Antimicrobial, immunomodulatory and hepatomodulatory effects of aqueous extracts of Petiveria alliacea root and leaf on growing pullets

Aim of study: To evaluate antimicrobial, immunomodulatory and hepatomodulatory effects of aqueous extracts of Petiveria alliacea L. (Guinea hen weed) root and leaf on growing pullets. Area of study: Abeokuta, Nigeria. Material and methods: Two hundred and eighty eight eight-weeks-old Isa-brown pullets were divided into eight treatments which consisted in birds administered root extracts or leaf extracts at four different concentrations (0, 15, 30 and 45 ɡ/L). Each treatment was replicated three times with 12 pullets per replicate. Main results: Oocyst counts was lower (p<0.05) in pullets administered root extract (78.42 opɡ) compared with leaf extract (103.58 opɡ). Oocyst counts was lower (p<0.05) in pullets administered 30 and 45 ɡ/L root or leaf extract compared with other treatments. Bacteria counts reduced (p<0.05) in pullets administered root extract compared with leaf extract. Pullets administered 45 ɡ/L root extract recorded lowest (p<0.05) bacteria count. Bursa and thymus weights increased in pullets administered root or leaf extract compared with the control. Pullets administered 30 and 45 ɡ/L root or leaf extract had highest (p<0.05) antibody titre against Newcastle disease vaccine 8.80, 8.86, 8.74 and 8.80 (log2) respectively. There was fatty infiltration in liver of control birds, while hepatocytes appeared normal in liver of pullets administered P. alliacea extracts. Research highlights: P. alliacea root and leaf extracts at 45 ɡ/L performed best as antimicrobial, immune-stimulating and hepato-protective agent in pullets. This study highlights P. allicaea as a valuable antimicrobial and immunostimulating agent in poultry production. Additional key words: oocyst counts; bacteria count; liver histopathology; herbal plants; poultry production; Guinea hen weed Abbreviations used: HI (haemagglutination inhibition); NDV (Newcastle disease vaccine); Authors’ contributions: Performed the experiments and wrote the paper: AMO. Supervised the work: OAA, LTE and RAS. All authors conceived and designed the experiments, analyzed the data and participated in critical revision of the manuscript. Citation: Oyeleke, AM; Adeyemi, OA; Egbeyale, LT; Sobayo, RA (2021). Antimicrobial, immunomodulatory and hepatomodulatory effects of aqueous extracts of Petiveria alliacea root and leaf on growing pullets. Spanish Journal of Agricultural Research, Volume 19, Issue 1, e0502. https://doi.org/10.5424/sjar/2021191-17300 Received: 31 Jul 2020. Accepted: 22 Feb 2021. Copyright © 2021 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Funding: The authors received no specific funding for this work Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Adetola M. Oyeleke: oyeadetola@outlook.com Spanish Journal of Agricultural Research 19 (1), e0502, 10 pages (2021) eISSN: 2171-9292 https://doi.org/10.5424/sjar/2021191-17300 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) OPEN ACCESS RESEARCH ARTICLE


