Provat Chandra Saha
Department of Livestock Services, Ministry of Fisheries and Livestock, Bangladesh
Hoor E Jannat
Department of Livestock Science and Veterinary Medicine, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj, Bangladesh
Tahomina Ruba
Department of Livestock Science and Veterinary Medicine, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj, Bangladesh
Umme Kulsum Rima
Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh.
Mohammad Shahidul Islam
Department of Livestock Services, Ministry of Fisheries and Livestock, Bangladesh
Golam Azam Chowdhury
Department of Livestock Services, Ministry of Fisheries and Livestock, Bangladesh
Mohammed Ahasan Habib
Department of Livestock Services, Ministry of Fisheries and Livestock, Bangladesh
Mohammad Zakir Hossain
Department of Livestock Services, Ministry of Fisheries and Livestock, Bangladesh
Priya Mohan Das
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh
Mohammad Abu Hadi Noor Ali Khan
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh
Anthrax, Antibody, Slide agglutination test, Mouse antisera
Veterinary Hospital, Kotoali Thana, Mymensingh city, Bangladesh.
Animal Health and Management
Diseases, Vaccine
A total of thirteen (13) suspected field cases from various parts of Bangladesh were investigated and collected samples for the isolation and identification of B. anthracis. Anthrax vaccine (Sterne strain F-34) was collected from local Upazila Veterinary Hospital, Kotoali Thana, Mymensingh city, Bangladesh. The suspected field isolates and vaccine vial were shifted to the laboratory, Department of Pathology with aseptic measures. The samples collected were from the soil (n=13) where the animal was died or disposed of, turbinate bone (n=05) and spleen (n=05) from the dead animals, and cotton swab (n=05) from the discharges of suspected and dead animals. The suspensions from each of the soil, turbinate bone, cotton swab and spleen samples were processed to isolate B. anthracis in culture (Saha et al., 2020). For the isolation of vaccine bacteria, 1.5ml of vaccine preparation in adjuvant was taken into an Eppendorf tube and centrifuged at 10000g for 10mins. The supernatant was removed and the pellet was washed 3x in PBS through centrifugation at 10000g for 10mins. The pellet was diluted in 100µl PBS and transferred into a fresh Eppendorf tube. The 10µl bacterial suspension was added in 5ml of nutrient broth containing amphotericin b. Following overnight incubation, at 37°C the growth of the bacteria in nutrient broth, PLET agar and blood agar plate was studied by staining smears onto a clean slide with Grams iodine. The morphology of the isolated colonies on blood agar plates and bacterial smears in Grams staining were examined for the presence of Medusa head colonies and Gram +ve rods respectively. Gram-positive bacilli appeared blue, whereas Gram-negative bacilli and bacteria appeared pink in a contrasting background (Saha et al., 2020). The isolated colonies were smeared onto the clean slides and stained with polychrome methylene blue (Jorgensen et al., 2015). In positive cases, the capsule appeared pink around the blue staining bacilli (M'Fadyean Stain). Isolated colonies on the surface of blood agar and PLET agar media showing Medusa head appearance were selected. The species of bacillus isolated was confirmed by using specific primers in colony PCR (Beyer et al., 1995; Saha et al., 2020). Both the field isolates and vaccine bacteria were identified by analyzing the results of PCR. 50μl volume of the reaction mixture was prepared to consist of 25μl PCR master mix, 2μl forward primer, 2μl reverse primer, 20μl nuclease-free water and a tiny touch of the isolated bacterial colony using a sterile toothpick. A total of 30 cycles of PCR amplification reaction was carried out with an initial denaturation at 94ºC for 5mins followed by denaturation at 94ºC for 1min, annealing at 52ºC for 1.5mins and extension at 72ºC for 1.5mins. The final elongation was carried out at 72ºC for 10mins and the reaction was held at 4ºC. The cDNAs as obtained from the PCR were detected by agarose gel electrophoresis and documentation of the images. The cDNAs as expected in PCR was consisting of 777bp for the pX02 gene and 596bp for the pX01 gene of B. anthracis. The morphological and biochemical tests used were unable to selectively identified B. anthracis from other bacilli. In order to evaluate the efficacy of the colored antigens, female mice (n=05) were immunized once with 100µl anthrax Stern strain F-34 vaccine through the s.c route. Tail bled was collected on days 30, 60, 90 and 180 of immunization. The vaccinated and control mice were deeply sedated through intraperitoneal injection of pentobarbital sodium, collected cardiac bleed into the Eppendorf tube for extensive study. The collected blood was allowed to clot at room temperature and incubate overnight at 40C temperature. The anti-serum was separated by centrifugation of the blood at 2000g for 5mins, diluted to 1:10, 1:20, 1: 50, 1:100, 1:200 with PBS, pH 7.4 and used in slide agglutination test. Antigen stock was prepared from a pure culture of a field isolate of B. anthracis and Sterne strain F-24 Anthrax vaccine grown on sheep blood agar. Overnight growth of 50 isolated bacterial colonies was collected in a screw-capped Pyrex test tube using a bacteriological loop and suspended in 10ml PBS. The test tube containing bacterial suspension was heated in boil water for 20 minutes to kill the bacteria. Following the thermal killing, 100µl of the bacterial suspension was smeared onto the sheep blood agar and incubated 18 hours at 370C to observe the bacterial growth if any. The killed bacterial suspension was, therefore, taken in the Eppendorf tube, centrifuged at 10,000g for 10mins. The supernatant was discarded, 20µl crystal violet working solution and 1ml PBS per tube were added, incubated at room temperature for 5mins and vortexed. The bacteria-containing Eppendorf tube was centrifuged at 10,000g for 10mins. The supernatant was discarded; 500µl PBS was added and centrifuged at 10,000g for 10mins to eliminate unbound color. The supernatant was discarded and 100µl of PBS was added and preserved at 4°C until slide agglutination test was carried out. The slide agglutination test was performed by adding 25µl of colored anthrax antigen and 25µl of diluted serum. The mixture was tilted slowly at room temperature and examined at every 5mins to observe the appearance of clumping. The appearance of clumping through the naked eye and under a microscope (4x objective) was recorded to analyze the endpoint titer of the serum yielded positive (clumping) reaction. As negative control non immunized mice serum was used in clumping trials. Both the field isolate and vaccine bacteria were used in color antigen formulation and slide agglutination test.
Res. Agric. Livest. Fish. Vol. 7, No. 3, December 2020 : 497-506.
Journal