Efficacy of Saccharomyces boulardii in mitigating necrotic enteritis induced by Clostridium perfringens in broiler chickens

Introduction

The global poultry industry plays a critical role in food and nutrition security by supplying affordable, high-quality animal protein to a growing population. Rapid intensification of poultry production through genetic improvement, balanced nutrition, enhanced biosecurity, and efficient management has substantially increased productivity worldwide. India has emerged as a major contributor, ranking as the world's second-largest egg producer and fourth-largest broiler meat producer (Honyal et al., 2025; DAHD, 2025) [1,2] . Despite these advances, enteric diseases remain a major constraint to broiler production, adversely affecting growth performance, animal welfare, and economic returns.

Necrotic enteritis (NE), caused by the Gram-positive, sporeforming, anaerobic bacterium Clostridium perfringens, is one of the most economically important enteric diseases in poultry. The disease occurs in both clinical and subclinical forms, resulting in mortality, impaired growth, poor feed conversion ratio (FCR), intestinal damage, and increased carcass condemnation (Shojadoost et al., 2012; Fathima et al., 2022) [3,4] . Broiler chickens between 2 and 6 weeks of age are particularly susceptible (Timbermont et al., 2011) [5] . Although C. perfringens is a normal inhabitant of the intestinal microbiota, factors such as coccidial infection, high-protein diets, poor litter quality, and high stocking density promote bacterial overgrowth and toxin production (McDevitt et al., 2006) [6] . Among the toxins produced, NetB has been recognized as a major virulence factor associated with avian NE (Keyburn et al., 2008) [7] . For many years, necrotic enteritis was effectively controlled through the routine use of antimicrobial growth promoters (AGPs). However, increasing concerns regarding antimicrobial resistance (AMR), food safety, and consumer preference have led to restrictions on AGP use, creating an urgent need for sustainable alternatives to control C. perfringens infections (Diaz et al., 2019; Abd El-Hack et al., 2020) [8,9] . Consequently, nutritional strategies incorporating probiotics, prebiotics, organic acids, phytogenic compounds, and exogenous enzymes have gained considerable attention for maintaining intestinal health and improving disease resistance (Moore, 2016; Bansal et al., 2021; Moore, 2024) [10,11,12] . Among these alternatives, probiotics have shown considerable promise in reducing enteric diseases by competitively excluding pathogens, producing antimicrobial metabolites, enhancing intestinal barrier function, and modulating host immune responses (Bermudez-Brito et al., 2012; Liao & Nyachoti, 2017; Yaqoob et al., 2022) [13,14,15] . Various bacterial and yeast probiotics have demonstrated beneficial effects against C. perfringens infection and improved productive performance in broiler chickens (Jha et al., 2020; Emami et al., 2021) [16,17] . Saccharomyces boulardii, a non-pathogenic probiotic yeast, has attracted considerable interest because of its unique biological properties and established safety profile. Unlike many bacterial probiotics, S. boulardii secretes proteolytic enzymes capable of degrading bacterial toxins, enhances intestinal barrier integrity, and modulates mucosal immune responses, thereby protecting the intestinal epithelium from pathogen-induced injury (Mumy et al., 2008; Pothoulakis, 2009; Rajput et al., 2013) [18,19,20] . Dietary supplementation with S. boulardii has also been associated with improved growth performance, feed efficiency, intestinal histomorphology, and overall gut health in poultry (Yaqoob et al., 2022) [15] . Despite these promising biological properties, studies evaluating the efficacy of S. boulardii against experimentally induced Clostridium perfringens-associated necrotic enteritis in broiler chickens remain limited. Most available reports have focused on Saccharomyces cerevisiae or mixed probiotic preparations, leaving limited evidence regarding the protective efficacy of S. boulardii under controlled challenge conditions (Caly et al., 2015) [21] . Therefore, the present study was undertaken to evaluate the efficacy of prophylactic and therapeutic supplementation with Saccharomyces boulardii in mitigating experimentally induced necrotic enteritis caused by Clostridium perfringens in broiler chickens by assessing growth performance, serum biochemical profile, intestinal pathology, intestinal morphometry, and bacterial colonization.

Materials and Methods

Experimental birds and management

A total of 100 healthy day-old Vencobb broiler chicks and reared for 38 days under standard management conditions. Birds were housed in separate floor pens bedded with rice husk litter and provided ad libitum access to feed and drinking water throughout the experimental period. Routine vaccination against Newcastle disease and infectious bursal disease was carried out according to standard poultry management practices.