Introduction
Commercial layer enterprise is a common subsection within Nigeria poultry industry. The productive performance of commercial layers to a great extent depends on health management during their growing phase (Olawumi, 2011). Pullets raised in proper health condition tends to reach and maintain optimal productive performance during their laying phase while pullets raised with health challenges may be inconsistent in production or become unproductive at the laying phase (Olawumi, 2011). Many health challenges encountered in poultry production arise from bacteria and coccidial infections (Adene, 1996).
Bacterial infection is of great economic importance in poultry production. Many poultry diseases result from colonization of the gut by harmful bacteria which are often characterized by high bacteria load (Albazaz & Buyukunal Bal, 2014). Coccidiosis is a crucial and common disease in poultry production (Ola-Fadunsin & Ademola, 2014). It is characterized by invasion of the gut by Eimeria parasites leading to tissues damage, haemorrhage, weight loss and death (Ola-Fadunsin & Ademola, 2014;Eke et al., 2016). Bacterial infections and coccidiosis significantly increase cost of production and causes huge economic loss in poultry industry (Ganguly & Praveen, 2016;Lawal et al., 2016). Overtime, these diseases have been prevented by the use of various conventional antibiotic and anticoccidial drugs (Aarestrup & Jenser, 2007;Thangarasu et al., 2016).
Emergence of drug resistant bacteria and harmful drug residue in animal products has led to restriction in use of conventional drugs (Diarra & Malouin, 2014;Dhama et al., 2015;Gonzalez-Ronquillo & Angeles-Hernandez, 2017). Moreover, there is increasing public awareness on harmful drug residue in animal products and necessity for food safety, leading to increase in demand for organic-based products (Biswas et al., 2010). These developments necessitate search for viable substitutes to conventional drugs for growth promotion, prophylactic, therapeutic and immune enhancement purposes, readily available at low cost without detrimental effects.
An alternative gaining considerable recognition is the use of medicinal plants which are residue-free, readily available and potent against diseases (Diaz-Sanchez et al., 2015). Several herbal materials have been studied for their prospective benefit as growth-promoting, prophylactic, therapeutic and immune-enhancing agents. Many of these studies have reported positive and promising results (Molan & Faraj, 2015;Nghonjuyi, 2015;Attia et al., 2017a,b;Navidshad et al., 2018). However, the use of medicinal plants in poultry production has not been fully established and investigation on herbal materials is still in progress in order to fully explore the benefits of available medicinal plants to the poultry industry.
Petiveria alliacea L. is a potent herb whose medicinal benefits to the poultry industry have not been explored. P. alliacea belongs to the family Phytolaccaceae, the herb is native to North, Central and Southern America and the Caribbean with wide spread introduced population in Nigeria (Schmelzer & Gurib-Fakim, 2008; USDA-ARS, 2020). The plant has been used successfully in folk medicine to treat various ailments and conditions (Brown, 1995;Randle et al., 2018). Scientific investigations showed that the plant possess antibiotic, anti-oxidant, anti-carcinogenic, anti-cancerous, anti-inflammatory and immuno-modulatory properties (Kim et al., 2006;Williams et al., 2007;Santander et al., 2012;Pacheco et al., 2013;Ekunseitan et al., 2016;Oluwa et al., 2017). Some researchers (Sobayo et al., 2017(Sobayo et al., , 2018aMuhammad et al., 2019;Odetola et al., 2019) have investigated the growth and performance enhancement abilities of P. alliacea on poultry species. However, there is no substantial information on therapeutic benefits of the plant to poultry species. Hence, this study investigated the antimicrobial, immunomodulatory and hepatomodulatory effects of aqueous extracts of P. alliacea root and leaf on growing pullets.

Material and methods
This experiment was carried out according to animal welfare guidelines of Animal Welfare Committee, Colle-ge of Animal Science and Livestock Production, Federal University of Agriculture Abeokuta, Nigeria. The study was carried out at the research unit of Livelihoods Support and Development Centre (SLIDEN AFRICA), Abeokuta, Ogun State, Nigeria.

Extraction of Petiveria alliacea root and leaf
Aqueous extraction was carried out according to the method outlined by Nodu et al. (2016). Fresh roots and leaves of P. alliacea were sourced from Kotopo area of Abeokuta, Ogun State, Nigeria. The roots and leaves were rinsed in clean water to remove adhering dirt. Using a sensitive weighing scale, 15, 30 and 45 ɡ samples of fresh roots and leaves were taken and each sample was blended separately in 1-L of clean water. Each blended mixture was separated by filtration using 0.1 mm sieve screen. The root and leaf particles were discarded while filtrate from each mixture was presented to experimental birds as drinking water according treatment.

Experimental management
A total of 288 eight-weeks-old Isa brown pullets were used for this study which lasted for 10 weeks. The pullets were arranged in a 2×4 factorial experimental layout in a completely randomised design. There were eight experimental treatments replicated three times with 12 birds per replicate. The treatments consisted in birds administered aqueous extract of P. alliacea root at 0 ɡ/L (control), 15 ɡ/L, 30 ɡ/L and 45 ɡ/L concentrations and leaf at 0 ɡ/L (control), 15 ɡ/L, 30 ɡ/L and 45 ɡ/L concentrations. The extracts were presented to pullets via drinking water on two consecutive days per week throughout the experiment. Extracts presented met and exceeded daily water requirement of the birds on all occasions of administration. Experimental birds were raised in a deep litter system and fed grower mash diet formulated according to National Research Council recommendation (Table 1). Pullets in all treatments were free of antibiotic and anticoccidial drugs throughout the experiment.

