EVALUATE THE EFFECT OF SOME PHYTOBIOTICS ON THE CONTROL OF NECROTIC ENTERITIS IN BROILERS CHICKEN

The harmful economic impacts on chicken production are caused by the bacterium Clostridium perfringens , which is also responsible for necrotic enteritis in chicken. The study's objective is to assess the beneficial benefits of a few phytobiotics as substitutes for antibiotics when treating necrotic enteritis and broiler performance. Seven treatment groups were created at random from 280 broilers. All groups, except the negative control group, faced C. perfringens type A strain seven days after receiving sporulated coccidia oocyst inoculations. G3 and G4 received a basal diet containing 1 and 2 gm of curcumin /kg of diet, respectively. G5 was given a control infection, while G6 received a basal diet containing 1 and 2 gm of propolis /kg of diet. The results revealed that a high concentration of curcumin 1&2 gm/kg as a feed additive had a significant detrimental effect on chicken growth performance. Moreover, the total count of C. perfringens in the cecum was significantly increased and severe histopathological alterations in the intestinal mucosa, including severe sloughing of villi epithelium were detected. On the other side, Propolis, when added to the diet at a dose of 1-2 gm/kg ration, protected the digestive system and ameliorated the pathological lesions induced by the concurrent infections with Eimeria and C. perfringens. Consequently, a considerable decrease in the overall number of C. perfringens in the cecum, and restoration of the normal histological structure of the intestine and the lesions were mild compared to curcumin groups.


INTRODUCTION
A healthy chicken's soil and intestines contain the spore-forming anaerobic gram-economic consequences (Skinner et al., 2010).
A class of substances known as phytobiotic additives, which are derived from medicinal plants and spices, can increase the secretion of digestive enzymes, improve feed conversion, boost immunity, have antioxidant effects, improve animal growth and weight gain, raise the standards of animal products (Ognik et al., 2020).
The most crucial requirement is the alternative use of antibiotics, in order to save lives and microbial diversity. An efficient substitute for antibiotic growth promoters is the use of phytobiotics as natural, nontoxic growth promoters (NGP) (El-Hack et al., 2020).
In the ileum of broilers, phytobiotics significantly reduced the overall counts of anaerobic bacteria (C. perfringens and coliform) (Vidanarachchi et al., 2006). Due to its antibacterial properties, turmeric (Curcuma longa), a Zingiberaceae family plant, is a natural herb, and can be a significant source of antibiotic alternatives. It is less toxic, leaves no traces, and is organic comparable to organic compounds or synthetic medicines (Khan et al., 2012). Curcumin is the substance in turmeric that is active. Anti-inflammatory properties of curcumin (Ammon et al., 1993), antioxidative (Osawa et al., 1995), anticoccidials (Abbas et al., 2010), and influencing the immune system (Yarru et al., 2009).
Consequently, avoiding the emergence of antibiotic resistance may be possible by incorporating turmeric into the feed for chickens. (Ali et al., 2020). Through the inhibition of C. perfringens and increased feed effectiveness, turmeric may be a significant contributor to the control of NE in poultry feed (Niranjan and Prakash 2008). Meanwhile, turmeric has improved resistance to intestinal diseases such as coccidiosis and necrotic enteritis (Kim et al., 2013).
Propolis, also known as "bee glue," is a waxyresinous material. The word "propolis" is derived from the Greek words: pro, which means in defense; and polis, which is for city. (Bankova et al., 2000).
Propolis and bee pollens are biologically active ingredients, which have immunemodulatory, antiviral, antibacterial, antifungal, analgesic, antiprotozoal, antioxidant, and anti-inflammatory properties in both people and animals. (Kačániová et al., 2013). In high concentrations, propolis can kill bacteria because it has antibacterial activity against various bacterial genera (Drago et al., 2000).
Phenolic acids, flavonoids and their derivatives are thought to be responsible for propolis ' antibacterial properties (Tosi et al., 2007). In addition, propolis is well known to contain amino acids, vitamins, proteins, minerals, flavonoids, and all of that are crucial nutrients for promoting poultry growth (Babaei et al., 2016). Silva et al. (2018) demonstrated that propolis' anti-inflammatory and antioxidant properties could be responsible for its effectiveness in the treatment of digestive disorders.
The experimental study sought to ascertain the C. perfringens prevalence rate in Assiut governorate private farms, evaluate the impact of propolis and curcumin as feed additives on the growth and presence of microbes in broilers infected experimentally with C. perfringens, as a means of preventing necrotic enteritis without using antibiotics.