Experimental design and induction of necrotic enteritis

The birds were randomly allocated into five experimental groups (T1-T5), each consisting of 20 birds. Group T1 served as the unchallenged control and received only the basal diet. Birds in groups T2, T3, T4, and T5 were subjected to experimental induction of necrotic enteritis. To predispose birds to necrotic enteritis, a tenfold dose of Livacox® Q coccidia vaccine was administered orally on day 13. On day 16, birds were subjected to overnight feed withdrawal as described by Gholamiandehkordi et al. (2007) [22] . Experimental infection was then induced by oral administration of Clostridium perfringens Type A culture (1 × 10⁷ CFU/mL) at a dose of 1 mL per bird for three consecutive days from day 17. 2 and confirmed spectrophotometrically at 600 nm (OD₆₀₀ = 0.756). The culture was subsequently diluted with sterile phosphatebuffered saline to obtain the desired challenge concentration of 1 × 10⁷ CFU/mL (Rathnapraba, 2021) [23] .

The treatment groups were as follows

Probiotic and antibiotic supplementation

The probiotic yeast Saccharomyces boulardii was incorporated into the feed at a concentration of 1 × 10⁸ CFU/kg feed. Enramycin was supplemented at 0.1 g/kg feed according to the experimental design. Prophylactic supplementation was initiated from day 1, whereas therapeutic supplementation commenced following the onset of clinical signs associated with necrotic enteritis.

Parameters studied Clinical observations and growth performance

All birds were monitored twice daily throughout the experimental period for the occurrence of clinical signs, morbidity, and mortality. Body weight and feed intake were recorded weekly. Body weight gain and feed conversion ratio (FCR) were calculated from the weekly data.

Serum biochemical analysis

Blood samples were collected from the heart and wing vein on 0 th , 7 th , 14 th and 21 st day post-infection. Serum was separated and stored at -20°C until analysis. Serum total protein, albumin, globulin, albumin-to-globulin (A/G) ratio, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were estimated using a fully automated biochemical analyzer following the manufacturer's instructions.

Gross and histopathological examination

Necropsy examinations were performed on all birds that died during the experiment as well as on representative birds sacrificed at day 24 (7 th day post-infection) and day 38 (21 st day post-infection). Gross lesions were recorded in the liver, spleen, kidneys, bursa of Fabricius, and intestinal tract (duodenum, jejunum, and ileum). For histopathological examination, tissue samples from the duodenum, jejunum, ileum, liver, spleen, kidneys, and bursa of Fabricius were collected in 10% neutral buffered formalin. Following routine tissue processing, paraffinembedded sections (4-6 µm) were prepared and stained with hematoxylin and eosin according to the method described by Luna (1968) [24] .

Intestinal morphometry

Jejunal and ileal tissue sections collected on day 24 and day 38 were subjected to morphometric evaluation. Villus height, crypt depth, and villus height-to-crypt depth ratio (VH:CD) were measured using an ocular micrometer to assess intestinal integrity and absorptive capacity.

Re-isolation and enumeration of Clostridium perfringens

Intestinal contents collected from the ileo-caecal region on day 24 and day 38 were used for re-isolation and enumeration of C. perfringens. Samples were enriched in Reinforced Clostridial Broth under anaerobic conditions and subsequently cultured on Tryptose Sulfite Cycloserine (TSC) agar and Sulfite Polymyxin Sulfadiazine (SPS) agar. Suspected colonies were confirmed by Gram staining, lecithinase activity on egg yolk agar, and stormy fermentation in litmus milk. For quantitative enumeration, one gram of intestinal content was serially diluted in sterile phosphate-buffered saline and plated on TSC agar. After anaerobic incubation at 37°C for 24 h, characteristic black colonies were counted and expressed as log₁₀ CFU/g of intestinal content.

Statistical analysis

All data were analyzed using a Completely Randomized Design (CRD) according to the procedures described by Snedecor and Cochran (1989) [25] . Results were expressed as mean ± standard error (SE). Differences among treatment groups were considered statistically significant at P<0.05.

