Coccidiosis is a difficult to control disease in poultry, because of the involvement of many species and their capability to become quickly resistant to anticoccidials. Pleurotus ostreatus is a medicinal mushroom. Anticoccidial effect of whole Pleurotus ostreatus (Fr.) Jacq. ex (Pleurotaceae) aqueous extract was investigated in vivo against Eimeria spp. Ninety six day-old broilers naturally infected with Eimeria spp was divided into 8 groups of 12 birds. Group A was infected untreated (negative control), group B was treated with Toltrazuril (positive control) while groups C-H were gavaged for 5 days with graded doses of Pleurotus ostreatus extract at 100, 200, 300, 400, 500 and 600 mg/kg, respectively. Body weight, oocyst output per gramme of faeces, lesion score, faecal score, hematological parameters and leucocytes differential counts, were assessed. The ED50 was determined using sigmoidal dose response curve. The acute toxicity study showed aqueous extract of P. ostreatus to be non-toxic even at high dose of 600 mg/kg. Groups B and G had highest average weight compared with the infected untreated group. Groups B, G and H significantly (P<0.05) reduced oocyst output compared with the untreated group. The therapeutic best-fit ED50 value for the extract was 417.3 mg/kg. Eosinophils counts decreased significantly (P<0.05) compared with infected untreated group. Packed cell volume slightly increased in all treated groups but the difference was not significant (P>0.05) compared with the untreated control. Pleurotus ostreatus therefore could be a potential source of new anticoccidial drugs which could find application in the control of avian coccidiosis.
Keywords: Avian coccidiosis; mushroom extract; phytomedicine; broiler
Introduction
Commercial poultry production is expanding geometrically every day and this is enhancing the provision of affordable and high quality proteins (Ghafoor et al. 2010). However, poultry farming is still faced with many gastrointestinal diseases like coccidiosis which are preventing its growth (Hafez 2011). The coccidial parasites develop and multiply in the intestinal epithelium causing tissue damage, blood loss, reduced feed consumption, poor absorption of nutrients, dehydration, retarded growth and death (Seifert, 2006). Anticoccidial drugs and feed additives have been used in the prevention and treatment of the disease but emergence of drug resistant strains of Eimeria and presence of drug residue in animal products have aggravated the problem (Abbas et al. 2011). Another effective control method of coccidiosis is the use of live Eimerial oocysts as vaccine (Shirley and Lillehoj 2012), however, use of live vaccine under poor production management system in broiler chickens may cause negative adverse effects resulting reduced flock performances. (Chapman 2000). As a result of the drawback in using live Eimerial oocysts as vaccine, attenuated vaccine containing Eimerial oocysts with mild infective ability have been developed, however, the cost of production is high and variation in Eimeria species and strains in different climatic regions is another bottleneck, rendering vaccine strains that stimulate immune protection in a zone ineffective in other domains. Due to the emergence of drug resistance and the disadvantages connected with vaccines, poultry farmers worldwide are depending on alternative method of control of poultry coccidiosis. Therefore, herbs and herb extracts may be highly beneficial as alternative natural medicines which contain substances of which resistance has not been found. Researchers have also been working on cheaper, non-toxic and effective treatment options for chicken coccidiosis (Zaman et al. 2011). In the light of this, more investigations are geared toward plants and their extracts for prophylaxis purposes and control of intestinal diseases caused by parasites. (Jung et al. 2011; Badar et al. 2011).
Mushrooms have been used because of their medicinal properties for five millennia (Borchers 1999). Glucans and proteoglycans found in mushrooms are potent sources of gastrointestinal, biologically active immune stimulants which concurrently produce enhancing effect on all important parts of immune system. Pleurotus species are called ‘Oyster’ mushrooms. Pleurotus ostreatus proximate analysis showed presence steroidal glycosides, carbohydrates, terpenoids and tannin in small amounts, whereas, cynogenic glycosides, alkaloids and flavonoids were absent (Iwalokun et al. 2007). P. ostreatus extracts were found potent against 89.8% Saccharomycee cerevisae (10.5 – 10.8 mm), Bacillus subtilis (7.6 – 7.8 mm) and Escherichia coli (7.6 – 8.2 mm) producing greatest effects against fungi, gram +ve and –ve organisms through agar well diffusion (Iwalokun et al. 2007). Phenolic content of P. ostreatus extract was reported high (24.012 mg AE/g) when methanol was used as solvent but low in aqueous extract (16.5468 mg GAE/g) with potent antioxidant attributes. Investigation with mushroom extracts has been found active in removing free radicals (Sanjay et al., 2015). In a study, Allen et al. (1998) discovered that compounds with antioxidant decreased the undesirable effect of E. tenella by improving the level of lipid peroxidation in the intestine. Report by Santos-Neves et al. (2008) indicated that Pleurotus species are consumable and are planted by commercial farmers all over the world. . In Nigeria, Pleurotus species has been used by traditionalists to cure constipation, fever, cold, stomach ache and headache (Akpaja et al. 2003; Ayodele et al. 2009). More recently, Ademola and Odeniran (2016) reported trypanocidal effect of Pleurotus sarjo-caju aqueous extract in mice infected with trypanosoma congolense. It has been reported that host animals treated with mushroom polysaccharides possess strong immunity against microorganisms like bacteria, fungi, viruses and parasites by promoting lysosomal enzyme operation, phagocytosis and interleukin-1 stimulation (Estrada et al. 1997). This study investigated the anticoccidial efficacy of Pleurotus ostreatus aqueous extract against Eimeria spp. in broiler chickens.