Data collection
Fourteen days prior to the end of the experiment, four birds were randomly selected from each replicate of all treatments. These birds were housed in a separate cage on treatment and replicate basis and administered Newcastle Disease Vaccine (NDV) lasota strain via drinking water for future serum antibody test. Routine administration of P. alliacea extracts was maintained Growing pullets administered aqueous extracts of Petiveria alliacea Faecal Eimeria oocyst count Anticoccidial effect was estimated by Eimeria oocyst counts in faeces of experimental birds. At the end of the experiment (18 weeks of age), dropping trays covered with aluminium foil were placed on the floor of each rearing unit. Fresh faecal droppings were picked from each replicate of all treatments into separate labelled sterile bottles using sterile forceps. The bottles were placed in ice and transported to the laboratory for egg count analysis using McMaster egg counting technique as described by Zajac & Conboy (2012).

Intestinal total bacteria count
Antibacterial effect was estimated by evaluating intestinal total bacteria count in experimental birds. At the end of the experiment, live bodyweights of birds selected earlier were determined after which they were slaughtered. Small intestines of the slaughtered birds were carefully isolated from the carcass and placed in separate labelled sterile bottles. The bottles were placed in ice and transported to the laboratory for bacterial count. Total bacteria count was carried out on mixed digesta from the small intestine using viable count method as described by Bassiri (2013). The colony-forming units (cfu) were expressed as logarithm to base 10.

Lymphoid organ weight and serum antibody against Newcastle disease vaccine
Immunomodulatory effect was estimated by evaluating weight of lymphoid organs and serum antibody titre against NDV.
Lymphoid organs (bursa, thymus and spleen) were carefully isolated from the carcass of slaughtered birds, weighed using a sensitive weighing scale and expressed as percentage of live bodyweight.
Serum antibody titre against NDV was determined by haemagglutination inhibition (HI) test. Blood (2 mL) was collected aseptically from each slaughtered bird into labelled plain bottles. The blood samples were allowed to clot and the sera separated into different labelled sterile bottles. The samples were transported to the laboratory for HI test using the method described by OIE (2008). The HI titre values were expressed as logarithm to base two.

Liver weight and histopathological examination
Hepatomodulatory effect was evaluated by liver weight and histopathological examination. Livers of slaughtered birds were weighed using a sensitive weighing scale and expressed as percentage of live bodyweight. The livers were fixed in 10 % formalin solution and processed for histopathological examination according to Slaoui & Fiette (2011). Hepatic changes were viewed with a BA410E Elite Research Compound Microscope and photomicrograph taken with aid of an Amscope MU900 digital camera.

Statistical analysis
The experiment was arranged in a 2×4 factorial experimental layout in a completely randomized design. Data collected were subjected to 2-way Analysis of Variance, significant (p<0.05) differences between treatment means were determined using Duncan Multiple Range Test as contained in Statistical Analysis Software (SAS, 2010) package.
The model of the study was: where Y ijk = output parameter, µ = overall mean, T i = i th effect of plant part (i = root, leaf), L j = j th effect of concentration of extraction (j = 0 ɡ L -1 , 15 ɡ L -1 , 30 ɡ L -1 , 45 ɡ L -1 ), (TL) ij = the interactive effect of plant part and concentration of extraction, k = k th observation in a treatment (i, j), and  ijk = residual error. Table 2 presents the main effects of the aqueous extracts of P. alliacea parts (root and leaf) and concentration of extraction on faecal Eimeria oocyst counts and intestinal total bacteria counts in growing pullets. Faecal Eimeria oocyst count was significantly (p<0.05) lower in birds administered root extract (78.42 opɡ) compared with bird administered leaf extract (103.58 opɡ). Intestinal total bacteria count was reduced (p<0.05) by administration of root extract compared with leaf extract. Furthermore, faecal Eimeria oocyst count reduced significantly from 197.50 opɡ in the control treatment to 21.33 opɡ at 45 ɡ/L concentration of extraction. Intestinal total bacteria count reduced significantly as concentration of extraction increased. Table 3 presents the interactive effect of aqueous extracts of P. alliacea parts (root and leaf) and concentration of extraction on faecal Eimeria oocyst counts and intestinal total bacteria counts in growing pullets. Eimeria oocyst counts were significantly (p<0.05) lower in pullets administered 15, 30 and 45 ɡ/L root and leaf extract than values obtained in the control treatments. Pullets adminis-tered 45 ɡ/L root extract and 45 ɡ/L leaf extract recorded lower (p<0.05) intestinal total bacteria counts (0.83 and 1.20 log10 (cfu/ɡ) respectively) than the control treatments (1.83 and 2.00 log10 (cfu/ɡ) respectively). Table 4 presents the main effects of aqueous extracts of P. alliacea parts (root and leaf) and concentration of extraction on liver weight, lymphoid organ weights and serum antibody against NDV. Bursa weight was significantly (p<0.05) higher in pullets maintained on 15, 30 and 45 ɡ/L concentrations than value obtained in the control treatment. The highest (p<0.5) thymus weight (0.48 %) was recorded in pullets maintained on 45 ɡ/L concentration while the lowest (0.37 %) was recorded in the control treatment. Spleen weight was higher (p<0.05) in pullets maintained on 45 ɡ/L concentration (0.15 %) compared with the control treatment (0.12 %). Serum antibody titre against NDV increased (p<0.05) from 7.05 (log2) in control birds to 8.83 (log2) in birds maintained on 45 ɡ/L concentration. Table 5 presents the interactive effect of aqueous extracts of P. alliacea parts (root and leaf) and concentration of extraction on liver weight, lymphoid organ weights and serum antibody against NDV. Bursa weights were statistically similar in pullets administered 15, 30 and 45 ɡ/L of P. alliacea root and leaf extracts but the values were higher (p<0.05) compared with the control treatments. Thymus weight was significantly (p<0.05) higher in pullets administered 15, 30 and 45 ɡ/L root extract and 30 and 45 ɡ/L leaf extract compared with the control treatments. Serum antibody titres against NDV were statistically similar in pullets administered 30 and 45 ɡ/L concentration of

