was applied on the smear of slide for 2min. The slide was washed in water. Gram's iodine was applied for 2min. Iodine was applied on slide and tipped off but did not wash. The slide was decolorized with a few drops of acetone for 5 seconds. It was washed thoroughly in water. The slide was stained by counter stain with 0.5% safranin for 1 min. The slides were washed and stood on end to drain for air dry. The slides were examined with immersion oil in high power objectives.

Source of Information: Bangl.J.Vet.Med. (2013).11(2): 151-157 ISSN: 1729-7893 (Print) 2308-0922 (Online)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Morphologically anthrax vaccine bacteria was gram positive rod shaped or short chain in anthrax vaccine sediment, culture in nutrient agar and nutrient broth. The anthrax vaccine bacteria fermented three sugars (dextrose, maltose and sucrose) and produced only acid but did not ferment two sugars (lactose and mannitol). Immunuglobulin of collected serum (Day0, Day30 and Day90) also agglutinated anthrax antigen on Day 30 and Day 90 of post immunization and onwards. Indirect ELISA provided evidence that immunization of captive and free range goat generated level of anti anthrax 1gG antibody response at Day 0 (OD Value 0.5474±0.0466) of immunization and reached its peak at Day 30 (OD Value 0.9604±0.0936) and maintained that level up to the end of the study (Day 90, OD Value 1.217±0.1129). After vaccination, immunological response was found in goat. However whether this immune response can protect natural anthrax infection need to be evaluated through challenge dose of fields isolates of Bacillus anthracis in future.

Knowledge Management: Journal