THERAPEUTIC POTENTIAL OF SOME ORGANIC ACIDS AND ESSENTIAL OILS AGAINST EIMERIA SPP . ISOLATES COLLECTED FROM BROILER CHICKEN FARMS IN ASSIUT

Different Eimeria isolates were collected and identified in 50 intestinal specimens. The therapeutic potential of the organic acids and essential oils blends against E. tenella was compared with the anticoccidial drugs toltrazuril and amprolium in broiler chickens. Results revealed that E. tenella was the most frequent identified Eimeria species (48 %) followed by E. acervulina (30 %) and E. mitis (28 %), while the least detected were E. mivati (2%) and E. brunetti (2 %). Organic acids medication reduced the clinical signs and mortality to 33.3 % compared to amprolium (40 %), Essential oils (40 %), and toltrazuril (53.3 %) medicated groups and the positive control (60 %). The best relative body weight gain percent during the experimental course was achieved by birds medicated with organic acids (40 %) and toltrazuril (40 %) followed by amprolium (30 %) and essential oils (30 %) medicated group and the least in the positive control (10 %). The severity of the cecal lesions was reduced similarly by 34 % in all the medicated groups; all scored 2.3 on average in comparison to 3.5 in the positive control. Both organic acids and essential oils were unable to reduce the output of oocysts by over 17% and 3%, respectively, as opposed to 37% in the toltrazuril and 20.6% in the Amprolium treated groups. Microscopic observations revealed their healing effect of on the cecal tissue (epithelium and inflammatory response). The results support the notion that integrating the organic acids and or essential oils may have the ability to induce positive therapeutic effects similar to some chemical drugs such as toltrazuril and amprolium.


INTRODUCTION
Chicken coccidiosis triggers nonstop economic losses and outbreaks despite the location specificity, type of lesion produced, and oocysts morphometrics (López-Osorio, 2020). The Eimeria life cycle, in general, is complicated and triggers a strong inflammatory reaction, oxidative stress, and lipid peroxidation, leading to tissue damage, diarrhea, impaired growth, increased susceptibility to other disease agents, and in severe cases, mortality (López-Osorio, 2020). E. tenella produces hemorrhagic typhlitis accompanied by white dots (schizonts and oocysts) that can be visible from the serosal surface (Schneiders et al., 2020). Although advanced immunological and especially molecular techniques are available, microscopic detection for parasitic elements, for its simplicity and specificity, remains the main diagnostic method for coccidiosis (Holdsworth et al., 2004). Various anticoccidial drugs are widely used to control coccidiosis comprised toltrazuril, amprolium, ionophores, and sulfonamides (Mustafa et al., 2021). Chapman, (1989) recorded the high efficiency of toltrazuril against E. tenella over the sulfaquinoxaline /pyrimethamine and Amprolium /ethopabate. Chemotherapy is the first choice for controlling coccidiosis outbreaks however many drawbacks were reported including drug residues and drug resistance. The international regulations prohibiting the use of chemical therapy on livestock intended for human consumption encouraged researchers to look for natural and secure alternatives (Muthamilselvan et al., 2016). Organic acids and essential oils are attractive natural alternatives to anticoccidial drugs. The anticoccidial alternatives depended on diminishing the oocyst shedding through retarding parasite invasion, replication, and development in the gut tissues. Some alternatives as essential oils (carvacrol, terpenoids, cinnamon bark, and anise oils) and phenolics react with cytoplasmic membranes stimulating coccidial cell death; reducing lipid peroxidation to an acceptable level; allowing epithelial repair; and reducing the intestinal permeability brought on by Eimeria species (El-Shall et al., 2021). Furthermore, essential oils are efficient antimicrobial agents against Gram-negative and Grampositive bacteria . Organic acids are efficient for feed and food preservation as well as having positive impacts on intestinal health and bird performance. Organic acids were reported as a favorable supplement for swine and poultry due to boosting animal physiology and immunity, gut protection, modifying microbiota, and reducing the gut pH (Mustafa et al., 2021). The organic acids such as short-chain fatty acids (majorly propionic acid, acetic acid, and butyric acid) can replace the antimicrobial growth promotors from broiler diets (Scicutella et al., 2021). In their undissociated forms, organic acids can enter the microbial cell and dissociate into H+ ions and anions (RCOO-) which run out the microbial energy and disrupt protein synthesis (Mani-López et al., 2012). Commonly, these alternatives were used in poultry diets as growth-promotors and natural immuno-stimulators (El-Shall et al., 2021). The effect of organic acids and essential oils as anticoccidial therapies has been not often investigated. So, this study highlights the therapeutic efficiency and the clinical outcome of 2 anticoccidial alternatives (organic acids and essential oils blends) against cecal coccidiosis in view of their effect on the performance and intestinal morphology of healthy and challenged broiler chickens