Materials and methods
Extraction of mushroom
Fresh cultivated Pleurotus ostreatus mushrooms were obtained from Forest Research Institute of Nigeria (FRIN). The Mushrooms were air-dried at 45ºC in a drier. The dried mushroom was grounded to a fine powder by a milling machine. Extract was prepared by maceration with intermittent shaking in distilled Water with a 10:1 solvent to dry weight ratio (Eloff, 1998). The extract was filtered using Whatman No.1 filter paper and a funnel and extract was administered to birds immediately. The mixture was filtered using a previously weighed filter paper. A brown filtrate was obtained. The residue was dried, weighed again and the difference in weight gave the amount of powdered mushroom dissolved in water (w/v). Appropriate aliquots of the aqueous extract were taken and diluted to prepare graded doses of the extract for the test.
Maintenance of experimental animals
The study was conducted with the permission of the University of Ibadan Animal Ethics Committee (UI-ACUREC/App/2015/056) and in line with the guidelines of the committee. Broiler chicks were purchased from Farm support® limited, Ibadan, Nigeria and fed ad libitum on an anticoccidial free proprietary broiler ration from Premier Feed limited (Top feeds), Ibadan, Nigeria. Water was given ad libitum. The chicks were raised on used deep litter system from a day old at the Teaching and Research Farm, University of Ibadan, Nigeria. The chickens were routinely vaccinated against Newcastle and Gumboro diseases. No antibiotic was administered throughout the study.
Toxicity test
Preliminary acute toxicity study was conducted by using 24 one-day-old broiler chickens that were divided into six groups of four chickens each. Each bird in groups A–F was individually gavaged orally with the graded doses (100, 200, 300, 400, 500 and 600 mg/kg body weight) of aqueous extract of Pleurotus ostreatus. The chickens were observed for 24 hours for any signs of toxicity (eye blinking, panting, lethargy, salivation and incoordination), including change in behaviour or death.
Parasite
Birds were naturally infected with mixed field Eimeria species. The field isolates contained the following species (differentiated on the basis of oocyst morphology, site of colonisation, pathology and clinical signs): Eimeria tenella, Eimeria acervulina, Eimeria necatrix, Eimeria maxima and Eimeria brunetti.
Experimental design
A total of 96, one-day-old Arbor Acres breed of broiler chickens of both sexes were reared as a single group and were adequately infected with field strains of Emeria spp. by 28 day of age. The birds were randomly assigned to 8 treatment groups (A–H) according to the oocyst level in faeces which ranged from 50,416 to 54,367 and faecal samples were assessed in triplicate for the number of oocyst per gram (OPG) of faeces prior to treatment. Group A was the untreated infected group (negative control), group B (positive control) was treated with toltrazuril (Keprocox® 2.5% ORAL, Batch No. 13G919) at a dose rate (25 mg/Kg) prescribed by manufacturer, groups C–H were gavaged with graded doses of P. ostreatus aqueous extract (100, 200, 300, 400, 500 and 600 mg/kg) from day 29 to day 33 (5 days treatment duration). Koinarski et al. (2005) estimated the period of oxidant insult induced by the coccidian parasites to be five days; hence the birds were treated for five days.