MATERIALS AND METHODS
Field Samples collection: 120 intestine samples from diseased and recently deceased broiler chickens were collected with various ages (22-40 days of age) suffering from diarrhea from private farms in (Assiut Governorate, Egypt) for isolation of C. perfringens. Isolation and detection of C. perfringens Willis (1977) states that the samples taken in tubes were incubated anaerobically for 24 hours at 37 o C using boiled freshly prepared and cooled cooked meat medium (Oxoid CM81). On a neomycin sulfate sheep blood agar plate, the inoculated liquid medium was spread into a loop (Cruickshank et al., 1975) and 48 hours of anaerobic incubation at 37 o C using an anaerobic gas jar. Egg yolk agar and sheep blood agar are placed on two plates., suspicious C. perfringens colonies were grown. The first plate was grown aerobically, while the second plate was grown anaerobically. Colonies with duplicate hemolytic zones on blood agar, which only grow under anaerobic and lecithinaseproducing conditions, were collected, cleaned up, and used in identification testing (Cruickshank et al., 1975). The biochemical tests were then used to analyze the biochemistry of isolated colonies with typical appearances. All isolates were stored on cooked beef medium for later purposes.
Multiplex PCR for detection of C. perfringens strain From the C. perfringens isolate mentioned by Sambrook and Russell, complete DNA was obtained (Russell and Sambrook 2002). To distinguish between the various strains of C. perfringens types D, C, B, and A, there were four primer sets, one for each of the organism's toxins (Table 1), according to Ahsani et al. (2010). Thermal Cycler was used to conduct the PCR ® (USA, Bio-Rad) 5 liters of 10x PCR buffer are contained in a 50-liter total reaction volume (pH 9.0, 50 mM KCl, 10 mM Tris-HCL), Deoxynucleotide triphosphates, 250 μM apiece,2 μL 50 mM MgCl2, 5U of Taq DNA polymerase, five liters of template DNA and one hundred pmol of primers. 35 cycles of DNA amplification were completed. Each cycle includes a step of 94°C denaturation for 45 seconds, synthesis at 72°C for 90 seconds, and a step of 55°C annealing for 30 seconds. The last extension step occurred at 72°C for ten minutes. Finally, Ethidium bromide and agarose gel (1.5% concentration) were used to electrophorese 7μ L of the amplified DNA product. UV illumination was used to visualize the amplified bands. A total of 280 broilers (Ross308, 14 days old) were divided into seven treatment groups at random. All treatments were repeated 5 times, with 8 chickens per pen. The experimental treatments included; G1 and G2 had a baseline diet of 1 and 2 grams of propolis /kg diet respectively; G3 and G4 were fed with a basal diet containing 1 and 2 grams of curcumin /kg diet respectively (pure curcumin powder); G5 control infected and G6 control negative were merely given a basic meal, and G7 was given with a "spiramycin+based diet.
Oral inoculations of 5x10 4 sporulated oocysts of coccidia Tenella were administered to all groups except the control negative group (Hofacre et al., 1998).
The isolated field strain of C. perfringens type A was administered orally to the same groups after 7 days by inoculating 1.0 ml of broth culture with 1.0 x 108 CFU/bird, as described by (Dahiya et al., 2005). After the bacterial challenge, the experiment was carried out for a further 7 days. Broilers in Group 7 fed on a spiramycin+based diet were treated with spiramycin (0.5 gm/liter distilled water), and the treatment started on the 3 rd day after bacterial infection (appearance of clinical signs) and continued for 3 days.
During the experiment, all treatments were subjected to comparable management procedures, such as lighting, feeding, and irrigation, with the exception of the special diet offered. Give broilers light for a full day. unlimited access to food and water.
Feed conversion ratio (FCR), feed intake (FI) and weight gain (WG) were measured weekly. All clinical signs, post-mortem lesions and mortalities were recorded.

Bacteriological Examination
At day 14 after infection, fresh intestinal contents from the cecum of slaughtered broilers were collected aseptically (the experiment's conclusion). Randomly, one bird was selected from each identical coop from each treatment group's 5 chickens. Dilute the sample in buffered peptone water to an initial dilution of 10 -1 . Spread the 10fold serial dilutions in duplicate on reinforced Clostridium agar for the calculation of C. perfringens. For 48 hours, every plate was incubated at 37 °C in a Gaspack anaerobic tank. Count the number of colonies on the fortified Clostridium agar plate and express it as log10 CFU / g of cecal content. The isolated colonies were then subjected to biochemical tests and Gram's stain for microscopic examination to confirm they were C. perfringens (Cruickshank et al., 1975).