Discussion
Reduction in Eimeria oocyst counts in faeces of pullets administered extracts of P. alliacea root and leaf at various concentrations indicated that the plant is capable of inhibiting Eimeria parasite replication in growing pullets. Several bioactive compounds effective against parasitic cells have been isolated from P. alliacea (Ekunseitan et al., 2016). Saponin which was found in the plant was reported to cause death of parasitic cells by disrupting their cellular structure (Wang et al., 1998). Alkaloid present in the plant was reported to prevent parasitic cell replication by damaging their DNA sequence leading to eventual cellular death (Wink, 2012). Sulphur compound found in garlic (allicin), which is similar to sulphur compound isolated from P. alliacea (Randle et al., 2018), has been reported to be toxic to a wide range of protozoan parasites (Al-Snaf, 2016). Moreover, Ekunseitan et al. (2016) reported the presence of tannins, flavonoids and phenolics in P. alliacea; these compounds were identified as strong antioxidants (Balan et al., 2018) which help in moderating lipid peroxidation processes within the gut, thereby reducing the intensity of Eimeria infection (Allen et al., 1998). These biological processes could have inhibited the Eimeria parasite's replication. Further reduction in oocyst count at higher concentrations of extraction indicated that Eimeria parasite replication is responsive to the concentration of phytochemicals present in the extracts administered to pullets. Lower faecal oocyst count recorded in pullets administered root extract compared with leaf extract suggested that the bioactive compounds are present at higher concentration in the root compared with the leaf. Although information about the influence of P. alliacea on Eimeria parasites in chickens is not available in literature, similar results were reported in studies involving other medicinal herbs. Combined aqueous extracts of Azadirachta indica A. Juss. and Khaya senegalensis (Desr.) A. Juss. reduced faecal oocyst count in broiler birds infected with Eimeria species in a dosage dependent manner (Gotep et al., 2016  oocyst shedding in broiler chickens infected with Eimeria parasite and treated with acetone extract of Moringa oleifera Lam. leaf. This study confirmed the antibacterial activity of P. alliacea by reduction in intestinal total bacteria count observed in pullets administered aqueous root and leaf extracts. This antibacterial activity can be attributed to the presence of various bioactive compounds identified as potent antibiotic agents in the plant. Ekunseitan et al. (2016) and Adesipo et al. (2017) isolated phytochemicals such as terpenoids, flavonoids, tannins, alkaloids, phytate, phenols, saponin, oxalate, carotenoids and sulphur-containing compounds from P. alliacea. The sulphur-containing compounds (benzyl-containing thiosulfinates) isolated from the plant have been reported to exhibit strong antimicrobial ability and as the major constituent contributing to the antimicrobial property of the plant (Kim et al., 2006;Randle et al., 2018). Moreover, flavonoids, tannins, alkaloids and phenols were reported to cause death of microbial cells by damaging their cellular structure (Cowan, 1999;Barbieri et al., 2017). The concentration-dependent reduction in intestinal bacteria count signified further suppression of bacteria growth and replication when higher concentrations of the bioactive compounds were administered. Lower intestinal bacteria count in pullets administered root extract compared with leaf extract implied that antimicrobial compounds were present in the root at higher concentration compared with the leaf. This corresponds with the finding of Kim et al. (2006), who stated that precursors (cysteine sulfoxides) to the major antimicrobial compound (dibenzyl trisulfide) in the P. alliacea are present at higher concentration in the root compared with the leaf. No previous study has presented result on antimicrobial effect of P. alliacea on poultry species. However, in an in-vitro study, Ekunseitan et al. (2016) concluded that extract of P. alliacea leaf inhibited some bacteria pathogen important to poultry species. Related results were presented in studies involving herbal materials having similar bioactive compounds as P. alliacea. Dietary supplementation of garlic powder reduced caeca Clostridium perfringens population in broiler chickens (Jimoh et al., 2013). Feeding garlic meal at 1000 mɡ/kɡ inclusion and neem leaf meal at 1500 mɡ/kɡ inclusion reduced intestinal bacteria count in broiler chickens (Sobayo et al., 2015).