Results

Growth Performance Parameters

Feed intake Feed intake did not differ among treatment groups during the first week (P>0.05) (Table 1), whereas significant differences were observed from the second week onward (P<0.05) (Table 1). During week 2, birds receiving prophylactic Saccharomyces boulardii (T4) exhibited the highest feed intake (297.60 ± 0.57 g/bird), followed by the enramycin-treated group (T3; 289.00 ± 0.41 g/bird), whereas T1, T2, and T5 showed comparable feed intake (281.50-282.10 g/bird). Following Clostridium perfringens challenge, feed intake declined in all challenged groups during week 3, with the greatest reduction observed in the therapeutic probiotic group (T5; 595.00 ± 0.41 g/bird) and the infected untreated group (T2; 599.75 ± 0.75 g/bird). The negative control (T1) maintained the highest feed intake during the challenge period (611.80 ± 0.27 g/bird). During the final week, T2 recorded the lowest feed intake (1072.13 ± 0.63 g/bird), whereas T3 maintained the highest intake among the challenged groups (1095.25 ± 0.49 g/bird), followed by T4 (1083.88 ± 0.83 g/bird) and T5 (1081.06 ± 0.54 g/bird).

Body weight

Body weight did not differ among treatment groups during the first week (P>0.05), whereas significant differences were observed from week 2 onward (P<0.05) (Table 1). During week 2, T3 recorded the highest body weight (414.50 ± 1.26 g), whereas T5 showed the lowest value (394.50 ± 1.26 g). Following bacterial challenge, body weight decreased markedly in the infected untreated group. During week 3, T2 recorded the lowest body weight (738.00 ± 2.55 g), while T1, T3, T4, and T5 remained statistically comparable (813.75-823.00 g). This trend continued during weeks 4 and 5, with T2 consistently exhibiting the lowest body weight (1215.00 ± 1.08 and 1989.00 ± 4.53 g, respectively). By the end of the experiment, birds in T4 achieved a final body weight (2230.15 ± 3.22 g) comparable to the negative control (2233.45 ± 2.11 g), whereas T3 and T5 recorded intermediate values.

Body weight gain

Weekly body weight gain did not differ among treatment groups during the first week but varied significantly from week 2 onward (P<0.05) (Table 1). Before bacterial challenge, T3 exhibited the highest body weight gain during week 2 (260.50 ± 0.50 g), whereas T5 recorded the lowest gain (244.25 ± 0.46 g). Following C. perfringens challenge, birds in T2 demonstrated a marked reduction in body weight gain, recording the lowest value during week 3 (328.90 ± 3.07 g), compared with T1 (421.23 ± 4.07 g), T4 (415.35 ± 3.90 g), and T5 (419.25 ± 5.28 g). During week 4, T1 maintained the highest body weight gain (547.00 ± 4.40 g), whereas T2 remained significantly lower (477.00 ± 2.38 g). By week 5, T4 (919.40 ± 4.79 g) and T5 (914.88 ± 4.36 g) recorded the highest body weight gains and did not differ significantly from each other, whereas T2 continued to show the lowest body weight gain (774.00 ± 5.05 g).

Feed conversion ratio:

Feed conversion ratio (FCR) did not differ among treatment groups during the first week (P>0.05) but differed significantly during weeks 2-5 (P<0.05) (Table 1). During week 2, T4 recorded the highest FCR (1.18 ± 0.01), whereas T2 and T3 showed the lowest values (1.10-1.11). Following C. perfringens challenge, FCR increased markedly in the infected untreated group, reaching 1.82 ± 0.02 during week 3 and remaining the highest during the final week (1.39 ± 0.01). In contrast, birds receiving prophylactic S. boulardii (T4) and therapeutic supplementation (T5) recorded the lowest FCR during week 5 (1.18 ± 0.01), followed by T3 (1.22 ± 0.01).

Serum Biochemical Parameters

Serum total protein concentrations did not differ among treatment groups before bacterial challenge (0 th day; P>0.05), whereas significant differences were observed on 7 th , 14 th , and 21 st day post-infection (P<0.05) (Table 2). On 7 th day, the infected untreated group (T2) recorded the lowest serum total protein concentration (2.95 ± 0.06 g/dL), which was comparable to T5 (2.99 ± 0.03 g/dL) but significantly lower than T1 (4.25 ± 0.08 g/dL), T3 (4.00 ± 0.02 g/dL), and T4 (3.96 ± 0.10 g/dL). On 14 th day , T2 (3.32 ± 0.05 g/dL) remained significantly lower than all other groups, whereas T5 (3.59 ± 0.07 g/dL) recorded intermediate values. By 21 st day, serum total protein concentrations increased in all treatment groups; however, T2 (3.60 ± 0.04 g/dL) remained significantly lower than T1 (4.33 ± 0.08 g/dL), T3 (4.19 ± 0.08 g/dL), T4 (4.10 ± 0.02 g/dL), and T5 (3.94 ± 0.07 g/dL). Serum albumin concentrations did not differ significantly among treatment groups throughout the experimental period (P>0.05).