Pathological Examination
Samples from cecum and jejunum of sacrificed broilers were collected at d 28 (end of the experiment) and were fixed in 10% neutral-buffered formalin. After being cleaned in Xylene and dehydrated in ethanol at increasing concentrations, after fixing the samples, paraffin wax was used to embed them. Cut paraffin sections of 5 m thickness were used for Hematoxylin and Eosin stain for general histological analysis (Fischer et al., 2008).
A histopathologist, who was unaware of the groupings, looked at the histological samples under a light microscope (Olympus, USA). Sections from the cecum and jejunum were photographed at low and high magnifications.

Statistical analysis
All information was presented as mean standard deviation (SD). Utilizing the Windows program (Graph Pad Prism 8.0.2), statistical analysis was carried out, "California USA, San Diego, GraphPad Software". To show the differences between groups that were significant, one-way analysis of variance (ANOVA) was utilized, then Tukey multiple comparisons for post hoc analysis, p < 0.05 were regarded as important. Using the Shapiro-Wilk normalcy test, whether or not the data supported the statistical approach's presumptions, and if they did not, ANOVA with the Kruskal-Wallis test and the Dunn's Multiple Comparison Post hoc test were both employed.

Field incidence of C. perfringens
Overall, 77 (64%) isolates of C. perfringens were detected from the total 120 samples collected from freshly dead and diseased broilers chickens suffering from severe diarrhea, severe congested, dilated and friable small intestine filled with gas and necrotic foci in the liver.

Multiplex PCR
PCR analysis of twenty representatives of C. perfringens isolates showed that all of them were genotyped as C. perfringens type-A because the only amplified gene was the alpha toxin, as shown in Fig.1.

Effect of phytobiotic on growth performance
Propolis and curcumin's effects on growth performance one week after a challenge with C. perfringens were obvious, as shown in Table 2. The difference in weight increase per day across the various groups was highly significant (p < 0.01, F = 5.07). Moreover, the weight gain of G4 (curcumin 2 gm) was highly significantly different when compared with G6 (control -ve) at p < 0.001 and G7 (antibiotic-treated) at p < 0.05. Regarding the feed conversion ratio, there was a highly significant difference between different groups in the feed conversion ratio "FCR" (p < 0.001, F = 6.42). Group 4 (curcumin 2 gm) was highly significantly different when compared with G6 (control -ve) at p < 0.0001, G7 (antibiotic-treated) at p < 0.001 and G2 (propolis 2 gm) at p < 0.05.

Clinical symptoms
From the fourth to the sixth day after the C. perfringens challenge, nearly all experimental groups, with the exception of the non-infected control, experienced bloody diarrhea. The extent of bloody diarrhea was less severe in the groups given rations supplemented with 1 and 2 gm of propolis /kg of ration than in the other groups, which had mortality rates of 2/40 and 1/40, respectively. While the mortality rates for the groups receiving rations supplemented with 1 and 2 gm of curcumin/kg of ration were 4/40 and 6/40, respectively. They also displayed severe bloody diarrhea, signs of dehydration, and lethargy. While the mortality rate was 0% in both the antibiotic-treated and control noninfected groups, it was 5/40 in the control infected group. Table 3 shows how propolis and curcumin affected the quantity of C. perfringens found in broiler caeca after being exposed to a C. perfringen type A strain, a high significant difference between different groups was detected (p < 0.001, Kruskal-Wall is statistic = 32.28). Table 3 Effects of propolis and curcumin on C. perfringens numbers in caeca of broilers after challenge with C. perfringens strain (log10 CFU/g of cecum). Histopathology

Postmortem examination
After 7 days of C. perfringens infection, the groups that received 1gm and 2gm of curcumin developed necrotic enteritis lesions. The jejunum and ileum appeared dilated, had a thin, friable wall, and were filled with gas, or contained green or redtinged, foul-smelling intestinal fluid mixed with debris. The lesions were mostly in the jejunum, ileum, and cecum ( Fig. 2 B, E, and F). One bird from the group that received 2 gm of curcumin had a ruptured cecum and the lumen was filled with dark red clotted substances that blocked the intestinal passage (Fig. 2F). The intestinal tract appeared normal in the control non-infected, 1gm, 2gm propolis-treated groups, and the antibiotictreated group, with no signs of congestion, gas, or foul-smelling from intestinal fluid ( Fig. 2 A, C, D, and G).

