Antibacterial and Immuno-Modulatory Effects of Probiotics and Phytobiotics in Spent Rhode Island Red Layers
Muhammad Mushtaq1, Muhammad Shuaib1*, Aamir Iqbal2, Abubakar Sufyan3, Muqader Shah6, Noor Ul Ain Nawaz4, Muhammad Ismail Khan5, Hamayun Khan6, Muhammad Shahkar Uzair1, Qudrat Ullah6 and Usman Zeb7
1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan.
2Department of Animal and Poultry Production, Faculty of Veterinary and Animal Sciences, Ponch University, Rawalkot, Azad Kashmir
3Department of Livestock and Poultry Production, Bahauddin Zakariya University, Multan, Pakistan
4Department of Pharmacy, City University of Science and Information Technology, Dalazak Road, Peshawar
5Department of Zoology, Islamia College, Peshawar
6College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan
7Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Pakistan
ABSTRACT
This research study was designed to evaluate the synergistic effects of phytobiotics and probiotics during the induced mix (Escherichia coli and Salmonella) infection in egg-type birds. Six different phytobiotic extracts with a constant dose level of probiotic were first evaluated through in-vitro screening by disk diffusion method against the resisted E. coli and Salmonella paratyphi. Based on in-vitro screening, phytobiotics were further evaluated by in-vivo trial. The potential of phytobiotics with probiotics was evaluated by evaluating the mortality, morbidity, intestinal microbiota count, and serum immunoglobulin (IgA, IgM, and IgG) during the induced mixed infection. Birds were inoculated with E. coli (O157:H7) and S. paratyphi at the rate of 1:1x109 cfu/ml each. Birds were divided into 12 groups having three replicates. Phytobiotics extracts at concentration of 1, 100, and 1000mg/L were used for all the three selected medicinal plants with probiotics @ 0.03ml/L (12×106 spores). The in-vitro evaluation of phytobiotics (Fenugreek, B. Lycium, and T. arjuna) extracts with probiotic (Bacillus clausii) showed more potential by inhibiting the growth of selected bacterial strains. It was noticed that phytobiotics and probiotics at 1000mg/L+0.03ml/L dose level showed improved microbial count, IgA, IgM, IgG, mortality, and morbidity against the infection as compared to the control and standard group. It was concluded from the present experiment that phytobiotics with probiotics possess antimicrobial and immune-modulatory properties.
Article Information
Received 05 March 2023
Revised 20 July 2023
Accepted 11 August 2023
Available online 22 March 2024
(early access)
Published 08 May 2025
Authors’ Contribution
MM, Study design, statistical analysis, and manuscript writing. MS, Statistical analysis, manuscript writing, and reviewing. NUAN, Performed phytobiotics exaction processes. AI, AS, MS, MIK, HK, MSU, QU and UZ, Manuscript writing and reviewing.
Key words
Bacillus clausii, Egg-type birds, Immunology, Microflora count, In-vitro, Poultry production, Rhode island red
DOI: https://dx.doi.org/10.17582/journal.pjz/20230305080346
* Corresponding author: [email protected]
0030-9923/2025/0003-1409 $ 9.00/00
Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
INTRODUCTION
The poultry industry of Pakistan is one of the vibrant sub-sectors of the Pakistan livestock industry and plays a vital role in fulfilling the demand for animal protein among the consumer of Pakistan. Advancements in poultry husbandry practices in feeding management lead to the incorporation of different feed additives during ration formulation. These feed additives include antibiotics, pro-pre-biotics, phytobiotics, exogenous enzymes, and a balanced diet, which act as growth promoters in poultry production (Mehdi et al., 2018). Several poultry husbandry practices in feeding management are reported by previous studies (Roess et al., 2013) that become the risk factors for transmitting of drug resisted infectious zoonotic-infection. Microbes are disease-causing agents in all living organisms. Structurally they are tiny in size but cause profound damage to the living body. Antimicrobial/antibiotic is the class of drugs that can kill or arrest the multiplication of these microbes. With the advancement of antimicrobial drugs against the different categories of microbes, the microbes developed resistance against these antimicrobial drugs shortly or passively to stabilize their survival in the environment. Escherichia coli infections are widely distributed among poultry of all ages and categories. Salpingitis (inflammation of the oviduct) due to E. coli infections could be also observed in growing birds. Extra intestinal pathogenic Escherichia coli (ExPEC) constitutes ongoing health concerns for women, newborns, elderly, and immunocompromised individuals due to increased numbers of urinary tract infections (UTIs), newborn meningitis, abdominal sepsis, and septicemia (Manges and Johnson, 2012). Recognizing and treating the zoonotic risk posed by ExPEC would greatly enhance food safety