Research Articles

2020  |  Vol: 5(6)  |  Issue: 6 (November-December) | https://doi.org/10.31024/apj.2020.5.6.1
Antibacterial activity of Cannabis sativa extracts against Salmonella Gallinarum isolated from Poultry

Yunusa Umar Abubakar1*Dalha Wada Taura2Abdulhamed Baba Njobdi2Patience O. Ihedigbo1Miracle Uwa Livinus3

1Department of Animal Health, College of Agriculture, Jalingo, Taraba State, Nigeria

2Department of Microbiology, Bayero University, Kano, Nigeria

3Department of Biochemistry, Bayero University, Kano, Nigeria

*Address for Corresponding Author

Yunusa Umar Abubakar

Department of Animal Health, College of Agriculture, Jalingo, Taraba State, Nigeria

 


Abstra​ct

 

Background: Salmonella Gallinarum is a non-motile gram-negative rod and host-specific bacteria for poultry with very high economic implications. Objective: This study was carried out to determine the antibacterial activities of Cannabis sativa plant extracts (petroleum ether and methanolic, seed and leave extracts) against Salmonella Gallinarum isolated from poultry. Materials and Methods: The antibacterial activity of the plant extracts was conducted by assessing the activity of the plant extracts on Salmonella Gallinarum isolated from poultry (chickens) using agar well diffusion method. Results: The antibacterial activity of C. sativa plant extracts against Salmonella Gallinarum ranged between 8.50±0.71 and 23.50±0.71mm. Petroleum ether seed extract had the highest antibacterial activity of 23.50±0.71mm at 2000µg/ml. The antibacterial activities of C. sativa plant extracts were in the order; PSE > PLE > MSE > MLE. All plant extracts inhibited the activities of Salmonella Gallinarum at varying concentrationThe minimum inhibitory concentration (MIC) of petroleum ether leave and methanol seed and leave extracts were 125µg/ml respectively while the MIC of petroleum ether seed extract was the least at 62.50µg/ml. The findings show significant antibacterial activity of C. sativa plant extracts against Salmonella Gallinarum (P<0.05). Conclusion: The findings of the present study shows that C. sativa seed and leave extracts can be used to discover bioactive natural products that may serve as leads in the development of new pharmaceuticals research activities that brings about breakthrough in the treatment of Fowl typhoid (FT) caused by Salmonella Gallinarum.

Keywords: Antibacterial, Cannabis sativa, poultry, Salmonella Gallinarum


Introduction

Salmonella is a bacterial enteric pathogen capable of infecting a guest host. Depending on the serotype of the infected host, Salmonella can induce various diseases, gastroenteritis type, and systemic infection, through the asymptomatic carriage (Youssef et al., 2017). Fowl typhoid (FT) caused by Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (Salmonella Gallinarum) is a severe systemic disease, producing high mortality rates (up to 90%) in poultry birds of all ages, thus causing heavy economic loss (Kumari et al., 2013; Lawal et al., 2015).

Chickens are the natural hosts for S. Gallinarum. However, in natural outbreaks, FT has been discovered in turkeys, guinea-fowl, quail, pheasants, ring-necked doves, ostriches, peafowl, sparrows and parrots (Shivaprasad, 2000). The susceptibility of ducks, geese and pigeons to S. Gallinarum varies, but all of these birds generally appear to be resistant (Kang et al., 2012). A greater percentage of females appear to remain as reactors, probably due to the sequestered nature of localised infection of the ovarian follicles. Salmonella Gallinarum could be cultured for up to 121 days from the faeces of experimentally infected rats (Badi et al., 1992).

Salmonella Gallinarum rarely cause clinical cases in people. Self-limiting enteritis is thought to be the most common presentation, although other syndromes have been reported. Their threat to immune-compromised individuals is still unclear (PHAC, 2010). A urinary tract infection caused by Salmonella Gallinarum was reported in a kidney transplant patient in 2017, and it is possible that some infections might have been missed (Ahamed et al., 2017). Organisms identified only as group D Salmonella have occasionally been recovered from people who were infected with HIV or had other immuno-suppressive conditions (Abuhasna et al., 2012).

Fowl typhoid disease can be transmitted orally (e.g., in food and water or by cannibalism) and via the respiratory tract (Basnet et al., 2008). The causative organisms may also enter the body at other sites, such as in wounds. Salmonella Gallinarum can be found in the feces of sick birds; however, they do not seem to colonize the intestinal tract in the absence of disease (Xu et al., 2018). Clinical signs of FT in chickens include anorexia, diarrhea, dehydration, and decreased egg production (Kwon and Cho, 2011). The morbidity and mortality of FT are highly variable in poultry and are influenced by host age, host susceptibility, nutrition, flock management, and virulence of Salmonella Gallinarum, which is a major factor affecting the severity of the disease (Lee et al., 2007). Due to the detrimental effect of virulent bacterial diseases to poultry production and the entire national economy, this research was embarked to determine the the antibacterial activities of Cannabis sativa plant extracts (petroleum ether and methanolic seed and leave extracts) against Salmonella Gallinarum isolated from poultry.

