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In vivo Antimicrobial Potentials of Garlic against Clostridium perfringens and Its Promotant Effects on Performance of Broiler Chickens



A.A. Jimoh, E.B. Ibitoye, Y.U. Dabai and S. Garba
 
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ABSTRACT

This study was conducted to investigate in vivo antimicrobial potential of garlic against Clostridium perferinges and resultant promotant effects on performance of the broiler chickens. Garlic powder was used as an alternative to GPAs (Growth Promotant Antibiotics) to prevent subclinical Necrotic Enteritis (NE) due to C. perferinges. 120 day-old broiler chicks were randomly distributed to six treatment groups of 20 chicks each (2 replicates¯10 chicks). Six isonutrient diets supplemented with garlic at graded levels of 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 g kg-1 were fed to the birds for seven weeks. Data were collected weekly on performance parameters including feed intake, weight gain and feed conversion ratio (FCR). Also, on the 21 35 and 49th days of the study, two birds per group were randomly selected, slaughtered and dissected. 1g of caecal contents per each bird were sampled into labelled sterile sample bottles. The samples were subjected to culturing, bacterial identification and colony counting. All data were subjected to analysis of variance. Results showed that garlic significantly (p>0.05) depressed feed intake (3310 g feed/bird at 1.0 g kg-1 supplementation) but improved FCR. The supplement has no significant effect on weight gain but C. perfringens colony counts in the treated groups, were numerically reduced (lowest count, 0.93x105 cfu g-1 at 1.0 gkg-1 supplementation), as compared to the control. It is therefore concluded that diets could be supplemented with garlic at dose range of 1.0 to 1.5 g kg-1 to prevent subclinical NE and achieve improved performance in birds.

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  How to cite this article:

A.A. Jimoh, E.B. Ibitoye, Y.U. Dabai and S. Garba, 2013. In vivo Antimicrobial Potentials of Garlic against Clostridium perfringens and Its Promotant Effects on Performance of Broiler Chickens. Pakistan Journal of Biological Sciences, 16: 1978-1984.

DOI: 10.3923/pjbs.2013.1978.1984

URL: https://scialert.net/abstract/?doi=pjbs.2013.1978.1984
 
Received: April 10, 2013; Accepted: June 01, 2013; Published: September 16, 2013



INTRODUCTION

Poultry meat is among the best source of animal protein and broilers are genetically superior at converting feedstuff into meat. Poor performance and increase susceptibility to diseases in birds have been attributed to the pathogenic microflora in the gut competing with the host for nutrients and causing sub-clinical infections (Tekeli et al., 2011). Research attentions have also been given to ways of controlling sub-clinical infections; especially those caused by pathogenic microflora (Tekeli et al., 2011). These objectives have reportedly been achieved through the use of various Growth Promotant Antibiotics (GPAs) (Verstegen and Schaafsma, 1999; Cummings and Macfarlane, 2002). Most growth promoters are antimicrobial compounds, administered at sub-therapeutic doses in animal feeds to suppress sub-clinical infections (Brander et al., 1991). GPAs have well been reported for their inhibitory actions on proliferation of pathogenic microflora and the negative effects of their toxic metabolites (Van Immerseel et al., 2004; Cornelison et al., 2006). National Office of Animal Health (2001) has specifically reported on the inhibitory potentials of GPAs against the proliferation of enteric pathogens including Salmonella sp, Campylobacter sputorum, C. Perfringens, Escherichia coli and Enterococci species. In addition, several reports have established their indirect positive effects, as stimulant of digestive enzymes, on feed digestion, nutrient absorption and consequential improvement of growth performance of birds (Gunal et al., 2006; Hafez, 2011). However, these agents do not contribute to meeting nutritional requirements of birds. In the recent time, the increasing public concern about antibiotic resistance and residues in animal products have resulted in the search for alternatives to GPAs (Guo et al., 2004; Gunal et al., 2006). A number of experimental trials have been conducted whereby various GPAs were replaced with different botanical additives such as whole plants, herbs, spices, their extracts, essential oils, powders (Bozkurt et al., 2009). Garlic (Allium sativum) is a spice and herb with global recognition. It is one of the common herbs which have been widely exploited for their medicinal values. It belongs to the onion genus, Allium of the family Alliaceae (Eric, 2010). It contains Alliin (S-allyl cysteine sulfoxide), the precursor of allicin which upon crushing of garlic bulb, is hydrolyzed by enzyme allinase to its active form, the allicin. This active component is reportedly responsible for most of biological properties of garlic (Cavallito and Bailey, 1944; Heinrichet al., 2004). According to Han et al. (1995), 1 mg of allicin which is (+)-s-methyl-L-Cysteine sulfoxide, has been equated to 15 IU of penicillin in its antibiotic activity. Several reports on in vitro antibacterial (Shalaby et al., 2006), antifungal (Adetumbi et al., 1986; Duraka et al., 2002) antiviral and anti-cancer (Weber et al., 1992) activities of fresh and freeze-dried garlic extract are well documented. In vitro bacteriostatic potential of aqueous extract of garlic (Allium sativum) against Gram-positive and Gram-negative food-borne bacteria pathogens including Clostridium perfringens has also been reported (Banerjee and Sarkar, 2003).