and positively impact human health (Mellata, 2013). Salmonella is a group of bacteria that causes typhoid fever, food poisoning, gastroenteritis, enteric fever, and other illnesses. Meat and eggs are known to be a source of human pathogens such as Campylobacter, Listeria, and Salmonella, which frequently leads to a food recall of the suspected contaminated products (Olugbenga et al., 2021). In humans, the main source of infection is the consumption of contaminated poultry meat and eggs. Because of its significant risk to public health, Salmonella virchow is one of five serovars that has been given priority by the European Union (EU) for investigations on poultry farms to control its entry into the food chain (Snow et al., 2007: Arnold et al., 2010). Resistance produced by microbes against the synthetic antimicrobials lead to human health hazard with low immunity, therefore, the photochemical (natural antimicrobials) from the plants’ kingdom got attention (Kazi et al., 2022). Biomolecules of plant origin and probiotics appear to be one of the alternatives for the control of these antibiotic-resistant human pathogens in producing animals/birds. Plant materials are used widely in traditional systems of medicine (Aziz et al., 2018). Plant extracts, also known as phytobiotics, have been exploited in animal nutrition, particularly for their antimicrobial, anti-inflammatory, antioxidant, and antiparasitic activities (Motoi, 2021). Biologically active components of plants are mostly secondary metabolites, such as terpenoids, phenolics, glycosides, and alkaloids (Baan and Hasan, 2022). These secondary metabolites may have a protective function in vegetal tissues. These compounds are assumed to be involved in plant defense and most of them may possess antimicrobial properties (Khameneh et al., 2019). Probiotics are beneficial live micro-organisms, which administration in the host confers one or more specified health benefits (Roess et al., 2013). Application of probiotics can result in structural and compositional alteration in intestinal architecture and microflora by improving the absorptive sites and reduction in pathogenic microbiota. It can help in the production of intestinal-lactic acid and hydrogen peroxide (H2O2), which further causes the inhibition/reduction in oxidation processes. This phenomenon of probiotic in the host intestinal site, resulting in the inhibition of aerobic pathogens (toxin amines and ammonia), promote the production of intestinal essential digestive enzymes, and vitamin-B complex and also stimulate the host appetite (Singh et al., 2004). As per previous studies by Guo et al. (2004) and Jamroz et al. (2003) that bioactive compounds of phytobiotics are considered potential agents by promoting beneficial intestinal-microbiota (probiotics) without influencing the growth of pathogenic microflora spp. Considering this beneficial aspect of phytobiotics compounds on the promoting gut-probiotics, it can provide an optimal precondition for effective protection against zoonotic infectious pathogens and involve in the host gut-immune/defense system (Wenk, 2003). Therefore, the present research study was designed to evaluate the synergistic effects of phytobiotics and probiotics during the induced mixed (E. coli and salmonella) infection in egg-type birds.
MATERIALS AND METHODS
Biochemical materials and phytobiotics extracts preparation
The phytobiotics (Berberis lycium bark, Fenugreek seeds, Terminalia arjuna seeds, Nigella sativa seeds, Withania coagulans seeds, Peganum harmala seeds) and probiotics (Bacillus clausii) materials were obtained from Forest Institute Peshawar and Veterinary Teaching Hospital (VTH) of College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan. Methanolic extracts of medicinal plants were prepared according to the standard protocol of Dabur et al. (2004). Clinical isolates of the poultry microorganisms [Salmonella paratyphi and Escherichia coli (O157:H7)] were used as challenged microbiota. The strains of the microorganisms were collected from the Microbiology Lab of the College of Veterinary Sciences, The University of Agriculture Peshawar, Pakistan.
In-vitro antibacterial activities of methanolic extract of phytobiotics and probiotic
The methanolic extracts of 6 phytobiotics with the combination of Bacillus clausii were screened against 2-bacterial strains, Escherichia coli and Salmonella. These challenged microbiotas were obtained from the Microbiology Lab of the College of Veterinary Sciences. Active cultures of microbiota for experimental purposes were prepared by transferring a loop-full of cells from previously stored stock cultures (maintained at 4°C on slopes of nutrient agar) to Mueller-Hinton broth (MHB) containing test tubes. The test tubes were incubated for 24 h at 37oC and 25oC, respectively, without agitation in the incubator. For achieving optical densities (OD) corresponding to 2x106 CFU/ml, the cultures were diluted with fresh MHB.
