Comparative effect of dietary supplements on the performance and severity of experimental Eimeria tenella infection in broiler chickens

The effect of the dietary supplements, sugarcane molasses (prebiotic), Antox® (probiotic) and Enflorax® (synbiotic), on gut health, performance and severity of infection with Eimeria tenella in broiler chickens was evaluated in this study. Ninety-day-old broiler chicks were divided into six groups (A, B, C, D, E and F). Groups B to F were challenged with Eimeria tenella (2.0 × 104 oocysts/chick). Groups C, D and E were supplemented from day old with prebiotic, probiotic and synbiotic, respectively, while F was administered amprolium. Groups A and B served as negative and positive controls, respectively. Feed intake and performance parameters were assessed weekly for each group. After infection clinical signs, morbidity and mortality rates were monitored alongside oocyst output, gross and microscopic caecal lesions. Infected chickens exhibited clinical signs 4 days post infection (dpi) with 100% morbidity in all infected groups. Infected groups showed significant (P < 0.05) drop in feed intake and weight gain from 3 to 5 weeks of age. Feed conversion ratio was highest in B but lower in the supplemented groups. Oocyst output in faeces were significantly lower (P < 0.05) in the supplemented groups compared with B. Macroscopic lesion scores 7 dpi were significantly lower in the supplemented groups compared with B, though group F had the lowest mean score. Histopathological examination of caeca tissues showed milder lesions in the supplemented groups. In conclusion, the supplements prebiotic, probiotic and synbiotic ameliorated the consequences of caecal coccidiosis in broiler chickens and therefore recommended for use in broiler production.