Materials and Methods

Collection and processing of plant sample

One kilogram (1kg) of dried leaf and seed of Cannabis sativa (Indian hemp) were obtained from Taraba State Command, National Drug Law Enforcement Agency (NDLEA), Jalingo, Taraba State. The samples were confirmed at the Department of Plant Biology, Bayero University Kano. The samples were grounded into powder using mortar and pestle as described by Mukhtar and Tukur (1999).

Extraction of plant materials

A procedure by Redfern et al. (2014) was followed for extraction using Soxhlet apparatus (Pyrax Company, United Kingdom). Petroleum ether and methanol solvents were used for the extraction. One hundred and eighty grams (180g) of the powdered plant leaf and seed material was filled in cotton sacked material and introduced inside the Soxhlet extractor. Following this, five hundred miles (500ml) of petroleum ether was also introduced into the soxhlet extractor. The side arm was lagged with glass wool, the solvent was heated using hotplate, the condenser on the isomantle beginning to evaporate moving through the apparatus to condenser, the condenser thin dripped into the reservoir containing the plant materials. As the level of solvent reached the siphon, it poured back into the flask and the circle continued until the plant material colour turned to colourless, and the extracted material poured into the beaker. The process was done for methanol solvent, with the same gram of the plant material. The extracts were left to air dry for the solvents to evaporate and the fraction was later obtained.

Phytochemical Screening

Phytochemical screening of petroleum ether and methanol seed and leaf extracts of Cannabis sativa was carried to find the presence or absence of active secondary metabolites such as alkaloids, tannins, flavonoids, saponin, glycoside, phenols and steroid/triterpenoid as described by Edeoga et al. (2005) with modifications by Abubakar et al. (2020a).

Collection of chicken samples

In this study, a total of 50 chicken visceral organs and intestinal contents were randomly pooled from freshly slaughtered diseased and healthy chicken of different abattoirs in Jalingo town, Taraba State, Nigeria. The samples were collected between September and November 2019, from five poultry abattoir. The collected samples were introduced into sterile polythene bag and transferred immediately in an ice park to the laboratory for bacteriological analysis. Following transport in ice parks (<8˚C) to the laboratory, visceral organs and intestinal contents samples were homogenised in buffered peptone water within 24h post collection from abattoirs as described by Abraham et al. (2019).

Bacteriological Study

Isolation and identification of bacterial isolate

About 2g of the homogenized chicken sample was immediately inoculated into Rappaport Vassiliadis Soya (RVS) peptone broth for selective enrichment of Salmonella organism of Veterinary Importance and was incubated at 37°C for 24hrs as described by OIE (2012). After 24hrs, one loopful of RVS peptone broth culture was streaked into MacConkey lactose agar (MLA) and blood agar (BA) and was incubated at 37°C for 24hrs and the groose colony was observed after 24hrs incubation and the result was recorded. The identification of the organisms was performed by the tests as described by Merchant and Packer (1997) and Cheesbrough (2006). On the basis of growth morphology and Gram staining characters, and biochemical tests the organism was isolated and identified.

Determination of antibacterial activity of Cannabis extracts

Salmonella Gallinarum isolates were subcultured onto sterile nutrient agar plates and incubated at 37°C for 24 hours. A sterilized wire loop was used to ensure absolute dilution of the subculture in normal saline (0.85% w/v) such that their turbidity matches with 0.5 MacFarland standards which give a mean of 1.0 × 108 cfu/ml microbial population density (Abdulrashid et al., 2018). Agar well diffusion method was used for the antibacterial assay as described by Nester at al. (2004). Mueller Hinton agar was prepared as specified by the manufacturer. The media was autoclaved at 121°C for 15 minutes, poured aseptically into sterile petri-dished and allowed to gel. A loopful of standardized isolate suspension was streaked evenly on each agar plate. A 0.004g of the leaf and seed extract were seperately dissolved in 1ml dimethyl sulphoxide (DMSO) to obtain 4000µg/ml stock solution of leaf and seed fractionated extracts of Cannabis sativa. From this, the working concentrations of 2000µg/ml, 1000µg/ml and 500µg/ml were made. Then 0.1ml of each fractionated extract was inoculated into three wells (6mm diameter) bore with a sterile cork borer to each plate. The fourth (4th) well contains a commercial oxytetracycline HCl (0.03 µg/ml) and served as positive control. The plates were then allowed to stand for 30mins on the table for pre-diffusion of the extract and were inoculated at 37°C for 24hrs. After incubation, the antibacterial activity of the extract was determined by measuring the mean diameter zones of inhibition produced by each of the extracts against the bacterial species and result was recorded in millimeter (mm).