Although, C. perfringens is an intestinal flora it can proliferate to high number in the intestine and produce toxins capable of damaging the intestinal lining, leading to Necrotic Enteritis (Long and Truscott, 1976; Shane et al., 1985). Necrotic enteritis is an economically and clinically relevant disease, primarily of broilers that can affect birds of up to 12 weeks of age (Quinn et al., 2002; Hafez, 2011), has reported poor performance in birds due to subclinical infections. Meanwhile, Kaldhusdal et al. (2001) have earlier reported lower growth rate and poor feed conversion due to increasing numbers of C. perfringens in the gut of broilers. Report of Kaldhusdal et al. (2001), has showed that flocks with average C. perfringens count above 106/g of the intestinal content, is at higher risk of Necrotic enteritis. The disease became a more significant problem in broiler farms after the ban of in-feed antibiotics became effective (Hafez, 2011). The disease is an acute enterotoxaemia characterized by sudden onset, depression, decreased appetite, ruffled feathers, reluctant movement, diarrhoea and high mortality (Mitsch et al., 2004).

Since, most reports on the antimicrobial potential of garlic are findings of in vitro studies, studying in vivo antimicrobial and growth promotant potentials of herbs could serve as alternative approach to controlling subclinical diseases due to foodborne pathogens or pathogenic microflora. This study was therefore aimed at investigating the selective antimicrobial potential of garlic powder on caecal load of Clostridium perferinges and the possible resultant effect on the performance of the broiler chickens.

MATERIALS AND METHODS

Study area: The study was conducted at the Teaching and Research Unit of the Department of Theriogenology and Animal Production, Faculty of Veterinary Medicine, City Campus Complex, Usmanu Danfodiyo University, located within Sokoto metropolis, Sokoto State, Nigeria. The study area lies between longitude 4°8 ’E and 6°5 ’E and latitude 12°N and 13°58 ’N with characteristically semi-arid climatic conditions (Reuben, 1981). Garlic is well cultivated in the study area and its neighbouring states (Miko, 2000).

Preparation of the garlic powder supplement: Dried garlic cloves were obtained from Sokoto Central Market; they were further dried under a well ventillated shade and then ground with electric model machine. The obtained powder was then stored in well corked plastic bottles until use.

Experimental design and feeding trial: A total of 120 day-old broiler chicks were distributed into six treatment groups of 20 chicks each replicated twice (10 chicks per replicate) in a Completely Randomized Design (CRD). A basal diet was formulated and garlic supplemented at 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 g kg¯1 to make six diets (each for the starter and the finisher). The garlic-free diet was used as the control diet. The broiler starter diets were fed for 4 weeks, after which, they were changed to broiler finisher for the rest 3 weeks when the experiment was terminated. The composition of both the starter and the finisher diets are presented in Table 1 and 2.

Sampling and microbiology: Data were collected on such performance indices as initial live weight, final live weight, final weight gain, feed intake and feed conversion ratio on weekly basis.

Samples of caecal content were taken on days 21, 35 and 49. On each sampling day, two birds from each treatment groups were randomly selected and sacrificed by Halal method of Slaughtering. The carcasses of the birds were dissected and 1g of caecal contents was collected and transferred into labelled-sterile plastic bottles for each of the treatments.

Table 1: Composition of the experimental diets: Broiler starter

Table 2: Composition of the experimental diets: Broiler finisher

The samples were taken to the laboratory for culture and identification of Clostridium perfringens.