Microscopic examination
The severity of microscopic lesions in the jejunum was different in each group seven days after C. perfringens infection (14 days after Eimeria infection). The severe lesions were in the form of eosinophilic pink necrotic debris mixed with the intestinal contents, sloughing and detachment of villi epithelium, or even complete destruction of villi in the control infected, 1 and 2gm curcumin-treated groups.
Additionally, mononuclear inflammatory cells were present in the lamina propria (Fig. 3B1, B2; E1, E2; F1, F2). Jejunum from the group that received 1 gm of propolis displayed mild lesions with some epithelial detachment and minimal infiltration of mononuclear cells (Fig. 3C1,  C2). In the 2gm propolis-treated and antibiotic-treated groups, the normal histological structures of the jejunum were roughly restored, and the lesions were noticeably less severe than in the control noninfected group. Additionally, the lamina propria appeared normal, composed of loose connective tissue, infiltrated with few lymphocytes. The intestinal villi epithelium was intact and consisted of simple columnar cells with goblet cells (Fig. 3A1, A2; D1, D2; G1, G2).
The degree of the cecum's pathological lesions 14 days after an Eimeria infection, 7 days later, the symptoms were comparable to those in the jejunum. Severe lesions with entire villi epithelium sloughing and detached epithelium accumulating in the intestinal lumen were visible on the cecal mucosa of control infected, 2gm curcumin-treated groups. Mononuclear inflammatory cells had infiltrated the area, particularly macrophages and lymphocytes, in the lamina propria and submucosa. Eimeria species were seen in various stages of development in the intestinal glands, submucosa, lamina propria, and villi epithelium. There was intestinal gland hyperplasia and enterocyte hyperplasia in some villi ( Fig. 4B1-B3; F1-F3). In the group treated with 1 gram of curcumin, there were mild coccidiosis lesions, some villi epithelium detachment, and intestinal gland and lamina propria Eimeria stages were also found ( Fig. 4E1-E3). On the other hand, the 2gm propolis-treated and antibiotic-treated groups virtually completely restored the normal histological appearance of the cecum. There were not many inflammatory cells in the lamina propria, and the villi epithelium was unharmed. Eimeria spp. developmental stages were not observed in the groups treated with 2 gm propolis or antibiotics. While 1 gm/kg propolis revealed mild pathological changes and a few stages of coccidia in the lamina propria (Fig. 4A1-A3; C1-C3; D1-D3; G1-G3).