Determination of Minimum Inhibit​ory Concentration (MIC)

The minimum inhibitory concentrations (MIC) of the petroleum ether and methanol, seed and leave extracts against the test organisms were determined using the broth dilution method (Clinical Laboratory Standard Institute, 2015). To each concentration of the extracts i.e. 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml and 31.25µg/ml, a loop full of culture adjusted to 0.5 MacFarland standard was inoculated into it and all the tubes were incubated at 37°C for 24 hours. The tube containing broth and extract serve as the positive control while the tube containing broth and inoculum serves as negative control. The lowest concentration of the extract that inhibited the growth of each organism was considered as minimum inhibitory concentration (MIC).

Minimum Bactericidal Conce​ntration (MBC)

This was conducted by the method described by CLSI (2015). To determine this, the tubes that showed no visible bacterial growth from the MIC tubes were streaked onto Mueller Hinton plates and incubated at 37°C for 24 hours. The lowest concentration in which no growth occurred was taken as the MBC.

Statistical Analysis

All data were analyzed using the statistical package SPSS (Version 20) and Microsoft Excel 2013. Descriptive statistics were carried out to determine the mean zone of inhibition of Cannabis sativa extracts against Salmonella Gallinarum and expressed in millimetre (mm). One Way Analysis of Variance and Duncan Multiple Range test were used to show significant difference between mean variables. The P < 0.05 were considered statistically significant.

Results

Phytochemical analysis of C. sativa plant extracts

The result of the phytochemical analysis of C. sativa plant extracts using petroleum ether and methanol solvents is presented in our earlier study (Abubakar et al., 2020b).

Cultural and biochemical characterization of S. Gallinarum

The result of the cultural and biochemical characteristics of the test organism indicated that S. Gallinarum was observed to be a Gram negative, rod-like and non-motile bacteria and reacted positive to oxidase, methyl red and hydrogen sulphide tests. Also, the bacterial isolate reacted negative to catalase, coagulase, Vogues Proskauer, indole, citrate, urease and Triple Sugar Iron tests (Table 1).

Table 1. Cultural and Biochemical characterization of S. Gallinarum

Test

Inference

Test

Inference

Microscopic Morphology

Rods

Oxidase

+

Gram stain

Vogues Proskauer

Catalase

Methyl red

+

Coagulase

Indole

Hydrogen sulphide

+

Citrate

Urease

Triple Sugar Iron

Motility

 

 

Positive (+), Negative (–)

Antibacterial activity of C. sativa extracts on Salmonella Gallinarum

The results from the sensitivity test showed that the plant extracts had remarkable activity against the tested microorganism with inhibition zones ranging between 8.50±0.71 and 23.50±0.71mm at various concentrations. Petroleum ether seed extract (PSE) had the highest activity of 23.50±0.71mm at 2000µg/ml while methanol leave exract had the least activity of 8.50±0.71mm at 500µg/ml. The antibacterial activities of C. sativa plant extracts were in the following order, PSE > PLE > MSE > MLE (Table 2).

Table 2. Antibacterial activity of C. sativa plant extracts against Salmonella Gallinarum

Plant extracts

Concentration (µg/ml) / Zone of Inhibition (mm)

2000

1000

500

Control (+ve) (0.03µg)

PSE

23.50±0.71a

19.50±0.71b

14.50±0.71c

29.10±1.51d

PLE

20.50±0.71a

16.50±0.71b

13.50±0.71b

32.00±1.00c

MSE

12.00±0.00a

10.50±0.71a

8.50±0.71a

23.00±1.51b

MLE

9.50±0.71a

9.50±0.71a

8.50±0.71a

22.00±0.75b

Abreviations: Petroleum ether seed extract (PSE); petroleum ether leave extract (PLE); methanol seed extract (MSE); methanol leave extract (MLE). Values are means ± SD of triplicate observation. Control (oxytetracycline HCl). Values (Means±SD) (n = 3) followed by different letters across the rows are significantly different at P<0.05.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of C. Sativa plant extracts

The MIC values ranged between 62.50µg/ml and 125.00µg/ml while the MBC values ranged between 125.00µg/ml and 250.00µg/ml (Table 3). Petroleum ether seed extract had he least MIC value of 62.50µg/ml at MBC of 125.00µg/ml. Petroleum ether leave, methanolic seed, and leave extracts had MIC values of 125µg/ml respectively.