I. Frequency of E. species in broiler chickens 1) Sampling
Multiple Eimeria isolates were collected from 50 representative intestinal specimens of different clinically-ill broiler farms (birds aged 10-40 days) and submitted (September 2021-March 2022 to the research laboratory of Avian and Rabbit Medicine Department, Faculty of veterinary medicine, Assiut University, with a history of bad feed conversion, depression, and or bloody diarrhea. The fresh intestines were partitioned into duodenum, jejunum, ileum, and two ceca. The contents of each part were directly examined for Eimeria oocysts and other Eimerian developmental stages through "unstained wet mount" using light microscopy (10X and 40X objectives, Olympus CH30, Olympus Optical Co., Ltd., Tokyo, Japan) (Garcia, 2001;Conway and McKenzie, 2007).
II. The therapeutic possibility of the organic acids and essential oils against cecal coccidiosis a. Eimeria isolate An Eimeria tenella isolate was obtained from the ceca of a 28-day-old broiler chicken flock with typical signs of cecal coccidiosis; bloody diarrhea, abnormal growth rate, hemorrhagic typhlitis, and mortality. For further E. tenella isolate identification, cecal tissue samples from these birds were fixed in 10% neutral buffered formalin and submitted to histopathological processing according to Talukder, (2007). The isolated oocysts were propagated, sporulated, and identified following the standard measures described by Ryley et al. (1967) and Levine, (1985), then stored at 4 o C until use.
Birds were raised following the ethical guidelines of Assiut University, Assiut, Egypt. The experimental groups accessed freely to feed and water and continuous constant lightening for 24 hrs throughout the experiment. Standard starter and grower pelleted rations were supplied without any anticoccidial medication.
At 23 days old, all birds, except the negative control group, were challenged with 4.1 x 10 4 sporulated oocysts/bird. Birds received the experimental drugs 48 hrs after infection for two consecutive days.
The anticoccidial potential of the experimental drugs was assessed on the 7 th day post-infection (30 days old) through clinical signs including the mortality ratio, the mean relative body weight gain percent (RBWG %), the mean cecal lesion score (MLS), the reduction % of the MLS (RLS), and the oocyst reduction % according to Haug et al. (2008), Conway and McKenzie, (2007), and Li et al. (2019).

Mortality %
The cumulative mortality during the challenge period was recorded and the mortality % was calculated; Mortality % = Nomber of initial chickens in the group x 100 The mean relative body weight gain percentage (RBWG%) All birds of each group were weighed prior to infection (initial BW) and at day 7 postinfection (final BW). The RBWG % between the negative control, positive control, and challenged-medicated groups was calculated as assigned by Li et al. (2019) via the following equation: The cecal lesion score After sacrification, the cecal lesions caused by E. tenella were scored in 7 birds/group grossly following Johnson and Reid, (1970) scale; from 0 to 4 depending on their severity, 0 being no lesions and 4 the most severe. The average lesion score (MLS) for each group was calculated and used for estimating the reduction % in lesion severity (RLS %) from the equation:

Cecal oocysts burden
Oocysts / gram cecal contents (OPG) were counted for each bird sacrificed for lesion score at 7 th day post-challenge. The average OPG of each group was for calculation of the oocyst's reduction percent as follow: % = Shedding of control challenged − Shedding of medicated Shedding of control challenged 100

Microscopic alterations of the cecal tissue
Cecal section from 5 birds per group were histopathologically processed according to Talukder, (2007) for judging the histological condition and findings for each treatment. Cecal tissue pieces (1 cm length) were excised during necropsy, rinsed briefly with normal saline, clamped on a piece of clean cardboard and fixed with freshly prepared 10 % neutral buffered formalin. The tissues were dehydrated, infiltrated, embedded in wax following standard histological practices, thin sectioned, mounted on a glass slide, and stained with hematoxylin and eosin.