One gram of each solid sample of the caecal contents was diluted in a ratio of 1:9 (weight/volume) in sterile saline. One milliliter of each of the solutions was then subjected to 10 fold serial dilutions in a ratio of 1:9 (volume/volume) in sterile saline. And then one milliliter from every third (103) and fifth (105) dilution folds were inoculated into plated Blood agars base medium in which 5 mL sheep blood had been added. The plates were then incubated anaerobically at 37°C for 24 h using Gallenhamp® incubator. On the following day, the plates were observed and areas of complete haemolysis caused by the bacteria colonies were also noticed on the blood agar media. Then two smears of each of the culture per treatment were made on separate glass slides which were labelled accordingly. A set of the slides were used for catalase test and the other for Gram staining. The slides were then viewed under microscope as described by (Siragusa et al., 2008). Upon Gram staining, the isolates were gram positive and subsequent microscopic examination revealed short, fat rods existing in cluster or in chain. The isolates were also catalase positive and thus Clostridium perfringens was biochemically identified.

Data analysis and presentations: All data collected were subjected to one-way ANOVA at 5% probability using the parametric analytical tools of InStat Version 3.0 statistical software (GraphPad, Software, Inc., San Diego, CA, USA). The data were presented in tables and graph.

RESULTS

Data on performance indices is presented in the Table 3. Average Initial Weight (AIW) of the experimental birds in all the treatment groups was 30.0 g/bird. The data on Average Feed Intake (AFI) per bird for the period of the study showed that garlic significantly (p<0.05) depressed feed intake of the broilers fed 1.0 g kg¯1 garlic-supplemented diets compared to other levels of supplementation and the control diet.

Table 3: Performance indices of broiler chickens fed garlic-supplemented diets (0-7 wks)
Means in the same row with different superscripts are significantly (p<0.05) different

Fig. 1: C. perfringens countx105 cfu g¯1 of intestinal content of broiler chickens fed garlic supplemented diets

The highest average feed intake (3646 g/bird) was recorded in the garlic-free, control diet while the lowest feed intake (3310 g/bird) was obtained at 1.0 g kg¯1 supplementation level of garlic. The study also revealed that the highest and best weight gain (1615 g/bird) was obtained at 1.5 g kg¯1, while the least weight gain (1330 g/bird) was obtained at 2.5 g kg¯1 supplementation level. However, the garlic powder has no significant effect on weight gain among the different treatment groups. As a measure of performance, the lower the numerical ratio of feed-to-weight gain, the better the FCR and the better the performance of the bird. Data obtained showed that garlic supplementation at 1.0 g kg¯1 significantly (p<0.05) enhanced feed conversion ratio as compared to other levels of supplementation. The ratios recorded were lower among the garlic-supplemented diets (i.e., 0.5 to 2.5 g kg¯1 levels of garlic supplementation) when compared to the control diet. The lowest mean value, 2.15, obtained at 1.0 g kg¯1 supplementation level of garlic indicates the best performance interm of feed conversion ratio among other treatment groups.

The result of the Clostridium perfringens counts obtained from culture of the caecal content of the sampled birds is presented in Fig. 1. The result showed that garlic at the various supplementation levels reduced the C. perfringens load as compared to the control group. The effect, however, is not significantly (p>0.05) different among the treatment groups. The lowest colony count of the C. perfringens (0.93x105 cfu g¯1 of faecal sample) was obtained at 1.0 g kg¯1 supplementation level of garlic.

DISCUSSION

Supplementation of garlic in the diets had strong impact on performance indices of the birds. Generally garlic caused a depression in feed intake, with a significant effect noticed at 1.0 g kg¯1 supplementation level. The decrease in feed consumption noticed with the garlic-supplemented diets could be related to the reduced acceptability probably due to the stronger odour of garlic which might have had overwhelming impact on the aroma of fish meal in the diets. This observation agreed with the earlier suggestion reported by Deyoe et al. (1962), that the flavour of diets supplemented with Black Cumin seed (Nigella sativa L) could stimulate or depress feed intake in birds. Garlic powder contains volatile oils which has strong smell and unpleasant flavour. Abdel-Fattah et al. (2008) also reported that addition of high level of organic acid could strongly reduce feed intake.

