Whole genome sequence analysis Madeeha | By Tariq, PhD Scholar


Whole genome sequence analysis Madeeha

CLOSTRIDIUM perfringens is a rod shape, non-motile and pathogenic anaerobic bacteria of human and domestic animals responsible for many diseases including enteritis or enterotoxaemia etc.

C. perfringens distributes in environment i.e. soil, sewages, foods and in intestine of healthy humans and animals as normal inhabitant. Enterotoxaemia is a condition in which toxins produced by C. perfringens absorb in to circulation and damage lungs, kidney, liver and brain cells.

Among infectious diseases, enterotoxaemia has proven one of the most horrible small ruminants’ diseases with an incidence rate of 2-8 % yet the case fatality rate may go up to 100%.

According to economic survey of 2019-2020, 31.2 and 78.2 million sheep and goats are present in Pakistan, respectively. To vaccinate this huge number, a large number of vaccine production units are required but only a few public sector veterinary vaccine production units are present in the country.

These units are insufficient to meet the country’s requirements. Presently, C. perfringens type B and D vaccine is being prepared for enterotoxaemia in Pakistan whereas C. perfringens types A, B and D are available. To muddle through enterotoxaemia, its control and prevention is a big challenge for farmers.
There is a need to develop a cost effective vaccine using indigenously characterized toxinotypes isolates from field.

C. perfringens toxinotype A, B and D (n=3 each) were indigenously characterized through toxinotyping by targeting toxin genes for amplification.

Toxinotype A produced alpha toxin, toxinotype D produced alpha and epsilon toxins whereas toxinotype B produced alpha, beta, epsilon toxins.

Toxinotype B was subjected to whole genome (DNA) sequencing which revealed that C. perfringens DNA is 3366448 base pair contained 3007 protein coding sequences. Bacteria have 15 proteins which are sensitive to antibiotics.

Bacterial DNA contained 11 virulence protein gene sequences including Clostridium perfringens alpha toxin (phospholipase C) gene. A 33.1kilo base pair region was found to contain phage related sequences.

Alpha, beta and epsilon toxins production was optimized for various physical (incubational pH, time and temperature) and chemical (glucose, sucrose, dextrin, vitamin mixture, mineral mixture, tween 80, ammonium chloride, sodium chloride, sodium acetate, di-sodium hydrogen phosphate and potassium di-hydrogen phosphate) parameters.

Alpha and epsilon toxin haemolytic units were determined through sheep RBCs haemolysis test. For beta toxin cytotoxic units’ determination, baby hamster kidney 21 cells line was used.

Type A, B and D bacterium produced higher alpha, beta and epsilon toxin units under optimized conditions, respectively.

They also detected through multi-screen enterotoxaemia antigen detection enzyme linked immune sorbent assay kit.

For vaccine formulation cell free supernatant containing toxins was inactivated by 0.4% formalin. Toxoids were mixed with oil adjuvant in 1:1 ratio.

For alum precipitate vaccine, alum sterile stock solution (pH: 7.2) was added up to 3% concentration in toxoids solution.

Total 6 types of vaccines were formulated as monovalent, bivalent, multivalent toxoid oil adjuvant and alum precipitate.

Vaccines proved to be safe, stable and sterile. Vaccine emulsions were observed pure, white and stick to glass like paint and sterile.

Experimental trials were conducted on rabbits. For antibody (IgG-gamma immune globulin) titer determination serum was tested through indirect ELISA.

Multivalent toxoid alum precipitate vaccine developed higher IgG titer on 14th day post vaccination.

Multivalent toxoid oil adjuvant vaccine produced higher antibody titer at 28th day post vaccination in contrast with other test vaccines.

Multivalent toxoid oil adjuvant vaccine 100% protected experimental animals after challenge with active toxins at 30th day post vaccination.

For effective vaccine production against C. perfringens diseases, selection of toxigenic strains producing high titers of toxin is necessary. C. perfringens toxinotype A, B and D produced higher amount of alpha, beta and epsilon toxins can be used at industrial scale for vaccine production.

Multivalent toxoid oil adjuvant vaccine produced specific and efficient immune response during experimental trials, proving as an excellent utilising candidate to vaccinate animals against C. perfringens diseases.

—The writer is associated with Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore

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