EXPERIMENTAL STUDIES ON COCCIDIA (EIMERIA TENELLA) INFECTED BROILER TREATED WITH AQUEOUS EXTRACT OF OYSTER MUSHROOM  

GHADA A.A.^*; DOAA A.H^**; REHAB R.A.^*** and RANIA I.M.^****

^* Department of Poultry Diseases, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, Egypt, Al-Mansoura Provincial Lab.

^** Department of Mycology, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, Egypt, Al-Mansoura Provincial Lab.

^*** Department of Parasitology, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, Egypt, Al-Mansoura Provincial Lab.

^**** Department of Pathology, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, Egypt, Al-Mansoura Provincial Lab.

Email: dr.ahmedsob7y@gmail.com

INTRODUCTION

Coccidiosis is an acute to chronic infectious disease caused by protozoal parasites of genus Eimeria which multiply in intestinal mucosa of chickens and produce severe tissue damage resulting in bloody diarrhea, reduce growth, weight and increase susceptibility to other pathogens. So, avain coccidiosis is a fatal disease which cost the industry millions of dollars annually (Champman and Shirley 2003, Lilleh et al., 2004). The cost associated with prevention via drugs in the feed, adds to the economic hardship of producer. Moreover, the increased drug resistant problems occurring in poultry production has heightened public concern. In some countries the use of drug in production has been severely regulated or banned altogether there for alternative control methods are being researched with great intensity, one such alternative receiving much interest is mushroom. Mushroom  a natural health promoting fungi such as (Pleurotus  ostreatus) and (Ganoderma lucidumare) used as food supplemments and medicines to improve various parameters of human and animal health and immue function in certain disease conditions (Chang and Mshigeni, 2001, Anthony and Joyce, 2007 and Fasuyi, 2007).

Mushroom like probiotics are natural ingredients that contain bioactive chemical substances or polysaccharides, protein, crude fibers, unsaturated fat, minerals  as (patassium, phosphorus, calcium, iron and zinc), vitamins, essential amino acids and organic acids that can be used as a good source of supplements and medicine to promote health and production (Jang and Briminghan, 1992; Chang and Mshigeni, 2001; Ogbe et al., 2005; Anonymous, 2007; Ezeokeke, 2008 and Selegean et al., 2009).Mushroom stablise microflora in GIT and prevents colonization of host cells by pathogens, also stimulates non specific host immune response or phagocytosis by macrophages (Sundu et al., 2006). There has been a recent upsurge of interest in mushroom not only as a healthy food which is rich in protein (Baross et al., 2008),butalso as a source of biologically active compounds of medicinal value which  includecomplementary medicinal dietary supplements for anticancer (Cheung et al., 2003), antioxidant (Chang and Miles, 2004), antimicrobial (Lindequist et al., 1990), antiviral (Brandt and Piraino, 2000 and Mothana et al., 2003) and anti inflammatory (Kim et al.,  2003  and  2004).

In addition, it has immunopotentiating (Reshetinkov et al., 2001) hypocholestroemic, (Ishikawa et al., 1984) and anticoocidial effect against Eimeria tenella (Ogbe et al., 2009; Naphade et al., 2010; Willis et al., 2012 and Hossain et al., 2013).

Infection with Eimeria is known to stimulate a protective immune response in chicken (Yun et al., 2000).The polysaccharide extract from mushroom (Pleurotus ostreatus) was shown to have immunomodulating effects in chicken (Selegean et al., 2009). Mushroom can reduce Eimeria tenella oocycts output in infected broilers, improve body weight gain and hematological changes that may occur (Ogbe et al., 2009).

This study was carried out to investigate the performance and health promoting effects of aqueous extracts of mushroom (Pleurotus ostreatus) on broiler chicks infected with Eimeria tenella.

MATERIALS and METHODS

1- Experimental birds: One hundred and forty chicks of 14 day age were obtained from a commercial hatchery. The chicks were weighed, divided into 7 groups (20 chicks each) and housed on wire cages labeled 1 to 7. All groups of birds were fed with standard ration.