Measurement
Birds from each treatment group were weighed individually on days 28/0 (pre-treatment), 32/4 (mid-treatment), and 35/7 (post-treatment) to determine the mean body weight. Pooled daily oocyst counts per group were undertaken. Individual oocyst counts were not feasible because the birds were housed in groups. A total of 10 g of faeces was collected daily beginning from day 28/0 to 35/7 post-treatment. The modified McMaster technique as described by Vassilev (2002) was used to estimate oocyst per gram of faeces. This was repeated three times and the mean value calculated. The method described by Youn and Noh (2001), was used to determine the extent of bloody diarrhoea daily from day 28/0 to 35/7 of age/treatment by assigning it one of five levels, from 0(-) to 4(++++). Zero was the normal status, while 1, 2, 3 and 4 represented less than 25%, 26-50%, 51-75% and over 76% bloody faeces, respectively.
Two broilers per group were randomly euthanized on day 28/0 (pre-treatment), day 32/4 (mid-treatment) and day 35/7 (post-treatment) and subjected to necropsy. Any coccidial lesions found in the intestine were scored from 0 to 4. Zero was no lesion status, while 1, 2, 3 and 4 represented small scattered petechial haemorrhages, numerous petechial haemorrhages, extensive haemorrhages and extensive haemorrhage that gives a dark colour to the intestine respectively, as described by (Johnson and Reid 1970). A score was assigned to each category and a mean calculated from the individual scores to give a global lesion score.
Blood samples (2 mL) were collected from the jugular veins of three chickens in each group on day 28/0, 32/4 and 35/7 post-treatment. The packed cell volume (PCV), red blood cells (RBC), total white blood cell (TWBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), Haemoglobin (Hb) concentration and mean corpuscular haemoglobin concentration (MCHC) were determined using Auto haematology analyzer (Minray BC-2800). Differential leucocyte counts were determined by microscopic examination of Giemsa stained blood smear (Jain 1986).
Statistical analysis
The bioactivity of various doses of the extract and the positive and negative controls were assessed by comparing the oocyst reduction values by one-way ANOVA and Tukey’s multiple comparison test. Weight gains and haematological parameters for each group for the different time points were also compared using one-way ANOVA and Tukey’s multiple comparison tests to ascertain if weights differed significantly over the period. All data analyses were performed using GraphPad Prism version 5.0 for Windows (San Diego, CA). Sigmoidal dose response curve was used to determine the ED50 of P. ostreatus aqueous extract using this formula:
Parasite survival on the 3rd day post-treatment =
(Initial number of oocyst on 1st day in treatment/Number of oocyst after treatment on 3rd day)/(Initial number ofoocyst in negative control/Number of oocyst on 3rd day in negative control)
Results
Yields of extract
The aqueous extract of Pleurotus ostreatus gave a yield of 14.77 g (11.36% w/w).
Acute toxicity study and effect of aqueous extract on weight.
In vivo preliminary acute toxicity study showed that aqueous extract of P. ostreatus was non toxic (no eye blinking, panting, lethargy, salivation, incoordination or death in any of the treated groups of broiler chickens) including the group treated with the highest dose (600 mg/kg body weight).
Anticoccidial effect of Pleurotus ostreatus extract on oocyst output and ED50
Administration of P. ostreatus aqueous extract on groups G (500 mg/kg), H (600 mg/kg) and Toltrazuril (25 mg/kg) on group B produced significant Eimeria oocyst reduction (P<0.05) on broilers infected with mixed species of Eimeria when compared with group A untreated. The Eimeria oocysts output level in untreated group A remains very high although, gradual reduction in oocyst output was observed during the course of the study. Oocyst reduction in group B treated with Toltrazuril was significant and parasite clearance occurred 48 hours post-treatment. There was parasites clearance in Groups H and G on day 4 and day 5 post-treatment, respectively. Anticoccidial effect of the extract on groups C – F was dose-dependent (Figure 1). The statistical parameter of the curve fitting analysis and the best-fit ED50 values for the aqueous extract was 417.3 mg/kg (Figure 2).
Weight gain
Changes in the body weight of broiler chickens infected with species of Eimeria pre- and post-treatment are shown in Figure I. The weight in all the groups treated with the extracts and Toltrazuril increased compared with the untreated control. However, there was no significant difference (p˃0.05) between the group treated with 600 mg/kg BW of extract of P. ostreatus and the other treated groups. The weight of broilers treated with Toltrazuril (25 mg/kg) was slightly higher than the groups treated with aqueous extract P. ostreatus (Figure 1).
Faecal score
Bloody diarrhea (51-75%) was observed in all treatment groups (A-H) on day 28 pre-treatment. Group A which was untreated passed out bloody diarrhoea consistently throughout the study. The faeces in Groups B (toltrazuril 25 mg/kg), G (500 mg/kg extract) and H (600 mg/kg extract) were observed to be normal on day 3, 5 and 4 post-treatment, respectively (Table 1).