Introduction
Coccidiosis, the most economically important parasitic disease of poultry, is caused by members of the genus Eimeria and its usually characterized by gastrointestinal disturbances, high morbidity and mortality (Fanatico, 2006;Engidaw and Getachew, 2018). Several species of Eimeria have been identified in poultry (Fernandez et al. 2003) including the recently discovered Eimeria zaria, E. nagambie and E. lata (Blake et al. 2021), of which E. tenella and E. necatrix constitute the most pathogenic species (Musa et al. 2010;Owai and Mgbere, 2010) causing caecal and intestinal coccidiosis, respectively (Jatau et al. 2012). Coccidiosis is transmitted through the ingestion of feed, water, litter, or other materials contaminated with sporulated Eimeria oocysts (Owai and Mgbere, 2010;Patrick and Mgbere, 2010). The disease causes substantial economic losses (Haile, 2018), according to an estimate, the poultry industry loses about 14.4 billion USD annually to coccidiosis (Blake et al. 2020). Growth and feed utilization are known to be severely hampered by the parasite. The most severe damage is said to be inflicted by the developmental stages of the parasite (Engidaw and Getachew, 2018). Sporozoites penetrate various regions of the host's epithelial cells in the gut lining depending on the species of Eimeria. Eimeria tenella is known to have a predilection for caeca epithelia and is particularly notorious because schizogony also takes place in the lamina propria and crypts of caeca epithelia, causing extensive tissue destruction, hemorrhages and drop in feed intake (Taylor et al. 2007;Engidaw and Getachew, 2018). The merozoites lyse infected host's intestinal epithelial cells to infect new epithelial cells of the intestine, destroying the epithelial cells and in the process interfere with digestion and impair nutrient absorption and caeca function with resultant losses in productivity which is associated with the disease. Again, trophozoites, a developmental stage of the parasite, also absorb nutrients from the host which further deepens production deficits (Allen and Fetterer, 2002).
The control of coccidiosis relies majorly on chemoprophylaxis (the use of drugs) to prevent disease and vaccination with live or attenuated vaccines along side biosecurity measures and improved management (Kheirabdi et al. 2014). The frequent use of these medications results in increased production costs and the emergence of drug-resistant strains of Eimeria species (Chapman, 1997a;Pop et al. 2015). The sulphonamides, amprolium and toltrazuril are still popular in managing clinical coccidiosis which is a testimony to their effectiveness against the disease (Hafez, 2008). However, following widespread reports of Eimeria resistance to most conventional anticoccidials, public health concerns about their tissue residues and various side effects (Kheirabdi et al. 2014); a safer method of coccidiosis control is desired. Also, the use of antimicrobials at sub-therapeutic levels as growth promoters in poultry is considered hazardous and gradually being phased out (Portugalia and Fernandez, 2012). Upon the realisation of the connection between what transpires in the lumen of the gastrointestinal tract and numerous homeostatic phenomena in the body, it is no brainer that considerable interests have been kindled in modulation of critical components of the enteric microenvironment-the gut microbiota (Barbra et al. 2016), with the hope of beneficially influencing immunity, secretory, motility, sensory and neuroendocrine responses among others (Kabouridis and Pachnis, 2015;Thaiss et al. 2016;Dinan and Cryan, 2017). As the importance of the gut microbiota in health and disease is increasingly recognized, interest in interventions that can modulate the microbiota and its interactions with its host has also soared (Markowiak and Slizewska, 2017;Quigley, 2018).
The dietary supplements, prebiotics, probiotics and synbiotics, have emerged as potential alternatives for growth enhancers, for their beneficial effects on health and performance (Trafalka and Grazy, 2004;Dankowiakowska et al. 2013); they are also reported to be safe, healthy and less hazardous compared with synthetic growth promoters (Onyimonyi and Onu, 2009). The mechanisms underlying probiotics' beneficial effects are thought to be associated with probiotics' antagonistic effects on pathogenic microorganisms through the release of antibiotic compounds, competitive adhesion to the mucosa and epithelium, strengthening of the gut epithelial barrier and immune system modulation among others (Collado et al. 2010).
Probiotics are live microbial organisms used as dietary supplements which have positive impact on the host animal's gut microbial balance and health (Hassan et al. 2012). Prebiotics are non-digestible feed elements that have favourable effects on the host by encouraging the growth and activity of one or a few beneficial bacteria in the colon (Gibson et al. 2010). Synbiotics, on the other hand, are probiotic and prebiotic combinations (Yang et al. 2009). The prebiotics create an ideal environment for the probiotic organisms to thrive, flourish and multiply (Sekhon and Jairath, 2010). Sugarcane molasses is formed during sugar crystallization process, as the sugarcane is being crushed (van-Niekerk, 1981). It is the condensed residue which remains after removal of most of the sugar from the concentrated sugar juice. Sugarcane molasses have been used in feeds basically as an energy source (Mahala et al., 2013), but they also serve other purposes in animal health (Takara et al., 2007). It is rich in minerals and bioactive and phenolic compounds and has demonstrated antioxidant (Kitts et al., 2012) andprebiotic properties (Lukman andSuchada, 2018).
The use of these supplements has been adequately explored in the poultry industry especially in improving performance of birds and protection against enteric diseases. However, few have considered their comparative effects on the terribly devastating protozoal infection of poultry, coccidiosis. This study was therefore designed to determine the comparative effects of a prebiotic, probiotic and synbiotic products on the performance and gut health of broiler chickens infected with Eimeria tenella.

Housing and management of experimental chickens
The birds were housed at the experimental Poultry Research Pen of the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University Zaria, in cages with wire-floored batteries raised to a height of 10 cm off the floor of the pen. Electric bulbs and large netted wire-meshed windows were used to maintain the recommended temperature (30-32 °C). Feed and water were provided ad libitum throughout the study period. Feeders and drinkers (3 each) were provided per group with a 200-W bulb in each of the 6 compartments to provide light and heat. Feeders and drinkers were washed on a daily basis to minimize risks of contamination. Faecal droppings were screened for coccidia oocysts using simple floatation technique as described by Dryden et al. (2005) before experimental infection.

Eimeria tenella parasite
The Eimeria tenella oocysts isolated and characterized by Jatau et al. (2016), maintained in 2.5% potassium dichromate in the refrigerator (+ 4 °C) at the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria, were used for this study. The oocysts were propagated by infecting 2-week-old coccidian free broiler chicks. Progeny oocysts were recovered from caecal tissues and contents of the infected birds collected during post-mortem 7 days post infection, sporulated and purified as described elsewhere (Conway and McKenzie, 2007). The propagated oocysts were then stowed in distilled water at + 4 °C till required, 2 weeks after.