2- Preparatio n of aqueous extract of Pleurotus ostreatus (according to Ogbe et al., 2009): Mushroom was washed in distilled water, sun dried then ground to powder using a mortar pestle and then blended. The mushroom powder was again sun dried for about 3hours and then stored in plastic polythene bags and kept at room temperature until required. A 20% w/v solution of aqueous extract of mushroom was prepared by soaking in hot water boiled to 100°C for 3 hours brining the concentration to 200mg/ml. The solution was sieved, solid matter discarded and the filtrate allowed to cool to room temperature befor use.

3- Experimental infection and treatment:

1^st group: Control group, non infected and non treated.

2^nd group: Infected with Eimeria tenella at a rate of 50.000 sporulated oocyst/ml per bird using an insulin syring introduced directly into crop of each bird at 14^th day of age. (Oocyst per gram (OPG) of feces was counted following McMaster technique.

3^rd group: Infected with Eimeria tenella as mentioned before at 14^th day of age and treated with aqueous extract of mushroom (200mg/ml) by day 6 post infection and continued for 7 days consecutively.

4^th group: Infected with Eimeria tenella at 14^th day of age as mentioned before and treated with amprolium (200mg/ ml drinking water) by 6 day post infection and continued for 7 days consecutively.

5^th group: Infected with Eimeria tenella as mentioned befor at 14^th day of age , by 6 day post infection and treated with both aqueous extract of mushroom (200mg/ml) and amprolium (200mg/ml) given for 7 days consecutively.

6^th group: Non infected with Eimeria tenella but received aqueous extract of mushroom (200mg/ml) at 14^th day of age and continued till the end of experiment.

7^th group: Non` infected with Eimeria tenella but received amprolium (200mg/ml) at14^th day of age and continued till the end of experiment.

1 - Determination of body weight: Body weight of broilers in all groups was monitored weekly using a weighting balance every morning prior to feeding.

2 - Collection of fecal sample and laboratory examination: The faeces of broiler chicks were collected at 21,28,35 day of age in plastic bags for parasitological examination. Oocysts per gram (OPG) were counted by McMaster's slide (Hodgson, 1970).

3 - The lesion score of E.tenella infestation:was carried out according to (Johonson and Reid 1970).

4 - Pathological examination: Clinical signs and post mortem findings were recorded on the experimental birds before and after the day of sacrifice (1 and 2 weeks post infection or post treatment). Specimens from the ceci were collected and fixed in 10% buffered neutral formalin solution, dehydrated in gradual ethanol (70-100%), cleared in xylene and embedded in paraffin, five micron sections were prepared and then routinely stained with hematoxylin and eosin (H&E)(Bancroft and Gambl 2008) and then examined microscopically.

5 - Statistical analysis: The obtained data were analyzed using the liner model programs of SAS (1990). The difference among means were tested using Duncan Multiple range test(Duncan, 1955).

RESULT 

1. Body weight record: The recurrent results in table (1) showed that at21 day of age (before treatment), second group (infected, untreated chicks) showed a significant decrease in body weight (465.50±1.5) compared to control 1^stgroup (uninfected, untreated) (540.50±4.5) and compared to all another groups (3^rd,4^th,5^th,6^th,7^th) which showed non significant difference between each other and between control 1^st group. At 28 days of age (one week PT.), 2^nd group (infected, untreated) showed a significant decrease in body weight (590.60±9.6) in comparison with control 1^stgroup (879.90±13.6) and in comparison with all another groups. Both 5^th group (infectd, treated with mushroom and amprolium) and 6^th group (uninfected, treated with mushroom) recorded the highest body weight (899.60±16.9,899.60±8.5), they showed non significant difference between each other and between control 1^stgroup (879.90±13.6), followed by 3^rd group (infectd, treated with mushroom) (886.20±3.9) then 7^thgroup (uninfected, treated with amprolium (870.40±3.9) which showed non significant difference  between each other and between control 1^st group. At 35days of age (2week PT.), 2^nd group showed significant decrease in body weight (800.70±8.5) as compared to control 1^stgroup (1189.5±14.4) and in comparison with all another groups. Fifth Group recorded the highest body weight (1378.5±18.5) which showed significant increase as compared to control 1^stgroup and all another groups, followed by 6^th group (1303.4±8.1) then 3^rd group (1299.7±2.4) which showed significant increase in body weight in comparison with control 1^stgroup. Wherease7^th and4^th groups (1220.4±5.2, 1208.8±6.9) showed non significant difference in body weight between each other and between contol 1^st group.