Lesion score
The lesion scores showed numerous petechial haemorrhages to extensive haemorrhages in the intestines in all the groups (A-H). Necropsy findings showed that the broilers were infected with Eimeria tenella (caecal lesions), Eimeria acervulina, E. Maxima and Eimeria praecox (duodenal and jejunal lesions). The untreated group (A) showed numerous petechiae haemorrhages in the duodenal and caecal surfaces (both serosal and mucosal) in pre-treatment, mid treatment and post treatment. Groups B treated with 25 mg/kg body weight toltrazuril and H treated with 600 mg/kg aqueous extract of P. ostreatus reduced lesions remarkably (Table 2).
Haematology
The mean PCV, Hb, WBC and RBC are shown in figure 3. The haematological values of broilers bear no direct relationship with the treatments as the values demonstrated considerable differences characterized by wide range values showing no significant difference (P>0.05) in all treated groups compared with untreated control. The PCV values obtained ranged 29.7-38.3%; Hb, 8.5-8.7g/dl; RBC, 1.9-2.7×1012/L and WBC, 189-233×109/L. However, packed cell volume (PCV) in all treated groups was slightly higher on day 4 post-treatment (33-38%) and decreased slightly (30.3-33.0%) on day 7 post-treatment compared with pre-treatment (29.7-34.7%) (Figure 4). Differential counts (Neutrophils, Lymphocytes, Monocytes, Basophils and Platelets) of broiler chickens naturally infected with mixed Eimeria species were not significantly different (P>0.05) in all treated groups when compared with untreated group, although eosinophil counts was significantly (P<0.05) higher in broilers treated 600 mg/Kg P. ostreatus aqueous extract post treatment (Figure 5).
Discussion
For many decades, conventional coccidiosis control methods have relied solely on anticoccidial chemical compounds and live vaccines which have played effective role in the control of the disease, but, their continuous administration has culminated in the development of drug resistant strains of Eimeria and an increasing concern over the presence of drug residues in animal products. These drawbacks have led to development of a kin interest in alternatives in the form of plant extracts containing many compounds which are safe, effective and cheaper (Abbas et al. 2012; Ademola and Odeniran 2016). Therefore, this study aimed to evaluate the anticoccidial activity of aqueous extract of P. ostreatus against field strains of Eimeria species of broiler chickens model.
Acute toxicity study of P. ostreatus aqueous extract proved to be safe and without any side effect at highest dose which could be the reason it is consumed by people of different races since ancient time. Harkonen (1998) showed that oyster mushroom was found to be non-toxic at high therapeutic dose in animals. Al-Deen et al. 1987 and Monika et al. 2006 reported LD50 of P. ostreatus to be 319 mg/Kg for oral route and 1143 mg/Kg for intraperitoneal route, respectively. Pleurotus ostreatus aqueous extract was shown in vivo to demonstrate anticoccidial activity in a dose-dependent manner in this study. The aqueous extract of P. ostreatus demonstrated an ED50 value of 417.3 mg/Kg on day 3 of post-treatment. The therapeutic potency of 600 mg/Kg extract in broilers infected with field strains of Eimeria species was 100% on the day 4 post-treatment and birds remained Eimeria-free throughout the experiment, while the group treated with toltrazuril demonstrated oocyst clearance on day 2 post-treatment. Allen et al. (1998) demonstrated that herbs with antioxidant components possess therapeutic activities against coccidial parasites. Tukey’s multiple comparison tests (post-ANOVA) revealed the relative Eimeria parasites level at different doses and level of significance within the groups. The significant (P<0.05) oocyst reduction in group H when compared with untreated group A could be due to P. ostreatus strong antioxidant components. Aqueous and methanolic extracts of P. ostreatus possess phenolics compounds with strong antioxidant activity (González-Palma et al., 2016). Rizwan et al (2016) reported that β-glucans derived from wheat had immunostimulatory effects on the broiler chickens with enhanced cellular and humoral immune responses. The β-glucans also had protective effects against Eimeria infection and significantly improved weight gains of chickens with few lesions and oocyst score. Fruit bodies of P. ostreatus contain branched β-1,3-1,6-glucan and linear a-1,3-glucan as the major components of cell walls (Synytsya et al. 2008). Hence, the anticoccidial effect observed could be due to the β-glucan in P. ostreatus. The difference in oocyst reduction time between toltrazuril and aqueous extract of P. ostreatus could probably be due to the components with anticoccidial properties that have not been separated and purified compared to synthetic drug where chemical compounds are purified. In a similar report, Hossain et al., (2013) reported that supplementation of 100 – 150 mg/Kg oyster mushroom in feed for extended period of time reduced the development of Eimeria tenella in broiler chickens. These results is similar to the report of Ahad et al. (2016) who reported the efficacy of aqueous extract of Ganoderma applanatum mushroom against Eimeria species oocysts and demonstrated that treatment with aqueous extract of G. applanatum produced a noticeable reduction in Eimeria oocysts output.