Experimental birds and infection
The study was performed in a 35-day period with experimental birds grouped into 6 having 15 birds each; each group was further divided into 3 replicates, containing 5 birds. A total of ninety (n = 90) Male commercial broiler chicks purchased at day old were randomly assigned into the 6 groups A, B, C, D, E and F. Group A was not supplemented or infected but served as the negative (noninfected) control. Group B birds were not supplemented but infected to serve as the positive (Eimeria tenella-infected) control. Groups C and D were supplemented with prebiotics and probiotics at 1 mL/L of drinking water while group E was supplemented synbiotic at 1 g/L of drinking water from day old till end of the study. Lastly, group F was not supplemented but administered Amprolium prophylactic treatment: 1 g/L of drinking water prior to infection and 0.25 g/L of drinking water thereafter for 5 and 7 days, respectively. All birds in the challenged groups B to F were gavage with 0.15 mL of water containing 2.0 × 10 4 Eimeria tenella-sporulated oocysts at 21 days of age as described by Kumar et al. (2014). All birds were fed a basal diet and water ad libitum. Feeds used were purchased from a commercial poultry feed distributor with the following composition ( Table 1). The second effect beside treatment is age, and its effects have been marked with other superscript letters and symbols in the tables.

Supplements/drug
Sugarcane molasses (prebiotic) used for the study were purchased from the local market in Zaria, and they comprised sugar, crude protein, crude ash, potassium, phosphorus, magnesium and sodium.
The anticoccidial drug "Amprolium 250 WSP," a saturated powder of Amprolium hydrochloride (HCl), water-soluble for use in drinking water of poultry, was used according to the producer's instruction.

Observation of clinical signs, morbidity, mortality and case fatality rates
Following infection onset of clinical signs, morbidity, mortality and case fatality rates were monitored, recorded and calculated for each group. The morbidity, mortality and case fatality rates were calculated as described by Bai et al. (2017):

Determination of feed intake, weight gains, feed conversion ratio and performance index
Live body weight and weight gain were assessed on a weekly basis while the feed intake, feed conversion ratio and performance index for each group were determined as described by Jahan et al. (2006) using the formulae below:

Faecal oocyst shedding
Faecal samples collected from each pen from 4 to 13 days post infection was kept in separate airtight plastic bags for determination of oocysts per gram (OPG) of faeces as described by Dalloul et al. (2003). The samples for each day were homogenized and stored at 4 °C until processed, and oocysts were counted microscopically using a McMaster counting chamber (Hodgson, 1970); all oocysts under the grid of each chamber in the McMaster were counted using a × 10 magnification. Results were then expressed as oocysts per gram of faeces (Dryden et al. 2005).

Caecal lesion scoring
At day 7 post infection, 3 chickens randomly selected from each group were humanely euthanized by cervical decapitation and the paired caeca were exteriorized for lesion scoring as described by Johnson and Reid (1970); caecal tissues were then harvested for microscopic examination.

Histopathology
Caecal tissues harvested were fixed in 10% formolsaline for 24 h. Fixed tissues were histologically processed according to the method of Bancroft et al. (1990). Tissues were dehydrated through ascending grades of alcohol (70%, 90% and 100%) for 2 h each. The tissues were then cleared in xylene for 2 h (1 h each, 2 changes). Tissues were impregnated/infiltrated in molten paraffin wax 2 h each (1 h each, 2 changes) and embedded in paraffin wax. The tissue blocks were sectioned at 5-micron thickness using Rotary microtome machine (Leica RT 25 made in England). Sectioned tissues were mounted on slides, dried Daily feed intake (g∕bird∕day) = Feed of fered − Feed lef t Number of chicks Feed conversion ratio = Feed intake during a period (g) Weight gain in that period (g) Performance index = Average live weight in kg FCR × 100 and stained using hematoxylin and eosin (H and E) stain. Photomicrograph of stained slides of the tissues was taken using Amscope Digital Camera for microscope version 3.0, made in Japan.