DISCUSSION
Growth and dissemination of bacterial resistance was thought to be facilitated by the excessive use of antimicrobials in animals to promote and prevent growth, which posed serious risks of transmission of resistant microbial strains to humans and the environment (Sharma et al., 2018).
According to our field survey, 77 (64%) of the 120 isolates of C. perfringens Type A were found in freshly dead and diseased broiler chicken, which had suffered severe diarrhea, a severely congested, dilated, and friable small intestine filled with gas, and necrotic foci in the liver. Those obtained results were to some extent like the outcomes of Eman et al. (2013), who found that 60% of the intestinal samples from commercial broilers in Egypt have necrotic enteritis were C. perfringens.
Our research revealed that the addition of 2 gm curcumin /kg ration as feed additives had a negative impact and significantly reduced weight gain and feed intake. The chickens in this group had higher feed conversion ratios than those in the other groups, as a consequence of the significant increase in the total count of C. perfringens in the cecum.
According to the current findings, supplementing the diet with 1 and 2 gm of curcumin /kg of feed did not protect the intestinal tract or stop the pathological changes caused by concurrent infections with Eimeria and C. perfringens. However, this 2 gm curcumin/kg feed had worsened the effects already present on the intestinal mucosa, accentuated the pathological lesions, and caused intestinal perforation in some birds. This might be explained by the study's use of a toxic high dose of curcumin (2gm/kg feed). High doses of curcumin have been discovered to irritate the stomach, cause diarrhea, and impair platelet function (Lao et al., 2006). Additionally, rats' stomach, cecum, and colon inflammation, hyperplasia, and ulcer incidence are all linked to turmeric consumption. It can also hinder the blood's ability to clot, which can make bleeding worse (Jiao et al., 2009).
Curcumin's antibacterial and antifungal properties have been the subject of numerous studies, but there is a lack of information on how it affects various microbial strains, particularly clinical isolates and MDR strains (Artur et al., 2020).
Sometimes, curcumin was taken at concentrations too low to assess its antimicrobial properties. Therefore, further investigation into the effects of curcumin on various microbial strains and species using standardized techniques is still necessary (Raorane et al., 2019).
A dose of 3 to 10 g/kg diet of turmeric powder had no appreciable positive influence on broiler chickens' performance in terms of growth (Sadeghi et al., 2012). Additionally, curcumin has a relatively poor bioavailability, and numerous studies have demonstrated that levels in the blood and extra-intestinal tissues are extremely low or even undetectable. Rapid metabolism, poor absorption, rapid systemic and elimination of chemical instability were thought to be the primary causes (Anand et al., 2007). Kermanshahi and Emadi (2007) reported that broilers feed intake, increase weight, and feed conversion rate were not significantly affected by turmeric supplementation. Additionally, our results showed that broiler groups dosed with 1 and 2 gm propolis/kg ration had significantly lower body weight, non-significantly lower feed intake, and significantly higher conversion rates compared to the control group. These outcomes support the findings of Mahmoud et al. (2013), who recorded that growth performance indicators in broiler chickens are significantly impacted by the addition of propolis to the diet. Also, Santos et al., (2003) claimed that broiler weight gain at 42 days was considerably inhibited by propolis. The reduced growth parameters could be explained as a result of the antiobesity properties of propolis extract, which decreased fatty acid synthesis in the liver (Koya-Miyata et al., 2009). Furthermore, Tatli et al., (2008 reported that propolis supplementation had no effect on FCR in broilers exposed to chronic heat stress. Also, Silici et al., (2006) reported that propolis had no negative health effects, but did not enhance the growth characteristics of quails in the first 35 days.
Age and length of supplementation have an impact on the effectiveness of propolis. Açıkgöz et al. (2005) claimed that feeding broilers propolis at a dose of 4000 mg/kg lowered their feed intake and weight gain when it was given between the ages of one day and three weeks, but not between four and six weeks, i.e. it would be effective in later production phases (at six weeks of age).
Propolis has a positive impact on the total bacterial count of C. perfringens compared to the curcumin-treated group, despite the poor performance parameters of those groups. Dietary propolis supplementation at a dose of 1, 2 gm/kg ration has a protective effect on the intestinal tract, ameliorates the pathological lesions caused by concurrent infections with Eimeria and C. perfringens, and restores normal histological structure of the intestine. The lesions were mild compared to curcumin groups. Babińska et al.
(2012) explained that these histopathology findings showed that broiler chickens given propolis had reduced organ damage compared to controls, caused by the infection with Salmonella enteritidis. Otherwise, propolis did not affect the reduction of aerobic bacteria and total coliforms against the control group (Mahmoud et al., 2014) Additionally, the 2 gm propolis treated group had superior protective effects, restored the normal appearance of the jejunal and cecal mucosa, and its protective effects were comparable to the administration of antibiotics. Similar outcomes were noted in laying hens raised under prolonged heat stress following the administration of 2 or 5 g propolis/kg diet (Tatli et al., 2008) and 3 g propolis/kg diet (Seven et al., 2011). Specifically against gram-positive and gram-negative bacteria, in vitro Propolis preparations demonstrated antibacterial activity. Its chemical composition, which includes flavonoids, phenolic acids, polyphenols, esters, amino acids, aromatic aldehydes, vitamins, and minerals, is credited with this activity (Schnitzler et al., 2010).
The control-infected group displayed severe necrotic enteritis symptoms, including severe bloody diarrhea, reduced feed intake, intestinal ballooning, and pathological necrotic lesions, along with a mortality rate of 5/40. Intestinal C. perfringens is associated with a reduction in the capacity to utilize food effectively, and/or the emergence of a different behavior that affects the intestinal microbiota (Granstad et al., 2020). Moreover, Stutz et al. (1983) reported an association between the increased numbers of C. perfringens in the gut and chicken growth retardation.
When compared to other groups, treatment with the antibiotic spiramycin has a positive impact on body performance and significantly increases weight gain and feed intake while significantly reduces feed conversion rate. Additionally, it significantly improved growth performance, restored the normal histological structure of the intestine, and had a strong reducing effect on C. perfringens counts. In comparison to the control non-infected group, the lesions were also noticeably less severe.
As a result of the increase in the overall count of C. perfringens, supplementation with a high concentration of curcumin and propolis had a significant negative impact on the growth parameters of broilers exposed to C. perfringens in an experimental setting. The intestinal tract was negatively affected by curcumin, which does not protect the intestinal tract but rather amplifies the negative effects brought on by C. perfringens infection in animal experiments.
The total number of C. perfringens comparison to the curcumin-treated group and the infected group, was considerably decreased in the propolis-treated group because propolis had a positive effect on the intestinal tract, restored the normal histological structure of the intestine, and the lesions were very mild.

Funding
Not applicable.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The study was conducted in accordance with the ethics committee's approval and the standards for the care and use of experimental animals, Faculty of Veterinary Medicine, Assiut University, Egypt.

Abbas
The effects of propolis