II. The therapeutic potential of the organic acids and essential oils against cecal coccidiosis The clinical observations
The therapeutic effect of organic acids and essential oils compared with the toltrazuril on the clinical signs and mortality % of the broiler chickens challenged with E. tenella weakness, feather-ruffling, depression, and mortalities. Signs were relatively less severe in the organic acids medicated groups. The mortalities were treatment dependent. The mortality was comparatively the lowest in the organic acids medicated group (33.3 %, 5/15) followed by the amprolium and essential oils medicated (40 %, 6/15). Toltrazuril medicated group revealed the highest mortality rate (53.3 %, 8/15) among the medicated groups. Mortality rate was 60 % (9/15) in the positive control group. The negative control group didn't exhibit any abnormal signs till end of the experiment.

The relative body weight gain percent (RBWG %)
The effect of medication using organic acids and essential oils on the RBWG % are showed in Table 2. At the 7 th dpi (30 days old), a significant improvement in the RBWG % was observed in the medicated groups compared to the PC group (P-value < 0.05). Nevertheless, the performance of the challenged-medicated wasn't as good as the negative control (P-value < 0.05). The organic acids medication expressed improving effect on the RBWG % (40 %, 0.4 ± 0.1) which was similar to the toltrazuril (40 %, 0.4±0.0), comparatively, medication with the essential oils and amprolium HCl induced somewhat a lower RBWG % (30 %, 0.3±0.0 each). There were statistically nonsignificant variations between the medicated groups during the challenge period (P-value ≥ 0.05)

The Cecal gross observations and lesion score
The gross observations, mean cecal lesions scores (MLS), and reduction % in the MLS (RLS) related to the tested therapies are shown in Table 3 and Figs. 6. On the 7 th day post challenge, birds belonging to the negative control group had no lesions. Whereas for the positive control group, birds had average scoring, 3.5.
All birds medicated with the organic acids, essential oils, toltrazuril, and amprolium HCl scored 2.3±2, 2.3±2, and 2.3±.1 on average, respectively, so they were capable of reducing the severity of the lesions by 34 % which was significantly lower than the MLS of the positive control (P-value ˃ 0.05). Ceca of the medicated groups showed numerous serosal and mucosal petechiae and slight thickening of the wall (+ 2). Ceca of the positive control group were very distended, engorged with blood, and contained hardened cecal cores (score +4).