Statistical analyses
Data obtained were analysed with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test, except for lesion scores which were analysed by Kruskal-Wallis non-parametric test, using GraphPad Prism version 8.0.2 (GraphPad software, San Diego, CA, USA) for windows. Two-way ANOVA was used with the treatment and age (week) effect to compare effects within and across the groups. Values of P ≤ 0.05 were considered significant. The analysed data are presented in tables and charts for easy comparison and understanding.

Clinical signs
Four days post infection (dpi), infected chickens showed signs of depression with whitish, mucoid diarrhoea and watery brown faeces. By the fifth day, feed intake dropped significantly in infected groups. Whitish, mucoid and brown faeces were seen with minute traces of blood in the infected groups. By 6 dpi, faecal droppings had become fully bloody (projectile) diarrhoea. Two mortalities occurred in the positive control group. Other signs observed were anorexia, somnolence, ruffled feather and weight loss. These signs appeared fairly milder in the amprolium treated and supplemented groups compared to the positive control.

Morbidity, mortality and case fatality rates
Morbidity was 100%. All inoculated chickens showed clinical signs of the infection in all groups. Mortality was 13.33% (two deaths were recorded in the positive control group; no other group had mortality). Case fatality was then determined, to be 8.33% in the affected group. Table 2 shows total feed intake per bird in all groups throughout the study period. Feed intake dropped after the infection at week 3 and gradually picked up again. There was significant (P < 0.05) difference between the feed intake in the non-infected (negative control) and the infected groups and, also, between the infected non-supplemented (positive control) and the infected but supplemented groups. The highest feed intake was recorded in the non-infected (negative) control group.

Live body weight
Live body weight gains in the supplemented groups were significantly (P < 0.05) higher than infected non-supplemented (positive control) group (Table 3) compared with the infected but supplemented groups. Among supplemented groups, maximum weight gain was shown by the prebiotic group which was supplemented with sugarcane molasses followed by the groups supplemented with synbiotic and probiotic. Live body weight was better in the non-infected (negative) control group compared with the infected groups at the end of the study except for group C, the prebiotic group.

Weight gain
Body weight gains in the supplemented groups were significantly (P < 0.05) higher than infected non-supplemented (positive) control group (Table 4). Among supplemented groups, maximum weight gain was noted in the prebiotic group which was supplemented with sugarcane molasses followed by the groups supplemented with synbiotic and probiotic products, respectively, throughout the study. The Table 2 Effect of prebiotic, probiotic and synbiotic supplementations on feed intake per bird in all groups in E. tenella-infected broiler chickens Means with different superscript letters ( a, b, c, d ) across rows and symbols (º , ⁕, #, *, " ) within columns are significantly (P < 0.05) different. Group A is the negative control, B is positive control (non-supplemented but infected) group, C is supplemented prebiotic and infected, D is supplemented probiotic and infected, E is supplemented synbiotic and infected while F was administered Amprolium hydrochloride prophylactic treatment before infection  Means with different superscript letters ( a, b, c ) across rows and symbol (•, º , ⁕, #, * , ") within columns are significantly (P < 0.05) different. Group A is non-supplemented uninfected (negative control), B is non-supplemented but infected (positive control) group, C is supplemented prebiotic and infected, D is supplemented probiotic and infected, E is supplemented synbiotic and infected while F was administered Amprolium hydrochloride prophylaxis  supplemented groups also gained more weight than the control groups prior to infection.

Feed conversion ratio
The results of FCR (Table 5) revealed that the FCR values of the prebiotic group were lower compared with other groups prior to infection but rose after infection. Furthermore, the supplemented groups showed lower (P < 0.05) FCR compared with the amprolium-medicated and infected (positive control) groups at weeks 3 and 4. Among the supplemented groups, the best FCR was observed in the prebiotic group.

Performance index
The analysis of weekly performance index showed higher performance in the supplemented groups. There was significant difference (P < 0.05) between uninfected (negative) control and the infected chickens 7 dpi in performance. The supplemented groups resolved the losses in production more rapidly by 14 dpi (week 5) compared even to group F, medicated amprolium prophylactic treatment. The prebiotic, synbiotic and probiotic groups showed remarkable improvement, akin with the negative control (Table 6). Figure 1 shows caecal macroscopic lesion scores by the method of Johnson and Reid (1970). Intestinal macroscopic lesions were completely absent in the negative control group, but present in the positive control, supplemented and medicated groups. The amprolium-medicated group (F) showed significantly (P < 0.05) lower lesion score than the supplemented and infected groups. Among the supplemented groups, the least mean lesion scores were seen in the prebiotic supplemented group.