The effectiveness of anticoccidia can further be evaluated from weight measurements of the chickens. Birds treated with the aqueous extract of P ostreatus, on average, had improved body weight at comparable levels to pre-treatment values which could be due to its anticoccidial, anti-stress, appetite stimulating and immune enhancing abilities even though the difference is not significant (P>0.05), while those in the untreated infected group demonstrated progressive reduction in body weights throughout the study. The reason might be due to the damaging effect of coccidial parasites in the intestine leading to loss of appetite and poor absorption of nutrients. In a related study, Dalloul et al. (2006) showed that lectin extract obtained from Fomitella fraxineo mushroom may be used as an effective growth enhancing and immune stimulating substance in Eimeria infected chickens. In another report, Ogbe et al., (2009) demonstrated in vivo anticoccidial effect of Ganoderma lucidium mushroom resulting in significant reduction in number of E. tenella oocyst shed in faeces and increased weight gain.
During necropsy, haemorrhagic lesions reduced in all treated groups especially at high dose of P. ostreatus extract. This might be as a result of reduction in the number of Eimeria sporozoites causing the lesions. Shaghun et al., (2014) reported wound healing activity of P. ostreatus in normal kidney cell lines. Untreated group was observed to retain numerous petechial haemorrhages throughout this study which may be due to the damaging effect of the coccidial parasites. The lesions observed during necropsy showed that broilers were infected with Eimeria acervulina, Eimeria maxima, Eimeria praecox (affecting upper intestine) and Eimeria tenella (caeca). The reduction in the number of sporozoites causing damages to the intestinal epithelium was responsible for the decrease in bloody diarrhoea in groups treated with extract. On the day 4 post-treatment bloody diarrhoea was absent in the group treated with 600 mg/Kg body weight of the aqueous extract. The haematological parameters of broiler chickens naturally infected with Eimeria species were not significantly (P>0.05) different in all treated groups but there was slight increase in packed cell volume. This may be due to reduction in number of sporozoites causing blood loss from the epithelial lining of the intestine and could also be due to strong amino acid constituents of P. ostreatus aqueous extract. Toghyani et al., (2012) reported in their study that supplementation of broiler diet with Oyster mushroom powder had no significant effect on haematological parameters when used as growth promoter. The absence of anaemia in the birds could be due to the compensatory increase in absorption of nutrients in greater parts of Jejunum and Ileum that were not infected. There was significant (P<0.05) reduction in eosinophil count of infected broilers treated with the extract. The reason could be because P. ostreatus possesses immune stimulating ability. Rose (1996) demonstrated that macrophages, dendritic cells, mast cells, natural killer cells, basophils and eosinophils were involved in the innate immune response to Eimeria infection. Other differential white blood cell count in pre-treatment, mid-treatment and post-treatment were not significantly different. Lymphocytosis was observed before the commencement of treatment and the groups treated with higher doses of the extract showed significantly lowered oocyst output which seemed to prevent lymphocytosis, which suggests that this phenomenon is a result of parasite load. Some authors have shown that mushrooms contain polysaccharides which can participate in stimulating the activities of various interdependent cell types such as T and B-lymphocytes, macrophages, natural killer (NK) cells, inducing secretion and production of cytokines and complements of T-cells and NK cells (Wang et al. 1997; Guo et al. 2003), which can be used as anticoccidia.
In conclusion, the aqueous extract of Pleurotus ostreatus mushroom demonstrated anticoccidial activity as observed in the inhibition of Eimeria oocyst output. Therefore, the use of the extract may be an effective alternative medicine for the treatment of coccidiosis in poultry production. Further studies should be carried out to determine the mechanism of action of Pleurotus ostreatus extract. Bioactivity guided fractionation could also give a better understanding of the active component(s) responsible for anticoccidial and immune enhancing activity.
Conflict of interest statement
The authors state no conflicts of interest. The authors are responsible for the content and writing of this article.