Table 3. MIC and MBC of C. sativa plant extracts on S. Gallinarum

Plant extracts

Concentration (µg/ml)

MIC

MBC

Petroleum ether seed extract (PSE)

62.50

125

Petroleum ether leave extract (PLE)

125.00

250

Methanolic seed extract (MSE)

125.00

125

Methanol leave extract (MLE)

125.00

250

Discussion

Findings of our previous study on the phytochemical constituents of C. sativa plant extracts revealed that C. sativa leaves and seeds extracts are rich source of alkaloid, flavonoid, tannins, phenol, steroids, glycoside, saponins and triterpenoid (Abubakar et al., 2020b). The presences of these secondary metabolites that are biologically active are responsible for the antibacterial activities of Cannabis Sativa. The selection of these plants was due to their bioactive constituents that produces some biological activity in man and animal (Simon et al., 2015). These compounds also serve defensive functions for the plant against insect attack, herbivores, and microbial infection (Patel, 2010). According to Sihoglu and Tepe (2015), plants have naturally occurring chemicals that are non-nutritive and for their self-protection. They have therapeutic values, protective or diseases preventive properties which can be synthesized into useful drugs (Heinrich et al., 2016).

The test organism, Salmonella Gallinarum was isolated and identified through standard cultural and biochemical techniques. Since the isolation and correct identification of Salmonella are very crucial for the characterization, the colonies having typical cultural characteristics were selected as presumptive for Salmonella serovars. Cultural morphology on Rappaport Vassiliadis Soya (RVS) peptone broth and Eosine Metheylene Blue (EMB) was in accordance with the findings of Park et al. (2012) and Ferdous et al. (2013). In this study, the colony characters of the isolate, the production of hydrogen sulfide gas with black color colonies on TSI agar was corresponded with the findings of others (Shrama and Katock, 1996; Hossain et al., 2006). In Gram’s staining, the morphology of the isolated bacteria was small rod shape, non-motile, Gram negative, Vogues Proskauer negative which also corresponded with morphological characters of Salmonella as described by several authors (Freeman, 1995; Jones et al., 1997; Hossain et al., 2006). Biochemical reactions reported during present investigation were classical findings of S. Gallinarum (Mir et al., 2015; Dey et al., 2016). The prevalence of Salmonella Gallinarum infections in chickens could be associated with the accumulation of Salmonella Gallinarum aerosols in the atmosphere of chicken barns that are inhaled by chickens into the respiratory tract.

Antibacterial activity of C. sativa plant extracts against Salmonella Gallinarum ranged from 8.50±0.71 to 23.50±0.71mm. Petroleum ether seed extract had the highest antibacterial activity of 23.50±0.71mm at 2000µg/ml. The antibacterial activities of C. sativa plant extracts were in the order; PSE > PLE > MSE > MLE. All plant extracts inhibited the activities of the test organisms at varying concentration. Considering the bioactive contents of C. sativa, as previously reported by Khan et al. (2010), the extracts can serve potent and effective therapeutic use. Furthermore, the susceptibility profle of S. Gallinarum to the plant isolates was an indication that the isolates can be harnessed as alternative treatment remedy for Salmonellosis and other bacterial avian infection (Marano et al., 2000; Liebana et al., 2013; Ferreira et al., 2014). The minimum inhibitory concentrations (MIC) of C. sativa leave extracts against Salmonella Gallinarum were higher than those of C. sativa seed extracts against Salmonella Gallinarum. The higher MIC of the leave extracts could be attributed to the higher prevalence of phytochemicals in the leave extracts than the seed extracts of the plant as recently reported by Kavitha et al. (2017).

Conclusion

The present study has indicated that petroleum ether seed and leave extracts had significant antibacterial effect against Salmonella Gallinarum. Thus, Cannabis sativa plant extracts are viable therapeutic agents for the treatment of avian diseases. Considering the high antibacterial activity of Cannabis sativa petroleum ether seed and leaf extracts against Salmonella Gallinarum, the researcher recommends that Cannabis sativa plants can be used to discover bioactive natural products that may serve as leads in the development of new pharmaceuticals research activities that brings about breakthrough in the treatment of Fowl typhoid (FT) caused by Salmonella Gallinarum. Further studies on the extracts of the whole plant using Mass Spectroscopy, and Nuclear Magnetic Resonance Techniques is recommended to elucidate the structure for drug synthesis.

Conflicts of Interest

The authors have no conflict of interest to be declared.

 

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