According to the standard protocol developed by Bauer et al. (1966), the antibacterial activities of extracts and probiotics were performed. The Mueller Hinton agar (MHA) plates were prepared by pouring 15ml of molten media into sterile petri-plates and allowed to dry for 5 min. The drying process was followed by swabbing 0.1% of inoculum suspension uniformly and allowed to dry for 5 min. The different concentrations of phytobiotics extracts with the probiotic and antibiotic discs were loaded at a distance of 24 mm from each other and 12mm from the plate edge. The plates were properly labeled, sealed with Parra-film, and inoculated at 37 °C for 24-48 h. To avoid any sort of contamination biosafety cabinet level II (ESCO, USA) was used. The zone of inhibition against the tested pathogens was recorded in millimeters (mm) after 48 h. About 10% DMSO-containing disc was taken as a negative control to compare the tested plates against the tested pathogens. The process was performed in triplicate and the triplicate average was analyzed as the mean.
In-vivo antibacterial activities of methanolic extract of phytobiotics and probiotic
The inoculum was prepared from a stock culture of E. coli and Salmonella typhimurium through a developed technique by Eman and Hoda (2008). A high concentration of the inocula (1.0x109) was prepared to increase the probability of establishing the disease condition in the experimental birds. One hundred and eighty (180) healthy laying birds of age 75 weeks were used for this experiment. The birds were obtained from the laying house of the Department of Poultry Science, The University of Agriculture Peshawar, Pakistan. The birds were assigned randomly to isolated cages in the house on a 16/08 light-dark cycle. The birds were allowed to acclimatize to their new environment for a week before inoculation and were tested to ensure that they will negative for E. coli and Salmonella. Feed and water were provided from the day the birds procure until the completion of the experiment.
Seventy-five weeks-old Rhode Island Red (RIR) birds were divided into 12 groups of 3 replicates (n=5). The phytobiotics extracts and probiotics incorporated in drinking water to the different treatment groups at the rate of 1mg/L+0.03ml/L (12×106 spores), 100mg/L+0.03ml/L (12×106 spores) and 1000mg/L+0.03ml/L (12×106 spores). The supplemented water was offered throughout the entire five weeks period of the experiment.
On the 4th day of the experiment, the volume of the inoculum was introduced into each bird as prescribed by Eman and Hoda (2008). All the groups were orally challenged with 1.0ml of E. coli and Salmonella paratyphi inoculum at a dose of 1:1 × 109cfu/ml except the negative control group.
For determination of performance indicators the birds were routinely observed for mortality if any due to microbe infections. The post-mortem was performed to identify the possible cause of mortality. The birds were physically observed for any signs of illness during the entire period of the experiment. The gut-microbiota count was performed according to the standard protocol developed by Barrow and Feltharn (1993) by using the standard plate count technique. The preparation of media for the count was performed according to the manufacturer’s specifications. About 1 ml ileum digesta was used for the count. The ileo-digesta was processed through serial dilution processing (10-3 dilution level) in sterile 15 ml test tubes (each tube consisting of 9 ml of 0.1% of sterile peptone water) and vortexed. Approximately 1 ml of the diluted digesta was pipetted/inoculated on plate count agar and MacConkey agar, following the incubation at 37oC for 24 h. The required/discrete colonies of microbiota on the plate were examined and counted by using a colony counter and expressed as log10 CFU/ml as results.
Immunoglobulin’s (Ig) determination
Immunoglobulin (Ig) was determined in egg-type birds according to the developed protocol of Delhanty and Solomon (1966), Yamamoto and Glick (1982), Martin et al. (1989), and Qureshi and Havenstein (1994). Sheep red blood cell (SRBCs) suspension was used for the determination of Ig-antibodies in egg-type birds. About 15 ml of blood was collected from healthy sheep reared at University Dairy Farm in an EDTA tube and washed with equal V/V with phosphate buffer saline (PBS). The packed cells at 2.5/0.25 V/V were taken in PBS followed by a washing process. Birds were immunized with 0.1-ml (0.25%) of sheep RBCs suspension. The blood was collected about 14-d of post-immunization and serum was collected by centrifugation at 4000 rpm for 10 mins for determination of serum antibodies against SRBCs.
Statistical analysis
The data were analyzed through a statistical package scientific analysis system (SAS) by using a complete randomized design (CRD) as described by Steel and Torrie (1981).
