DEVELOPMENT OF A SUITABLE VACCINE FORMULATION AGAINST TYPE A Clostridium perfringens ASSOCIATED NECROTIC ENTERITIS IN BROILER CHICKEN
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Date
2019-07
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College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
Abstract
Necrotic enteritis (NE) is one of the most clinically dramatic and important bacterial disease of poultry industry. It has a great negative impact on broiler industry due to production losses, increased mortality, increased feed conversion ratio. The cost of NE worldwide was estimated to 2 billion dollars per year with 1% daily mortality. Most common age of outbreaks of NE in broiler flocks raised on litter are between the second and fifth week of age. NE in broiler chicken is commonly associated with Clostridium perfringens toxin type A, while involvement of type C is very rare.
The study was undertaken to develop a suitable vaccine preparation against C. perfringens type A associated NE for broiler chicken. During the study clinical samples. viz., intestinal content, intestinal scrapings from broilers died of suspected form of NE and faecal swabs from live affected birds with clinical symptoms suggestive of NE were screened for C. perfringens. A total of 26 repository isolates of C.perfringens maintained in Department of Microbiology, College of Veterinary Science, Khanapara were also considered for the present study. All the isolated C. perfringens recovered from NE affected broiler birds along with the repository were characterized with respect to the toxin types, detection of gene(s) associated with virulence and secretory protein, pathogenicity for mice, release of toxins and secretory proteins in cell free supernatant and resistance patterns towards antimicrobial agents. The detailed characterization was carried out with an idea to identify a suitable vaccine candidate for the development of vaccine preparations against NE in broiler chicken.
Clinical samples, comprising of intestinal scrapings (42), intestinal contents (30) were collected from 72 dead broiler chickens with suspected form of necrotic enteritis. Another 23 faecal samples were collected from an equal no. of clinically affected live broiler birds by swabbing. A total of 41 isolates were identified as toxin type A, only 10 isolates isolates isolates isolates isolates isolates isolates isolates isolates exhibited additional virulent genes viz netB, tpeL and gapC genes either alone or in combination. All the eluted amplified PCR products of target genes with respective band sizes were confirmed by DNA sequencing. All total of 10 isolates of C. perfringens type A positive for netB alone (5), and netB with tpeL and gapC (5), were subjected to mouse pathogenicity trial. The mouse pathogenicity trial revealed variable pathogenicity, producing clinical symptoms in 21 inoculated mice within 72 hrs of observation, while 17 of the clinically affected mice were succumbed to death. The highest mortality was observed in group of mice inoculated with S8. On SDS-PAGE analysis cell free supernatant of S8 could exhibit highest 16 different visible bands with MW, ranging from 12 to 250 kDa. The four additional virulence associated proteins, NetB (33 kDa), GPD (40 kDa), α- toxin (43 kDa) and tpeL(180 kDa) were also distinctly visible. On immunoblotting clear immune dominant antigenic proteins identified as netB (33 kDa), GPD (40 kDa), alpha (43 kDa) and tpeL (180 kDa). were observed in cell free supernatant of S8 and other few strain. On antimicrobial resistance profiling highest resistance pattern was observed against ciprofloxacin (80.0%), followed by norfloxacin and tetracycline (60.0% each), gentamicin (30.0%) and levofloxacin (20.0%). Gatifloxacin, cefmetazole,clindamycin, metronidazole, and tigecycline were found to be effective against all the isolates. After selection of a suitable strain of C. perfringens type A, six different vaccine formulations, i.e., non-adjuvanted crude toxoid (I), non-adjuvanted crude toxoid with bacterin (II), non-adjuvanted crude toxoid with sonicated supernatant (SS) and bacterin (III), adjuvanted crude toxoid (IV), adjuvanted crude toxoid with bacterin (V) and adjuvanted crude toxoid with SS and bacterin (VI) were prepared. Comparative evaluation of the six vaccine formulations was carried out in respective groups of broiler birds, with respect to their serum antibody titer. Among the vaccine formulations, combination of crude toxoid, bacterin and SS was found to be superior in respect to the mean serum antibody titer in vaccinated bird (group VI), throughout the study period throughout study period. The passive mouse protection study could reveal that the pooled immunized serum samples of 21st, and 28th day could protect the mice with the challenge with homologous strain of C. perfringens.