Microscopic findings
The histological lesions in the ceca observed on 7 th day post challenge are represented in Table 5 and Fig. 8. Normal histological appearance of the cecal sections from the control negative birds was observed. Cecal sections of the positive control birds displayed severe mucosal destruction which was represented in loss of plica peculiars, epithelial necrosis to complete villus and cryptal loss, necrosis of goblet cells, and diffuse mucosal hemorrhages. Diffuse eosinophilic and mononuclear cells infiltration in lamina propria and submucosa, and submucosal congestion.
Numerous various developmental stages of E. tenella inhabited the mucosal layers (epithelium and lamina propria). The toltrazuril medicated group showed short to atrophied plica, fewer localized inflammatory cells infiltration, hyperplasia and distension of goblet cells. Necrosis of villar, cryptal, and glandular epithelial. Mucosal and submucosal layers were markedly thickened and edematous. Fewer congestion was noticed. The amprolium medicated cecal sections revealed epithelial regeneration, marked submucosal edema, relatively few inflammatory cells infiltration, and most of the parasitic stages were destructed. To the organic acids medicated group, cecal sections displayed histological changes similar to the toltrazuril medicated except little epithelial damage. Goblet cells were minimally hyperplastic. More diffuse inflammatory cells infiltration in the lamina propria and submucosa.
Although extensive parasitological stages in the essential oils medicated group, the epithelial lining of the cecal sections was in a good intact condition. Epithelial hyperplasia and interepithelial eosinophil and mononuclear inflammatory cells infiltration occurred. Goblet cells were within normal range. The parasite mainly localized subepithelial in focal areas. Less thickened submucosal layer which was diffusely infiltrated with mononuclear inflammatory cells and no congestion. Although there was no interaction between organic acids and essential oils and coccidial challenge, the therapy improved the cecal tissue. All the medicated groups revealed increase in the surface area of the ceval villi in comparison to the negative and positive control groups as explained in table 5.   The RBWG is expressed as the mean ± standard errors (%). The differences between the groups are statistically significant (ANOVA, P-value < 0.001). Means within columns sharing similar superscripts within a column are not significantly different (P-value ≥ 0.05).      Lesion score +2 in the organic acids blend, essential oils blend, toltrazuril, and amprolium HCl (respectively) medicated groups showing petechiae, which were apparent on the serosal surface, slight thickening of the cecal wall, noticeable loss in the mucosal corrugations, and somewhat normal contents.