Table I. In-vitro antibacterial activities of different concentrations of methanolic extracts of phytobiotics probiotic B. clausii and Ciprofloxacin.
|
Phytobiotics |
Methanolic extract+ B. clausii |
Mean±SE (mm in radius) |
|||
|
E. coli |
Salmonella |
||||
|
Ciprofloxacin |
40 μl |
20.56±0.86 |
22.65±0.58 |
||
|
B. lycium |
0.02 μl + 5 μl |
8.00 ±0.28e |
9.00±0.28e |
||
|
0.04 μl + 5 μl |
8.70 ±0.11ed |
9.40±0.11ed |
|||
|
0.06 μl + 5 μl |
9.46±0.12d |
9.96±0.12d |
|||
|
T. foenum- graecum |
0.02 μl + 5 μl |
9.00±0.57d |
12.00±0.28c |
||
|
0.04 μl + 5 μl |
11.00±0.57c |
13.00±0.57b |
|||
|
0.06 μl + 5 μl |
13.00±0.28a |
17.00±0.57a |
|||
|
T. arjuna |
0.02 μl + 5 μl |
7.00±0.57f |
7.00±0.28g |
||
|
0.04 μl + 5 μl |
9.00±0.57d |
8.00±0.28f |
|||
|
0.06 μl + 5 μl |
12.00±0.57b |
9.00±0.57e |
|||
|
N. sativa |
0.02 μl + 5 μl |
1.50±0.11h |
0.00±0.00h |
||
|
0.04 μl + 5 μl |
1.60±0.05h |
0.00±0.00h |
|||
|
0.06 μl + 5 μl |
1.60±0.15h |
0.00±0.00h |
|||
|
W. coagulans |
0.02 μl + 5 μl |
0.00±0.00i |
0.00±0.00h |
||
|
0.04 μl + 5 μl |
0.00±0.00i |
0.00±0.00h |
|||
|
0.06 μl + 5 μl |
0.00±0.00i |
0.00±0.00h |
|||
|
P. harmala |
0.02 μl + 5 μl |
2.00±0.28h |
0.00±0.00h |
||
|
0.04 μl + 5 μl |
2.43±0.08h |
0.00±0.00h |
|||
|
0.06 μl + 5 μl |
3.63±0.08g |
0.00±0.00h |
|||
|
P-value |
0.0212 |
0.0121 |
|||
B. clausii, Bacillus clausii; B. lycium, Berberis lycium; T. arjuna, Terminalia arjuna; N. sativa, Nigella sativa; W. coagulans, Withania coagulans; P. harmala, Peganum harmala
Means in column with different superscript are significantly different at α=0.05
RESULTS
Methanolic extracts of different phytobiotics and probiotics showed variable antibacterial activity against selected bacterial pathogenic strains as compared to the standard group of ciprofloxacin (broad spectrum antibiotic) and control (DMSO). Based on the zone of inhibition, the phytobiotic extracts and probiotic against the challenged pathogenic strains, the direct-proportional trend was observed that the increased sensitivity against these tested bacteria (E. coli) with increasing the extract level in all treatments except the Withania coagulans. Similar findings were recorded for Salmonella in the group of B. lycium, Fenugreek and T. arjuna extracts with the probiotic. The other phytobiotics failed to show any response against the challenged bacterial strains. The highest sensitivity (zone of inhibition) of phytoprobiotics was noticed in fenugreek + probiotic at a level of 60+5 µl (13.00 mm), followed by T. arjuna + probiotic @ 60 + 5 µl (12.00 mm) and fenugreek + probiotic @ 40 +5 µl (11.00 mm) against the E. coli. In the case of Salmonella as challenged bacteria, methanolic extract of fenugreek with probiotic @ 60, 40, and 20 + 5 µl showed the highest (17.00, 13.00, and 12.00 mm) sensitivity as compared to other phytoprobiotics (Table I). Based on the in-vitro antibacterial screening of phytoprobiotics extracts against the multi drugs resistance (MDR) E. coli and Salmonella, the selected plant extracts with probiotics were further evaluated for antibacterial activities in-vivo tests. Phytoprobiotics combination was further evaluated as an antibacterial agent by observing the mortality and morbidity in the challenged experiment. The highest mortality was observed in positive control as compared to the standard group of quanlone group of antibiotics (Ciprofloxacin). Indirect correlation/proportion was observed in phytoprobiotics combination groups regarding mortality. The lowest mortality was observed in the standard group of ciprofloxacin, followed by the phyto-pro-biotics combo-groups at the highest concentration (3.33 %). Less morbidity was observed in the negative control group while the highest morbidity was observed in the positive control. Ciprofloxacin-treated group morbidity was very low (+), while in phytoprobiotics combination treated groups high morbidity was observed for Ph(Fg)E+Bc at the rate of 1mg/L+0.03 ml/L and 100mg/ L+0.03 ml/L. Low morbidity was observed in B. lycium + probiotic (Ph(Bl)E+Bc) treated group at the rate of 1000mg/L+0.03 ml/L was (+) compared to the positive control group (Table II). It reflects that medicinal plant extracts with probiotic supplementation have antibacterial properties. Intestinal microflora count was found significant (P<0.05) variable data (Table III). Significantly the highest count was recorded in positive control and as well as in treatment groups with a low concentration of extract combination with probiotics for each E. coli and Salmonella inoculation. We observed the indirect trends of phytoprobiotics (increasing level) with intestinal microflora count (decreasing). The count for selected microflora was recorded lowest in the antibiotics treatment group, followed by phytoprobiotics groups at the highest concentration (1000mg/L+0.03ml/L), 4.15, 3.41, 5.12, 5.42, and 5.81 (CFU/g) for E. coli and 4.35, 5.35 and 5.81 (CFU/g) for Salmonella, respectively. Serum humoral immunity response was estimated through
Table II. In-vivo synergistic effects of different concentrations of methanolic extracts of phytoprobiotics on zootechnical indicators of egg type birds during induced infection.
