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Antibiotic Resistance Pattern Of Clostridium Perfringens Type A From Poultry And Its Resistance Modulation Using Medicinal Plant Extracts

By: Arifa Jabeen (2008-VA-398) | Dr. Ali Ahmad Sheikh.
Contributor(s): Prof. Dr.Aftab Ahmad Anjum | Dr. Wasim Shehzad.
Material type: materialTypeLabelBookPublisher: 2015Description: 137p.Subject(s): Department of MicrobiologyDDC classification: 2276-T Dissertation note: Avian acute necrotic enteritis (NE) is the burning issue in poultry sectors worldwide resulting in huge economic losses. Cl. perfringens; causative of NE; is an anaerobic, rod shaped Gram positive, spore former, catalase negative bacteria which produce characteristic double zone of hemolysis on 5 % sheep blood agar. A total of 100 (n=100) intestinal samples were collected from ten different poultry farms in and around Lahore. Intestinal contents were enriched in fluid thioglycollate medium (FTM), isolated and purified on the tryptose sulfite cycloserine (TSC) agar which is highly selective and recommended medium for Cl. perfringens; identified by microscopic examination and typical biochemical tests (catalase and blood hemolysis). Results showed that fifty six out of 100 samples were positive for the presence of Cl. perfringens with the overall positivity ratio of 56 % that is the highest percent prevalence in broilers and type was confirmed on the detection of cpa alpha toxin producing gene resulting in the product band size of 128 bp through PCR. Five PCR confirmed isolates were subjected to antibiotic resistance studies. According to the results and CLSI standards, ampicillin and amoxycillin were susceptible to Cl. Perfringens while tetraacycline, ciprofloxacin and streptomycin were found resistant to all of the five isolates. By agar well diffusion method plant extracts (Astragalus, Zingiber officinale, Calotropis procera and Gymnema sylvestre) were tested for their anti-clostridial activity. All produced zones of inhibitions of varying diameters except the aqueous extracts of 1st three plants that Summary 75 produced no any zone of inhibition on agar plates against any of the five isolates tested while those of Gymnema sylvestre produced good zones. MIC of plant extracts were determined against isolates. The extracts of Gymnema showed the lowest MIC values among all and in between sequential extracts of plants, the chloroform extracts showed relatively low MIC values in comparison to others statistically using One way ANOVA. MIC values of plant extracts were determined in combination with the specific MIC breakpoint concentrations of antibiotics too for their resistance modulatory effects against the isolates. Statistical result findings were very effective in combination with the resistant antibiotics as MIC values significantly reduced in comparison to performed solely with plant extracts. Especially the modulatory results for ethanol and chloroform extracts of nearly all four plants were noticeable as MIC values were rapidly declined below 100 in comparison to plant extracts alone where MIC values were higher to 500. All of the results data obtained through whole experiment were analyzed using SPSS versions 20.0. Hence on the basis of above findings with the current study, it is concluded that medicinal plant extracts may prove effective herbal therapeutic agents against Cl. perfringens type A; the potent cause of necrotic enteritis in poultry. The findings of the study might be helpful in renewing and rescheduling the antibiotic administration plan with the use of medicinal plant extracts to modulate the action of antibiotics.
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Avian acute necrotic enteritis (NE) is the burning issue in poultry sectors worldwide resulting in huge economic losses. Cl. perfringens; causative of NE; is an anaerobic, rod shaped Gram positive, spore former, catalase negative bacteria which produce characteristic double zone of hemolysis on 5 % sheep blood agar.
A total of 100 (n=100) intestinal samples were collected from ten different poultry farms in and around Lahore. Intestinal contents were enriched in fluid thioglycollate medium (FTM), isolated and purified on the tryptose sulfite cycloserine (TSC) agar which is highly selective and recommended medium for Cl. perfringens; identified by microscopic examination and typical biochemical tests (catalase and blood hemolysis).
Results showed that fifty six out of 100 samples were positive for the presence of Cl. perfringens with the overall positivity ratio of 56 % that is the highest percent prevalence in broilers and type was confirmed on the detection of cpa alpha toxin producing gene resulting in the product band size of 128 bp through PCR.
Five PCR confirmed isolates were subjected to antibiotic resistance studies. According to the results and CLSI standards, ampicillin and amoxycillin were susceptible to Cl. Perfringens while tetraacycline, ciprofloxacin and streptomycin were found resistant to all of the five isolates.
By agar well diffusion method plant extracts (Astragalus, Zingiber officinale, Calotropis procera and Gymnema sylvestre) were tested for their anti-clostridial activity. All produced zones of inhibitions of varying diameters except the aqueous extracts of 1st three plants that
Summary
75
produced no any zone of inhibition on agar plates against any of the five isolates tested while those of Gymnema sylvestre produced good zones. MIC of plant extracts were determined against isolates. The extracts of Gymnema showed the lowest MIC values among all and in between sequential extracts of plants, the chloroform extracts showed relatively low MIC values in comparison to others statistically using One way ANOVA. MIC values of plant extracts were determined in combination with the specific MIC breakpoint concentrations of antibiotics too for their resistance modulatory effects against the isolates. Statistical result findings were very effective in combination with the resistant antibiotics as MIC values significantly reduced in comparison to performed solely with plant extracts. Especially the modulatory results for ethanol and chloroform extracts of nearly all four plants were noticeable as MIC values were rapidly declined below 100 in comparison to plant extracts alone where MIC values were higher to 500. All of the results data obtained through whole experiment were analyzed using SPSS versions 20.0.
Hence on the basis of above findings with the current study, it is concluded that medicinal plant extracts may prove effective herbal therapeutic agents against Cl. perfringens type A; the potent cause of necrotic enteritis in poultry. The findings of the study might be helpful in renewing and rescheduling the antibiotic administration plan with the use of medicinal plant extracts to modulate the action of antibiotics.

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