DESCUSSION
Chickens' coccidiosis led to significant economic losses, globally, due to the capacity of Eimeria spp. to exploit the host digestive micro-environment for growth and survival (Balta et al., 2021). In addition to the widely spread drug resistance of Eimeria species, phasing out the use of anticoccidials is a global target that started by the US Food and Drug Administration which introduced its first ban on an agricultural drug in 2005 followed by European countries, in 2006. As a result, numerous studies looked for less harmful alternatives for the general public's health (Muthamilselvan et al., 2016;Canal et al., 2022). So, the current study verified the Eimeria species in the clinically-ill broiler chickens in Assiut, Egypt, during September 2021 -March 2022 and concentrated on the therapeutic potential of the organic acids and essential oils blends against the most frequent Eimeria species during the study period.
The relative higher frequency of E. tenella, regardless of the short study period, may be attributed to that all the examined birds were introduced to the diagnostic labs due to an acute clinical disease outbreak. Also, many poultry breeders associate coccidiosis with the bloody diarrhea and mortality. This high frequency of E. tenella, E. acervulina, and E. maxima may be a warning sign of probable endemicity and need to be ascertained in large-scale studies. The high detection % of E. mitis call for the concern of subclinical coccidiosis that leads to impaired feed conversion, as feed costs 70% of production cost (Haug et al., 2008).
Even though different Eimeria spp. screening scale and clinical condition of the examined birds, partially parallel results had been reported in earlier studies done. In Assiut, Ahmed, (2018) found that E. tenella was the most common while E. brunetti was the least recorded. Diverse results were recorded by Mohamed et al. (2021), they stated that E. acervulina was the most prevalent followed by E. tenella, while E. mitis was the least verified species.
The further histopathological identification of an E. tenella within cecal sections of a chicken sample with heavy oocyst intensity, revealed the unique large sized secondary stage meronts (schizonts) characteristic to E. tenella arranged in clusters within the cecal mucosa and submucosa. The noticed large multiple (40-55 × 30-40 µm) in the current study were previously described by Tyzzer, (1929) and Fernando et al. (1983) in E. tenella infection in poultry where large second-generation meronts develop in the lamina propria within displaced epithelial cells.
Susceptibility of Eimeria spp. to organic acids and essential oils has been demonstrated by several records through studying infection level in chickens after their feed or water supplementation and in vitro parasite susceptibility (El-Shall et al., 2021s). Both were considered a potential alternative to anti-biotic growth promotors, AGPs, (Mustafa et al., 2021;Canal et al., 2022). Organic acids are natural organic compounds with acidic properties, the most public example are the short-chain fatty acids, SCFAs, (commonly propionic acid, acetic acid, and butyric acid). SCFAs are formed after carbohydrates fermentation by the beneficial intestinal microbiota (Rawi et al., 2021). Organic acids have the ability to retard the flourishment of pathogenic and opportunistic bacteria, e.g., Salmonella and E. coli, in the bird's feed and gut, impacting positively on performance (Nguyen et al., 2020). Organic acids acidify the microbial cell cytoplasm due to anions (RCOO -) and H + ions accumulation. Essential oils are volatile phytocompounds which obtained via distillation or mechanical cold pressing. Essential oils have antibacterial, antioxidant, antiparasitic, anti-inflammatory, antidiarrheal, and antimycotic properties. Using Organic acids blend or essential oils blend is more advantageous than single component in broiler chickens. The combination optimizes a synergistic effect between them and their most bioactive elements, hence become more effective than individual acid or oil (Polycarpo et al., 2017;Canal et al., 2022). The characteristics of each element will define the final effects of each oil and acid, at the same time, different combinations will result in different properties. Lately, branded marketable blends, as a water and feed additive of Organic acids and Essential oils, have been established and developed in broiler chickens reared without antibiotics (Canal et al., 2022).
Due to being an important pathogen in the poultry industry and the most encountered species in the current study, an E. tenella isolate was used for studying the therapeutic potential of Organic acids and Essential oils as anticoccidials. The clinical signs, mortality, RBWG %, MLS, RLS, oocysts shedding reduction %, and scoring the histopathological findings in the cecal tissue were assessed after concurrent therapy. Blends of organic acids (included orthophosphoric, propionic, acetic, lactic, citric, and formic acids) and essential oils (included cinnamaldehyde, carvacrol, anethol, terpinene, carvaone, menthol, and eucalyptol oils) were studied as anticoccidial therapies against E. tenella for 2 successive days and compared with the toltrazuril 5 % suspension.
Organic acids blend successfully diminished the clinical signs of coccidiosis and decreased the mortality rate to 33.3 % compared with 40 % amprolium HCl and 40 % in the Essential oils, and 53.3 % in the toltrazuril medicated groups. All gave better results than the PC (60 % mortality). This agreed with Balta et al. (2021).
The best RBWG % at the end of the trial was achieved in birds treated with the Organic acids (40 %) which equaled RBWG % in the toltrazuril medicated group, signifying a candidate of better weights during cecal coccidiosis outbreaks, with no significant differences for birds treated with the amprolium HCl (30 %) and essential oils (30 %). This came in line with Balta et al. (2021) and Canal et al. (2022).
Both the organic acids and essential oils blend behaved equally as the toltrazuril and the amprolium HCl in reducing the severity of cecal lesions induced by E. tenella in the challenged broiler chickens (34 %). This reduction % in lesions severity was higher than that obtained by Canal et al. (2022) (19.3 %) after using essential oils blend against E. tenella in broiler chickens. Balta et al. (2021) recorded the success of Organic acids blend in reducing the E. tenella induced cecal lesions.
Compared to birds from the positive control (13.8 x10 6 OPG) 7 dpi, chickens medicated with organic acids blend, toltrazuril, and amprolium HCl had non-significantly lower (P-value ≥ 0.05) oocysts burden in the cecal content (8.7 x10 6 and 11.4 x 10 6 OPG, respectively), being able to reduce it by 37 %, 20.6 %, and 17 %, respectively. When contrasted with those obtained from the Essential oils treatment (13.4 x10 6 OPG) and positive control, it can be concluded that the difference in the results was not significant. The failure of Essential oils blend to diminish oocyst count per gram cecal content refer to the indirect antiparasitic effect of these compounds against E. tenella. That wasn't in concurrence with the report of Canal et al. (2022) which stated that Essential oils blend able to redusce oocysts shedding by 94.92%. This objection could be conveyed to the difference in medication regimen as in the current study either Organic acids or Essential oils were applied for brief time (2 days) during the prepatent period of the parasite challenge (2 days postinfection). Most previous studies used organic acids and essential oils as feed or water additives during the bird life time.
The present results demonstrated that supplementing an organic acid and or essential oils blend during coccidiosis outbreak can share in enhancing recovery from infection.