|
Groups |
Treatment (Extract+Probiotic) |
Mixed infection (E. coli + Salmonella) |
Means±SE |
Morbidity (Severity index) |
|
|
Feed intake (%) |
Mortality (%) |
||||
|
Negative control |
No infection, No treatment |
-- |
94.69±0.39a |
0.00±0.00b |
+ |
|
Positive control |
Infection without treatment |
1.0x109 |
64.54±1.89i |
10±0.00a |
++++ |
|
Standard |
Ciprofloxacin (2mg/kg) |
1.0x109 |
90.60±0.80b |
0.00±0.00b |
+ |
|
(MP(Tf)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
70.59±0.95gf |
6.66±3.33ba |
+++ |
|
100mg/L+0.03ml/L |
1.0x109 |
77.16±0.61d |
6.66±3.33ba |
+++ |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
81.76±0.52c |
3.33±3.33ba |
++ |
|
|
(MP(Bl)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
68.78±1.11gh |
6.66±3.33ba |
++ |
|
100mg/L +0.03ml/L |
1.0x109 |
72.25±0.51ef |
3.33±3.33ba |
++ |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
80.39±0.54c |
3.33±3.33ba |
+ |
|
|
(MP(Ta)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
66.83±0.56ih |
10±0.00a |
+++ |
|
100mg/L+0.03ml/L |
1.0x109 |
73.32±0.53e |
6.66±3.33ba |
+++ |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
74.80±0.87ed |
3.33±3.33ba |
++ |
|
|
P-value |
0.0120 |
0.0214 |
-- |
||
MP(Fg)E, methanolic extracts of phytoprobiotics (Trigonella foenum-graecum)
MP(B1)E, methanolic extracts of phytoprobiotics (Berberis lycium)
MP(Fg)E, methanolic extracts of phytoprobiotics (Terminalia arjuna)
Means in column with different superscript are significantly different at α=0.05. Severity Index: +, very low morbidity; ++, low morbidity; +++, milled morbidity; ++++, sevier morbidity.
Table III. Synergistic effects of different concentrations of methanolic extracts of phytoprobiotics on gut microflora count during induced infection.
|
Groups |
Treatment (Extract+Probiotic) |
Mix infection (E. coli +Salmonella) |
Means (Log10 CFU/g) |
|
|
E. coli |
Salmonella |
|||
|
Negative control |
No infection, No treatment |
-- |
5.21b |
4.51bc |
|
Positive control |
Infection without treatment |
1.0x109 |
8.4a |
7.48a |
|
Standard |
Ciprofloxacin (2mg/L) |
1.0x109 |
4.15c |
3.41c |
|
(MP(Tf)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
7.54a |
7.25a |
|
100mg/L+0.03ml/L |
1.0x109 |
6.89 ab |
6.74ab |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
4.35c |
5.12b |
|
|
(MP(Bl)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
7.87a |
7.09a |
|
100mg/L +0.03ml/L |
1.0x109 |
6.45ab |
6.48ab |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
5.35b |
5.42b |
|
|
(MP(Ta)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
7.42a |
7.52a |
|
100mg/L+0.03ml/L |
1.0x109 |
6.75ab |
6.27ab |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
5.81b |
5.81b |
|
|
P-value |
-- |
-- |
0.0001 |
0.0001 |
Bc, Bacillus clausii, For other abbreviations, see Table II.
Means in column with different superscript are significantly different at α=0.05
the detection/measured the titer of IgM, IgA, and IgG in egg-type birds (Table IV). Significantly increased level of serum immunoglobulin titer was recorded in the positive control group as compared to negative control and treatment groups (post infection). Significant serum humoral immunity response was recorded in the antibiotic standard group (1.95 IgM, 1.85 IgA, and 2.15 IgG), followed by the highest concentration of phytobiotics extract with probiotic (1000mg/L+0.03ml/L) treatment groups.