Bursa and thymus are primary lymphoid organs in chicken responsible for the production and orientation of immune cells, T-lymphocytes and B-lymphocytes (Teo & Tan, 2007). Spleen is a secondary lymphoid organ useful in filtering out pathogens in the blood (Lewis et al., 2019). Changes in weight of lymphoid organs could affect the ability of animals to maintain productivity during sanitary challenges (Eyng et al., 2015). Increase in lymphoid organ weight correlates with enhanced proliferation of immune cells which represents better immunity (Teo & Tan, 2007). The observed increase in lymphoid organ weights of pullets administered extracts of P. alliacea root and leaf suggested an increase in activities within these organs triggered by the immunomodulatory ability of the plant. It also indicated better proliferation of immune cells thus an enhancement in immune system. The concentration-dependent increase in antibody titre against NDV seen in pullets administered extracts of P. alliacea root and leaf also signified the ability of the plant to strengthen the immune system by increasing antibody production against foreign bodies. The immune-enhancement ability of P. alliacea can be attributed to bioactive compounds present in the plant. Phenols, part of the compounds found in P. alliacea, were found to maintain structural integrity of immune cells (Awaad et al., 2010). Flavonoids and terpenoids enhance humoural and cellular immunity and regulate the endocrine and circulatory markers of health (Abdulkarimi, 2011;Kamboh et al., 2015). Dibenzyl-trisulfide found in P. alliacea was also identified as an immunomodulatory compound (Alegre & Clavo, 2007). Earlier studies found that dibenzyl-trisulfide increased thymic weight in mice and histological analysis on the thymus revealed there was proliferation of cells (Williams et al., 1997(Williams et al., , 2002. In in-vitro and in-vivo studies water extract of P. alliacea was reported to enhance lymphocyte, interferon and interleukin production (Randle et al., 2018). Quadros et al. (1999) also reported an immune-stimulatory effect of P. alliacea on mice infected with Listeria monocytogenes. No literature was found on immunomodulatory effect of P. alliacea on poultry species however, studies on garlic which possess similar bioactive compound as P. alliacea have produced similar results (El-katcha et al., 2016;Gautam et al., 2017).
In the present study, hepatoprotective activity was displayed by P. alliacea by maintaining the structural integrity of hepatocytes. Antioxidant compounds such as flavonoids, saponin, terpenoids and tannins (Zhang et al., 2015) found in P. alliacea were reported to stabilise reactive oxygen species thereby inhibiting oxidative damage and degenerative changes in hepatocytes (Ojo et al., 2015). The result also suggested hypolipidemic effect of the plant considering hepatic fatty infiltration observed in liver of control birds which was absent in P. alliacea treated birds. Flavonoids and saponins found in P. alliacea were reported to prevent excessive fat synthesis by moderating activities of enzymes involved in lipogenesis within the liver (Borradaile et al., 2003;Patel et al., 2012) which might be responsible for the observed hypolipidemic property. However, Odetola et al. (2019) reported that P. alliacea root meal at high supplemental levels (1500, 2000 and 2500 ɡ per 100 kɡ feed) induced hepatocellular necrosis in broiler chickens but these signs were absent at lower supplemental levels (500 and 1000 ɡ per 100 kɡ feed). This means that methods of preparation which can affect stability of active compounds in plants (Poojary et al., 2017) as well dosage of administration can influence hepatoprotective activities of P. alliacea.
In conclusion, 45 ɡ/L concentration of P. alliacea root and leaf extracts performed best as anticoccidial, antibacterial and immune-stimulating agent without impairing liver health. The study encouraged further studies to determine effect of P. alliacea on individual Eimeria and bacteria species affecting poultry birds.