Faecal oocyst shedding
The result of the faecal oocyst shed (Fig. 2) revealed a pattern relatively similar to that of mean lesion scores among different groups. Oocyst output were significantly (P < 0.05) lower in the Amprolium-medicated group compared with positive (infected) control group. The Amprolium-medicated group also had a much lower oocyst output compared with the supplemented groups. Among supplemented groups, the maximum reduction in oocyst counts was seen in the prebiotic group followed by the probiotic and synbiotic groups, respectively. Oocysts were detected in faeces from 6 dpi to the end of the study, with the highest output occurring at 8 dpi after which it began to decline steadily. Means with different superscript letters ( a, b, c ) across rows are significantly (P < 0.05) different. Group A is non-supplemented uninfected (negative control), B is non-supplemented but infected (positive control) group, C is supplemented prebiotic and infected, D is supplemented probiotic and infected, E is supplemented synbiotic and infected while F was administered Amprolium hydrochloride prophylactic treatment before infection  Table 6 Effect of prebiotic, probiotic and synbiotic supplementations on performance index in E. tenella-infected broiler chickens Means with different superscript letters ( a, b, c ) across rows and symbol (•, º , ⁕, #, * ) within columns are significantly (P < 0.05) different. Group A is non-supplemented uninfected (negative control), B is non-supplemented but infected (positive control) group, C is supplemented prebiotic and infected, D is supplemented probiotic and infected, E is supplemented synbiotic and infected while F was administered Amprolium hydrochloride prophylactic treatment and infected