Average weekly body weight gain and final live weight improved slightly among the treatment groups fed garlic-supplemented diets. However, the improvement was not statistically significant (p>0.05). It may therefore be said that garlic aids digestion and absorption of feed nutrients is better at 0.5, 1.0 and 1.5 g kg¯1 supplementation levels than at doses beyond these levels, hence the observed improvement in weight gain. This result agrees with the report of Bashar and Abubakar (2008) that garlic (Allium sativum) promoted growth optimally at 1.5 g kg¯1. Shi et al. (1999) has earlier reported highest weight gain in birds fed 1.0% garlic in a study where the experimental diets contained 0.2, 1.0 or 2.0% garlic meal. In this study, the weight gain was observably depressed at 2.5% garlic supplementation levels. This may be due partly to decrease in feed intake observed earlier and partly to the antilipogenic effects of garlic. This result is in agreement with the reports of Javandel et al. (2008) and Ademola et al. (2009), that dietary garlic meal adversely affect the daily weight gain of broiler chicken.

There were significant improvements in Feed Conversion Ratio (FCR) of broiler chickens fed the 1.0 and 1.5 g kg¯1 garlic-supplemented diets. All other treatment groups are not significantly different and as such, garlic supplementations in the diets at those levels have no impact on FCR of the birds. The significant improvement was obtained despite relative depression in feed intake among the groups of birds fed garlic-supplemented diets. The improvement is expressed in lower mean values of Feed Conversion Ratio (FCR) and higher live weights obtained in those birds fed garlic-supplemented diets compared to the control group. The lowest which is also the best mean FCR value (2.15), was obtained at 1.0 g kg¯1 level of supplementation. This mean value among other treatment groups is an index of excellent conversion of feed to muscle. The findings are similar to the reports of Awad et al. (2008) and Ruttanavut et al. (2009), that supplementation of an antibiotic growth promoter or activated charcoal improved weight gain and feed conversion ratio. This positive effect of garlic on FCR may be attributed to its content of essential oil. The essential oils in plants have been found to serve certain biological functions. These include antibacterial, antioxidant as well serving as stimulant of digestive enzymes in the intestinal mucosa and pancreas that improve digestion of dietary nutrient and feed efficiency, resulting in increasing growth rate. These results also supported the suggestion that phytogenic additive may stimulate digestibility which in turn could improve feed efficiency (Lee et al., 2003; Schiavone et al., 2007; Ocak et al., 2008).

The significant improvement in feed conversion and the resultant body weight gain of broilers as obtained in this study may be attributed to the antimicrobial effects of the active ingredients in garlic on intestinal microbes competing with the host for available nutrients or producing toxic bacterial metabolites. Findings of early in vitro studies on inhibitory potentials of garlic on some bacterial pathogens including Bacillus cereus, E. coli, Staphylococcus aureus and Clostridium perfringens have shown varying levels of effectiveness (Onyeagba et al., 2004; Shalaby et al., 2006). Ross et al. (2001), reported that the antibacterial activity of garlic could mainly be attributed to the allicin, (+)-S-ally-L-cysteine sulfoxide, produced by the enzymatic activity of allinase on alliin after crushing or cutting the clove. According to the report of Tekeli et al. (2011), poor performance and increase susceptibility to diseases in birds have been attributed to enteric microbial competition with the host for nutrients, liberation of toxic microbial metabolites and inhibition of the binding of the bile acids to the pertinent substances, thereby decreasing the digestion of fat and fat-soluble vitamins. In this study it was observed that the garlic supplement reduced Clostridium perfringens load among the treatment groups fed garlic-supplemented diets compared to the control group. At 1.0 g kg¯1 supplementation dose, the best of antimicrobial and growth promotant potentials of garlic were evident with the lowest colony count of Clostridium perfringens (i.e., 0.93x105 cfu g¯1 of faecal sample) as well as the best performance indices obtained. However, the antimicrobial effect has no statistical significance (p>0.05) among the treatment groups. Although the real mode of antimicrobial action of garlic is yet to be extensively studied or clearly understood, Banerjee and Sarkar (2003), have reported in vitro bacteriostatic potential of aqueous extract of garlic (Allium sativum) against Gram-positive and Gram-negative food-borne bacterial pathogens including Clostridium perfringens. The mode of action of garlic as potential growth promotant may be related to any of the modes reported about general antibiotics used as growth promoter or feed additives (CAFA, 1997; Ranjhan, 2001).

CONCLUSION

The findings of this study showed that garlic depressed feed intake but improved feed conversion ratio significantly both at 1.0 and 1.5 g kg¯1 supplementation levels. Also, garlic supplementation at all levels reduced the caecal load of C. perfringens and numerically improved weight gain of the birds. Therefore, garlic could be supplemented at low dose range of 1.0 to 1.5 g kg¯1 in broilers= diets. These findings may therefore be used as basis for limiting the supplementation level of garlic in broilers diets in order to achieve positive growth response.

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