Table IV. Synergistic effects of different concentrations of methanolic extracts of phytoprobiotics on immune status during induced infection.
|
Groups |
Treatment (Extract+Probiotic) |
Mixed infection (E. coli +Salmonella) |
Means (g/L) |
||
|
IgM |
IgA |
IgG |
|||
|
Negative control |
No infection, No treatment |
-- |
1.43c |
1.51c |
1.62c |
|
Positive control |
Infection without treatment |
1.0x109 |
2.98a |
3.15a |
2.74a |
|
Standard |
Ciprofloxacin (2mg/L) |
1.0x109 |
1.95b |
1.85c |
2.15b |
|
(MP(Tf)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
2.52a |
2.4b |
2.65a |
|
100mg/L+0.03ml/L |
1.0x109 |
2.05b |
1.94b |
2.14b |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
1.75b |
1.84b |
1.92bc |
|
|
(MP(Bl)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
2.41a |
2.55b |
2.32ab |
|
100mg/L +0.03ml/L |
1.0x109 |
2.04ab |
2.14b |
2.05b |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
1.98b |
2.00c |
1.95bc |
|
|
(MP(Ta)E+Bc) |
1mg/L+0.03ml/L |
1.0x109 |
2.56a |
2.41b |
2.43ab |
|
100mg/L+0.03ml/L |
1.0x109 |
2.15ab |
2.21b |
2.14b |
|
|
1000mg/L+0.03ml/L |
1.0x109 |
2.02b |
1.98c |
2.00bc |
|
|
P-value |
0.0425 |
0.0415 |
0.0471 |
||
For details of groups and treatments, see Table III.
Means in column with different superscript are significantly different at α=0.05
DISCUSSION
The present research work mainly focused on the potential of medicinal plants to overcome microbial resistance and the prevention of E. coli infection without any immunosuppression. The highest sensitivity (zone of inhibition) of phytoprobiotics was noticed in fenugreek + probiotic at a level of 60+5 µl (13.00 mm), followed by T. arjuna + probiotic @ 60 + 5 µl (12.00 mm) and fenugreek + probiotic @ 40 +5 µl (11.00 mm) against the E. coli and Salmonella. This inhibition might be due to polyphenols, tannins, flavonoids, and saponins’ activities. Polyphenols, flavonoids, and saponins cause bacterial cell wall membrane disruption and leakage of the cell wall/cell membrane (Negi, 2012; Sung and Lee, 2008; Cushnie and Lamb, 2005; Francis et al., 2002; Ikigai et al., 1993) while tannins affect the microbial cellular metabolism by inhibiting the enzyme, phosphorylation and the electron transport system (Cowan, 1999: Scalbert, 1991). Or Probiotics have antimicrobial enzymes/proteins i.e. serine protease, clausin or reuterin (Bouhss et al., 2009: Kazan et al., 2005: Talarico et al., 1989) which utilized glycerol as a substrate to enhance the antibacterial effects (detoxification of pathogenic toxin). In this regard, the inhibition of pathogens might be due to phytobiotics that have high glycerol compounds (Vira et al., 2018) which are significantly utilized by proteolytic enzymes (Gabrielle et al., 2016) from probiotics to arrest the growth of microbiota. So, phytoprobiotics comically work together against the pathogens by the described above phenomenon. The findings of the present study are in line with the results of Fadareabcd et al. (2022) who documented that combination of probiotics with phytobiotics significantly arrests salmonella growth in an in-vitro study. The best antibacterial activities of petroleum ether extract of fenugreek seed against E. coli at the highest concentration (250 mg/ml) was 17 mm (zone of inhibition) while 10 mm inhibition was recorded in methanolic fraction as reported by Mawahib et al. (2015). The present investigation is supported by the work of Qureshi et al. (2015) who investigated the ethanolic extract of fenugreek seed at the rate of 0.5 mg/ml against E. coli and found 2.1mm inhibition. An antimutagenic and chemo preventive study was carried out by Chatterjee et al. (2013) in mice by using skin papilloma as a model. They observed that a water-based extract of fenugreek seeds at the rate of 20 g/ml showed strong inhibition against the mutagens in different strains of Salmonella. Nandagopal et al. (2012) evaluated the antimicrobial activities of the different organic solvents fenugreek seed extract against the different microbes including salmonella. Aneja et al. (2012) worked on the different solvent extraction of T. arjuna against the different bacteria with a main focus on E. coli. They notice the antibacterial activities of T. arjuna against the E. coli (14.6 mm of inhibition zone) as reported by the present study. Significant antibacterial activity of the methanolic extract of fenugreek seed against E. coli was reported by Dash et al. (2011). Screening of methanolic and acetone extract of fenugreek and coriander against the various gram-negative bacteria including Salmonella was performed by Dash et al. (2011). They revealed the significance of fenugreek as an antibacterial agent and concluded that the extraction should be used to develop a novel broad spectrum of the herbal antibacterial formulation. The ethanolic fraction of B. lycium was carried out by Hussain et al. (2011) against the different microbes including E. coli. The ethanolic fraction was found more effective as an antibacterial against E. coli including all other tested bacteria. The investigation of the present regarding