Table 1: Mean values of body weight of the 7 groups of broilers under experiment.

Value are means ± standard error, means with different letters at the raw differ significantly at (P <0.05).

1 - Ooycsts per gram count: The analysis of results in table (2) showed that, at 21 day of age (befor treatment): The oocysts output was (47100±458.25) 2^nd group, (45000±1414.21) 3^rd group, (45700±422.95) 4^th group and

2 - (44200±840.64) 5^th group. At 28 day of age (one week post treatment), 2^nd group showed high significant increase in OPG counts (70000±1626.17) while 3^rd, 4^th, 5^th groups showed significant decrease in oocysts count (7553±2.33, 2567±1.86, 2067±1.7) respectively as compared to control untreated 2^nd group, at the same time 3^rd, 4^th and 5^th groups showed non significant difference in oocyst count between each other. At 35 days of age (2week post treatment), 2^nd group showed significant increase in oocysts count (60000±4216.37) while all treated groups (3^th, 4^th, 5^th) showed significant decrease (50±2.58, 2±0.36,1±0.2) respectively as compared to control 2^nd group, as well as, these groups showed non significant difference in oocyst count between each other.

Table 2: Mean values of OPG count of broilers

Value are means ± standard error, means with different letters at the raw differ significantly at (P <0.05).

3. Mortality rate and morbidity:

No mortality occurred in any of the experimental groups till the end of the experiment while on day 5 PI. all the birds in the infected groups (2, 3, 4 and 5) appeared dull, weak, loss of appetite, their feces became bloody and watery and Eimeria tenella oocysts were detected in their feces. These symptoms nearly disappeared in chicks of third group at (one week post treatment) and disappeared completely at (two week post treatment.). While in chicks of fourth and fifth groups the symptoms disappeared completely at (one week post treatment). On the other hand, in chicks of second group the symptoms continued till (two week PI.) then decreased gradually.

4. Pathological findings

The ceci of control (group 1): “Non-infected, non-treated” showed neither gross nor microscopic abnormalities along the experimental periods (Fig 1). However, the ceci of (group 2): “Infected,non-treated”macroscopically showed extensive hemorrhages in the cecal lumen and petechiation on the mucosa and serosa, particularly at 1 week PI and hemorrhagic cecal core on 2 weeks PI. Microscopically, the cecum revealed severe necrosis, destruction and desquamation of the lining epithelium with the presence of developmental stages of Eimeriaincluding mature schizonts (Fig 2) and gamonts (Fig 3) in the enterocytes and lumens with extensive extravasated erythrocytes, 1 and 2 weeks PI. Sometimes, the necrotic mucosa was heavily infiltrated or replaced with lymphocytes and few heterophils besides hyalinization of the muscular coat (Fig 4). Hemorrhages, edema and necrosis were seen in the submucosa particularly near the muscular layer. Almost all the epithelial lining of the cecal mucosa were invaded with different developmental stages of coccidia (schizonts and gamonts), 2 weeks PI (Fig 5). The submucosa was edematous and severely infiltrated with lymphocytes, macrophages and few heterophils.