Discussion
Coccidiosis remains a major limiting factor challenging the optimization of the poultry industry in spite of the numerous research and management endeavors geared towards curbing the menace. Caecal coccidiosis caused by Eimeria tenella is a potent threat to global poultry production and food security group exhibited the least severity in clinical signs as expected since amprolium is a standard anticoccidial drug. The milder severity seen in the supplemented groups compared with the infected (positive) control group may have resulted from a possible resistance to colonization of caecal epithelium or a more rapid immune response. Sugarcane molasses like most prebiotics, rich in sugars reportedly compete for sugar receptors with enteric pathogens and thus prevent their adhesion and subsequent colonization (Iji and Tivey, 1998;Awais et al. 2011) which consequently enhance their elimination with the flow of digesta (Fernandez et al. 2002). Rolfe (1991) stated that one of the means probiotic organisms (which are contained in Antox and Enflorax) exert their protective effects on a host is through the "competitive exclusion" of pathogenic organisms, by adhering to the mucosal surface to prevent establishment of pathogens on the mucosa. It may be inferred, therefore, that the clinical signs were less severe in the supplemented groups because these supplements resisted Eimeria tenella-sporulated oocysts from colonizing the caeca of the broiler chickens. Also, morbidity rate recorded in this study was 100%, all inoculated chickens succumbed to the infection in all groups while mortality rate recorded was 13.33% in the infected control group only. This is consistent with the findings of Fanatico (2006), who stated that morbidity in coccidiosis is very high. Also, antioxidant properties of the supplements may have generated oxidative stress on the parasites and neutralized reactive oxygen species (Alia et al. 2019), accounting for the greater survival rate seen in supplemented groups. Holdsworth et al. (2004) specified that the World Association for the Advancement of Veterinary Parasitology (WAAVP) needs the determination of live body weight, weight gain and feed conversion ratio to appraise the severity of infection. A direct correlation exists between these performance parameters and the severity of a coccidia infection. The term "performance' is used by poultry producers and researchers all over the world to judge level of success in terms of financial returns and management, and this objective is assessed through various parameters like live body weight gain, weight gain, feed intake, feed conversion ratio, performance index, and so on.
In the present study, feed intake was found to be significantly higher (P < 0.05) in the uninfected (negative) control than in the infected groups from 3 to 5 weeks post infection. Eimeria is known to cause depression and injury to intestinal tissues thus discouraging feed intake (Taylor et al. 2007;Engidaw and Getachew, 2018). Among infected groups, the infected (positive control) group showed a significantly lower feed intake (Table 2) than others. The better feed intake in the supplemented groups that administered sugarcane molasses, Antox and Enflorax, is not unrelated to the aforementioned resistance to Eimeria colonization and less severity in clinical signs hitherto.
Live body weight gained in Table 3 shows that the sugarcane molasses group had the highest final mean live weight gain (2245.23 ± 29.46 g) at the end of the study at week 5 which was slightly higher (numerically) than the negative control (2215.30 ± 37.40 g) though not statistically significant. Compared with the negative control group, there was significant fall in live body weight gain from 3 to 5 weeks in chickens infected Eimeria tenella, similar to the trend observed in the weekly weight gain assessment (Table 4). Coccidiosis is known to exert such effects in infected chickens (Taylor et al. 2007;Engidaw and Getachew, 2018). Overall body weight gain of the supplemented and negative control groups was significantly higher (P < 0.05) than the infected non-supplemented group. The considerable increase in mean live weight gain in supplemented groups  191 Tropical Animal Health and Production (2022) 54: 191 as compared to the infected control group could be attributable to suppression of inflammation in the intestinal mucosa, the implication of which is higher nutrient absorption across the intestinal wall and improved feed conversion ratio in the groups (Gotep et al. 2016).
Weight gain assessed on a weekly basis shows the body weight gain from 3 to 5 weeks of age were significantly reduced in all infected groups (Table 4) compared with the uninfected (negative) control. The positive control group had the least body weight gain in that period. Amproliummedicated group recorded better weekly weight gain than the positive control group as should be expected. Since Amprolium is a standard anticoccidial drug. Variations in body weight gain in broiler chickens challenged with coccidiosis have been linked to changes in maintenance requirements, malabsorption of feed and reduced feed intake (Zhang et al. 2016). When birds are infected with coccidiosis, their nutritional needs are known to increase within the first week of infection (Cornelissen et al. 2009;Cox et al. 2010). Increased metabolic expenditures for healing injured tissues, immune system stimulation and diminished capability to use and transform nutrients into energy are only a few of the documented maintenance requirements (Chen et al. 2016;Grenier et al. 2016). The body's innate immune system can also create extra collateral damage, such as fever and inflammatory reactions, which further deplete resources, reduce nutritional stores and increase catabolism (Klasing and Iseri, 2013;Iseri and Klasing, 2014). These account for the severe losses in weight gain recorded in the infected chickens.