the antibacterial activities of T. arjuna is supported by the finding of previous researchers Ramya et al. (2008) that the plants possess antibacterial efficacy against E. coli. Similar findings were observed as reported by the present study. The study of Gulfraz et al. (2007) reported the B. lycium activities against gram-negative bacteria including E. coli, and they do notice that the methanolic extract of B. lycium has good antibacterial activities as it possesses some phytochemicals. Findings in this study indicated a similar pattern to results obtained by (Rees et al., 1993) where the phytobiotics extracts included in the mixed culture of E. coli and probiotics selectively affected the bactericidal on E. coli. Mortality and morbidity in the treated groups were significantly different from the positive control. All the probiotics containing medicinal plants showed good results by reducing mortality and morbidity in the experimental trial against the positive and standard groups. Reduction in mortality and morbidity during induced pathogenic infection in egg-type birds might be due to the incorporation of probiotics with phytobiotics that resulting in reducing the pathogenic/disease/health stress by improving animal welfare (Yazhini et al., 2018; Vase-Khavari et al., 2019) or this improvement is due to the phytogenic and probiotic effects on the health status of GIT of the birds. Healthy GIT improves digestion and inhibits the adhesion of pathogens by decreasing the inflammation at the site of infection (Mahmood et al., 2015). So, it revealed that mortality was controlled due to healthy GIT with mild morbidity due to mixed infection. The findings of the current study are in agreement with the previous researcher (Sokale et al., 2019: Bortoluzzi et al., 2019) that necrotic enteritis (NE) in poultry production leads to high mortality and morbidity which can be significantly reduced or minimized with the use of probiotics in poultry production. The results of the present study are in line with the findings of Motawe et al. (2014) that aflatoxin-induced chicken significantly recovered by using probiotics in their diet. As E. coli and Salmonella infection is considered as cross-infection (zoonotic infection) between animals and humans. Therefore, the birds were challenged with a mix of infections of E. coli and Salmonella in the present study at the dose rate of 1.0x109. To know the beneficial effects of phytobiotics with the combination of probiotics in drinking water against the induced mix infection were carried out in egg-type birds. As per the results of the present study, the combination of phytobiotics methanolic extracts with probiotics at the highest level of incorporation in drinking water of egg-type birds showed a significant reduction in the intestinal microflora count as compared to positive and other treatment groups. The possible phenomenon behind the reduction of pathogenic intestinal microflora might be attributed to the different protection, activation, and immunomodulatory activities of phytobiotics and probiotics. Bioactive compounds of phytobiotics are considered potential agents by promoting beneficial intestinal microbiota (probiotics) without influencing the growth of pathogenic microflora spp. This can provide an optimal precondition for effective protection against zoonotic infectious pathogens and involve in host gut-immune/defense system (Wenk, 2003). Probiotics help in the alleviation/inhibition of inflammatory reactions of pathogenic microflora and improved the host gut health immune system by modulating the cytokines expression (Wang et al., 2017; Lee et al., 2012) or secretion of antimicrobial substances, modulation of GIT-immune response, adherence to the spaces at intestinal mucosa, and improved intestinal epithelial barrier function (Broom and Kogut, 2018; Tejeero et al., 2012; Lin et al., 2008). Probiotics can reduce pathogenic activities either by lower the pathogenic-phospholipase activities or by blocking pathogens’ adherence space (Ohashi and Ushida, 2009; Fuller, 1991). Probiotics reduce the pH of the gut through the production of volatile fatty acids that lead to prohibiting the growth of pathogens by reducing the pathogenic-phospholipase activities (Chichlowski et al., 2007; Marteau et al., 1997; Zentler et al., 1984). These are all mechanisms of phyto-pro-biotics resulting in a reduced load of pathogenic bacteria and improving gut health status. Assessment of phytobiotics and probiotics as antibacterial agents, significantly reduced the load of E. coli and Salmonella in the intestinal digesta as presented in the current is supported by the results of Faisal et al. (2019). That basal diet supplemented with pro-phyto-biotics significantly reduced the pathogenic strains of bacteria in intestinal digesta. The findings of the present study are in agreement with the results of (Li et al., 2018) who documented that birds fed with probiotics can result in a decrease in ileocecal pathogens. Decreased pathogenic load in intestinal digesta in the present study by phyto-pro-biotics