The ceci of group (3): “Infected and treated with Mushroom” showed slight lowering in the lesions of group (2). Bloody contents and petechial hemorrhages on the mucosa were also visualized. Microscopically, the developmental stages of Eimeria were evident in the lining epithelium of the cecum similar to those described in the infected chickens besides necrotic mucosa and lymphocytes infiltrations, 1 and 2 weeks PT (Fig 6). The mucosa of the cecum was slightly intact and the submucosa was infiltrated with lymphocytes and few macrophages, 2 weeks PT. Some hyperplastic changes were seen in the cecal epithelium and crypts. These findings in this group were presented as regenerative attempts. The lamina propria and submucosa showed edema and focal fibrosis. Mild hyperplasia of the lymphoid follicles of cecal tonsils was also seen. Meanwhile the ceci of  group (4): “Infected and treated with amprolium” showed nonspecific changes, particularly at 2 weeks PT. Microscopically, the ceci showed intact mucous membrane with few developmental stages of Eimeriawith numerous lymphocytes and macrophages infiltrations, particularly in the crypts, 1 week PT (Figs 7 and 8). Partial desquamation of the lining epithelium was also detected. However, the ceci at 2 weeks PT showed intact intestine with mild mucinous degeneration and few round cells infiltrations. Severe hyperplasia in the lining epithelium was noticed with no evidence of coccidial stages (Fig 9) besides congested blood vessels and thick eosinophilic membrane coated the luminal surface of the lining epithelium. The ceci of group (5): “Infected and treated with both amprolium and Mushroom” were apparently normal particularly at 2 weeks PT. Microscopically, the lesions of such group were completely ameliorated with intact mucosa, complete absence of the developmental stages of Eimeria and few lymphocytes infiltrations in the submucosa (Fig 10). Few birds, sacrificed 1 week PT, showed few) degenerated or necrotic gamonts in the lining epithelium of crypts (Fig 11). The cecal mucosa was focally regenerated with hyperplastic lining epithelium in both sacrificed periods. The submucosa and lamina propria were infiltrated with round cells of mostly lymphocytes besides hyperplasia in the lymphocytes of the cecal lymphoid follicles and tonsils (Fig 12). The ceci of groups (6,7): Showed neither gross normicroscopic abnormalities along the experimental periods.

Figs. (1-4): Cecum from groups:

Fig.(1), (group1): shows normal intact mucosa and lymphoid follicles. H&E x 100.

Fig.(2), (group 2): shows several schizonts (arrows) in the enterocytes. H&E x 400.

Fig.(3),( group 2): shows numerous gamonts (arrows) in the enterocytes. H&E x 400.

Fig.(4), (group2): shows severe necrosis and lymphocytic infiltrations (arrow) with hyalinization of muscular coat (irregular arrow). H&E x 200.

Figs. (5-8): Cecum from groups:

Fig.(5), (group 2): shows developmental stages in almost all the intestinal epithelium (arrows). H&E x 200.

Fig.(6), (group 3): shows developmental stages of Eimeria in the lining epithelium and numerous lymphocytes infiltrations. H&E x 200.

Fig.(7), (group 4): shows few developmental stages of Eimeriain the lining epithelium of crypts (arrows) with numerous lymphocytes and macrophages infiltrations (arrowhead). H&E x 200.

Fig.(8): A higher magnification of fig (7). H&E x 400.

Figs. (9-12): Cecum from groups:

Fig.(9), (group 4): shows severe hyperplasia in the lining epithelium (arrows) with no evidence of Eimeria. H&E x 400.

Fig.(10), (group 5): shows intact mucosa with no evidence of developmental stages (arrow) and lymphocytes infiltrations (arrowhead). H&E x 100.

Fig.(11), (group 5): shows few degenerated gamonts in the lining epithelium of crypts (arrows). H&E x 400.

Fig.(12), (group 5): shows hyperplasia in lymphocytes of the lymphoid follicles (arrow). H&E x 200.

Scoring of histopathological findings in the ceca.