Another parameter, the feed conversion ratio (FCR), determined weekly was significantly lower in the prebiotic group prior to infection when compared with other groups. This is not surprising, given that sugarcane molasses is rich in sugars which readily supplied glucose, alongside crude ash and proteins. Sugarcane molasses is also known to have rich antioxidant properties (Valli et al. 2012); likewise, probiotic organisms are known to bestow potent antioxidant rich properties to their host (Serban, 2015;Tian et al. 2017). Upon Eimeria challenge, the rise in FCR seen from 3 to 5 weeks of age in all infected groups is consistent with coccidiosis (Alia et al. 2019). However, the supplemented groups, having had higher resistance to colonization, feed intake and a less severe inflammation with destruction of caeca tissues, showed a much lower FCR compared with the infected control.
The results of the performance index point to a significant (P < 0.05) difference between the negative and positive controls with amprolium-medicated groups. A significant difference also existed between the negative control and amprolium-medicated groups and the supplemented groups. Although Geier et al. (2009) reported that prebiotics did influence intestinal microbiota and not performance of broiler chickens, several studies have reported significant increase in performance and live weight gains (Hildalgo et al. 2009;Ndelekwute et al. 2010;Habibu et al. 2014). Gultemirian et al. (2014) and Hwangbo et al. (2013) attributed the better performance to increased short chain fatty acid production in the caeca and lowered intestinal pH which enhanced resistance to pathogens. Moreover, prebiotic ingredients (inulin, oligofructose, fructo-oligosaccharides and maltedextrin) present in the synbiotic supplement enhanced the performance of that group. Overall performance was better in the supplemented groups compared with the infected (positive) control, akin with the uninfected (negative) control suggesting that the supplements amended production inefficiencies typical of caecal coccidiosis.
No oocysts were detected in the faeces of birds in the negative control group, whereas oocyst shedding in faeces rose steadily from the sixth to eighth day post challenge in the infected groups and began to drop gradually; thereafter, oocyst shedding in the supplemented groups was significantly lower (P < 0.05) compared with the positive control group. Among supplemented groups, birds which received sugarcane molasses shed fewer oocysts while those medicated amprolium hydrochloride shed the least oocysts during the same period (P < 0.05). Oocysts were detected in the faeces of infected chickens up to 13 dpi. Shedding of oocysts in faeces of infected chickens even after the clinical disease is common in coccidiosis (Conway and Mckenzie, 2007;Engidaw and Getachew, 2018). The effectiveness of probiotic organisms in reduction of oocyst shedding is well documented (Dalloul et al. 2003;Caballero-Franco et al. 2007;Lee et al. 2011;Fatoba et al. 2018). Since Eimeria is an intracellular parasite, it has to infiltrate host cells by first sticking to epithelial surfaces in order to reproduce. Probiotic bacteria that have colonized the gut may compete for adhesion sites and receptors on epithelial cells. This prevented Eimeria oocysts from infiltrating and penetrating, as well as their reproducing and shedding oocysts. In broiler chickens, probiotics are said to have an immunoregulatory effect on the local immune system and increased resistance to Eimeria (e.g. reduced oocyst shedding) and also increased antibody production against E. tenella (Lee et al. 2007). Antioxidant-rich properties of the supplements must have played a key role here as well, generating reactive oxygen radicals that may have resulted in toxic insult to the parasite with subsequent oxidative damage and death.
The data with regard to macroscopic caeca lesion scores and histopathology by day 7 post infection indicates that E. tenella provoked significant injuries to the intestinal mucosa. The lesions seen in the caeca of E. tenella-infected broiler chickens fed prebiotic, synbiotic and probiotic showed significantly (P < 0.05) lesser mean lesion scores and histopathology compared to the positive control. Severe destruction of caecal mucosal layer and penetration of epithelial cells with resultant desquamation of caecal epithelium due to the attachment and invasion by E. tenella are characteristic of the disease (Zulpo et al. 2007;Bould et al. 2009). Histopathological lesions in E. tenella infection include the loss of caecal villi, necrosis and haemorrhages of caecal mucosa (Adamu et al. 2013;Sharma et al. 2015;Abdelrazek et al. 2020) as seen in the present study. The reduction in caeca lesion scores and histopathology by the supplements may be attributed to lower intestinal pH and conditions appropriate to increase useful microflora (Taherpour et al. 2012). The amprolium-medicated group naturally had the least mean lesion score. Lesions in coccidiosis are said to depend on degree of inflammation and injury to gut health and often include thickened intestinal wall, blood-tinged exudates, petechial hemorrhages, hemorrhagic enteritis, profuse bleeding in the caeca and necrosis (Engidaw and Getachew, 2018). The use of probiotic organisms have been widely confirmed to reduce microscopic lesions in chickens infected with coccidiosis (Ritzi et al. 2014;Chen et al. 2016).
In conclusion, the standard drug (Amprolium) offered protection against severe clinical manifestations, mortality and equally reduced performance deficits consistent with caecal coccidiosis compared with the positive (infected) control. However, it did not enhance overall productivity in the infected chickens as much as the prebiotic, probiotic and synbiotic supplemented groups as seen in 13 obtained in weekly weight gain, live body weight gain, feed conversion ratio and the performance index. The dietary supplements offered adequate preservation of gut health following challenge with Eimeria tenella, as seen in mean caeca lesion scores, histopathology and faecal oocyst shedding as much as prophylactic treatment with Amprolium hydrochloride which points to their ability to enhance resistance to Eimeria colonization. Therefore, the use of these supplements is recommended for broiler production.