combination is in-line with the results of Guo et al. (2004) who documented that plant extracts with probiotics feeding to broiler chicks, significantly increased the total viable count of beneficial microflora like lactobacilli and reduced the number of pathogenic microbiota in meat-type birds. Immune-globulin (Ig) is to be considered the first isotype of antibodies that are produced by mature B-cell in the primary humoral immune system in response to the antigenic agent. In response to antigenic stimulation or signals received by helper T-cells in the immune system, it promotes the production of Ig by B-cells to defend the host body from the pathogenicity of antigens (Zhangke et al., 2021). The presence of immune globulin in the host serum is the indicator of long-term exposure of the host (animals or humans) to foreign antigens. As per the present results of the project, phyto-pro-biotics significantly affect the serum Ig level in egg-type birds after induced mixed infection (E. coli + Salmonella) as compared to the positive control group. The highest supplementation of phyto-pro-biotics in the drinking water of egg-type birds significantly activated the humoral immune response against the induced foreign antigens. This might be attributed to the reduce the number of pathogenic microflora in the intestinal mucosa by phyto-pro-biotics which results in less response of induced pathogenic agents to stimulate the host humoral immune response to promote more production of Ig B-cells (Zhengke et al., 2021). Supplementation of probiotics in the meat type bird’s ration significantly promotes the immune system response (Ig) (Sefcova et al., 2020). Immunoglobulin-G in the present study indicated that birds were long-term/chronic exposure to different pathogenic agents or switching of acute to chronic inflammation, resulting in the activation of B-cells by the pathogenic agent differentiate into plasmocytes which later on make the part of plasma cells that are responsible for the secretion of IgG in the plasma to neutralize the pathogenic agent toxicity/count and become long-lived plasma memory B-cells (Chen et al., 2017). The findings of the present study are in line with the results of Wenk (2003) who documented that phytobiotics bioactive agents promote gut-beneficial microbiota which leads to a remarkable reduction in the pathogenic microflora. Pathogens cause an inflammatory reaction at the intestinal level which results in low or upset growth performance and alteration in immunological response by the intestinal immune system due to high count. Probiotics help the host immune system (Ig) to inhibit the inflammatory reactions (modulation in expression of cytokines) and also secret antimicrobial substances, modulation in GIT immune responses (Ig), promote the intestinal mucosal response and epithelial barrier function in the response of pathogenic-inflammatory reactions (Broom and Kogut, 2018; Wang et al., 2017; Lee et al., 2012; Tejeero et al., 2012; Lin et al., 2008). The synergistic effects of phytoprobiotics can also stimulate the intestinal immune response (Faisal et al., 2019). The findings of the present suggested that incorporation of phyto-pro-biotics in the egg types of birds leads to reduce the count of induced pathogenic agents which would be considered insufficient to promote/stimulate the intestinal immune system to produce a more specific immune response (Ig). The findings of the present study are in-line with the Zeng et al. (2015) results that essential oils from the plant kingdom reinforce the poultry birds’ immune system to promote the rate of lymphocyte proliferation and phagocytosis and also improve the serum immunoglobulin. All these observations encourage the assumption that these additives may favorably affect gut functions, but the number of in vivo studies in poultry is still limited.
CONCLUSION
Based on the in-vitro and in-vivo analyses, Trigonella foenum graecum, Berberis lycium and Terminalia arjuna with Bacillus clausii (probiotics) possess strong antibacterial and immunomodulatory effects against multi drug resistant Salmonella and E. coli infection.
ACKNOWLEDGEMENT
We acknowledge the Department of Poultry Science for the provision of an experimental shed and the college of veterinary sciences, The University of Agriculture Peshawar, Pakistan for the provision of a laboratory facility.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
IRB approval
The experimental work was approved by the Advanced Studies and Research Board (ASRB) (No: 5214-A/UAP, Dated: 31/12/2019), The University of Agriculture Peshawar, KP, Pakistan
Ethical statement
The experimental procedures used in the study were according to the guidelines of the Ethical Review Committee of the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar. Proper approval was taken by the aforementioned authority before the start of the experimental trial.
Statement of conflict of interest
All authors thoroughly go through the research materials and they have no conflict of interest.
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