++++       Serious   +++ Severe              ++ Moderate           + Mild         ± rare      - Nil

DISCUSSION

The present study investigated the anticoccidial efficacy of Oyster mushroom in relation to body weight, OPG count of faeces, morbidity, mortality, score lesion and pathological lesions. All broilers were infected (100%) by 7days PI., showed clinical signs of weakness, reduced appetite and bloody diarrhea. Regarding to the body weight, there was a significant decrease in the body weight of infected, untreated birds (2^ndgroup); while the infected chickens which were treated with mushroom (3^rd group) or with both mushroom and amprolium (5^th group) showed a marked increase in body weight. Those results  agreed with that obtained by (Guo et al., 2003; Ogbe et al., 2009 and  willis et al., 2012) who found that mushroom have polysaccharides that stimulating the activities of T and B lymphocytes, macrophage and natural killer cells (NK) inducing production and secretion of cytokines and complement, so it controls certain parasitic diseases. Among the reason why the treated broilers gained weight more than the non treated birds could be due to that aqueous extract of mushroom like amprolium may affect or prevent development of E.tenella stages. It appeared that this wild mushroom may contain compounds that are active against E.tenella. It was found to be non toxic in animal toxicity studies even when used at high therapeutic dose (Harkonen, 1998 and James, 2002). As well as the mushroom extract and amprolium stimulate appetite so the uninfected broilers which was treated with mushroom (6^th group) or treated with amprolium (7^th group) performed even high body weight.

Large numbers of oocysts were detected in the faeces of challenged birds that had received the primary infection challenge but not treated (2^nd group). That data clearly showed that birds were adversely affected by this protozoan while broilers in treated groups (3, 4 and 5) showed the lowest oocyst count. Those results demonstrate the effectiveness of the mushroom in reducing the shedding of oocysts and agreed with those obtained by (Ogbe et al., 2009; Willis et al., 2010 and willis et al., 2012) who showed that diet supplementation with aqueous extrat of mushroom (FMG) exhibited a reduction in oocyst excretion and mortality in Eimeria challenged broiler chicks. Mushroom have bioactive compounds or polysaccharides are known to play vital roles in enhancing health. They block colonization of the intestine by pathogens, thereby improving their elimination from the body (Guo et al., 2003 and Elmusharafa et al., 2006) and also it contains organic acids, resin and glycosides which include steroid are known to have therapeutic use against microbes and parasites (Anon 2007; Deihk et al., 2007).

Regarding to histopathological findings the cecum of infected, untreated chicks (2^nd group) showed severe necrosis, destruction and desquamation of the lining epithelium with presence development stages of Eimeria sometimes the necrotic mucosa was heavily infilterated with lymphocytes, haemorrhages, oedema and necrosis were seen near the muscular layer. Those result agreed with that obtained bySoomro et al. (2001); Ogbe et al. (2005) and Enas (2011). There were reduction in those lesions in broilers infected and treated with mushroom (3^rd group) and the lesions were ameliorated in broilers of (4^th group) (treated with amprolium) and (5^th group) (treated with both amprolium and mushroom). The regeneration of cecal mucosa was observed and there were decrease in the number of oocysts of E. tenella in epithelial at 2 week PT. From those results, it could be demonstrated that both amprolium and mushroom lead to improve the pathological finding induced by E. tenella as mushroom lead to improve innate immune responses against coccidiosis. Those finding, however, correspond to those recorded by (Naphade et al., 2010 and Hossain et al., 2013).

CONCLUSION

Treatment with aqueous extract of oyster mushroom lead to improve body weight more than amporilum, while treatment with amprolium lead to the reduction of faecal oocysts and improve pathological lesions more than oyster mushroom. On the other hand best results were obtained when we used oyster mushroom mixed with amporilum.

ACKNOWLEDGMENT

It is a great pleasure to record our independence to Prof. Dr. Mohamed Hamed Mohamed, Professor of Pathology, Faculty of Veterinary Medicine, Zagazig University for his great help throughout the period of study reading and comments on histopathological specimen.

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