Although numerically lower albumin concentrations were recorded in T2 and T5 following bacterial challenge, the differences were not statistically significant at any sampling interval. Serum globulin concentrations were comparable among treatment groups before infection (P>0.05) but differed significantly on 7 th , 14 th , and 21 st day post infection(P<0.05) (Table 2). On 7 th day, T1 (2.07 ± 0.17 g/dL), T3 (1.95 ± 0.11 g/dL), and T4 (1.99 ± 0.19 g/dL) recorded significantly higher globulin concentrations than T2 (1.31 ± 0.10 g/dL) and T5 (1.11 ± 0.10 g/dL). A similar trend was observed on 14 th day, with T2 (1.47 ± 0.08 g/dL) and T5 (1.62 ± 0.09 g/dL) recording significantly lower globulin concentrations than T1, T3, and T4. By 21 st day, T1 (2.04 ± 0.13 g/dL) and T3 (1.94 ± 0.19 g/dL) maintained significantly higher globulin concentrations than the remaining treatment groups, whereas T2 (1.38 ± 0.19 g/dL) recorded the lowest value. No significant differences were observed in the albumin-toglobulin (A/G) ratio throughout the experimental period (P>0.05). The A/G ratio ranged from 1.09 ± 0.05 to 1.10 ± 0.17 before infection and from 1.04 ± 0.16 to 1.76 ± 0.23, 1.08 ± 0.13 to 1.28 ± 0.13, and 1.15 ± 0.12 to 1.75 ± 0.35 on 7 th , 14 th , and 21 st day post-infection, respectively. Serum cholesterol concentrations were comparable among treatment groups before bacterial challenge (P>0.05) but differed significantly on 7 th , 14 th , and 21 st day post-infection (P<0.05) (Table 2). On 7 th day, T2 recorded the lowest cholesterol concentration (101.98 ± 0.57 mg/dL), followed by T5 (118.28 ± 0.38 mg/dL), T4 (123.11 ± 0.65 mg/dL), T3 (128.00 ± 0.76 mg/dL), and T1 (134.75 ± 0.64 mg/dL). Similar differences were observed on day 14, with cholesterol concentrations ranging from 112.37 ± 0.73 mg/dL in T2 to 141.00 ± 0.47 mg/dL in T1. On 21 st day, T1 (145.30 ± 0.40 mg/dL) maintained the highest cholesterol concentration, whereas T2 (131.16 ± 0.33 mg/dL) recorded the lowest value. No significant difference was observed between T4 and T5 on 21 st day. Serum alanine aminotransferase (ALT) activity did not differ among treatment groups before infection (P>0.05) but differed significantly on 7 th , 14 th , and 21 st day post-infection (P<0.05) (Table 2). The infected untreated group (T2) consistently recorded the highest ALT activity, with values of 52.83 ± 0.47, 50.13 ± 0.50, and 46.78 ± 0.49 IU/L on 7 th , 14 th , and 21 st day, respectively. In contrast, T1 recorded the lowest ALT activity, whereas T3, T4, and T5 showed intermediate values throughout the post-infection period. Serum aspartate aminotransferase (AST) activity was comparable among treatment groups before infection (P>0.05) but differed significantly on 7 th , 14 th , and 21 st day post-infection (P<0.05) (Table 2). The infected untreated group recorded the highest AST activity, whereas T1 maintained the lowest values throughout the study. T3, T4, and T5 exhibited intermediate AST activities at all postinfection sampling intervals. Serum creatinine concentrations did not differ among treatment groups before bacterial challenge (P>0.05) but differed significantly following infection (P<0.05) (Table 2). The infected untreated group recorded the highest creatinine concentrations, whereas T1 maintained the lowest values throughout the post-infection period. Birds in T3, T4, and T5 exhibited intermediate creatinine concentrations at all sampling intervals.

Gross Pathological Findings

No gross lesions were observed in birds of the negative control group (T1) throughout the experimental period. On the 24 th day, birds in the infected untreated group (T2) developed characteristic lesions of necrotic enteritis. The small intestine, particularly the jejunum and ileum, was markedly distended with gas and exhibited thin, friable intestinal walls. The duodenal and jejunal mucosa showed severe congestion, multifocal hemorrhages, ulceration, and adherent fibrinonecrotic pseudomembranes, producing the characteristic "turkey towel" appearance. Blood-tinged intestinal contents and fibrinous casts were frequently observed within the intestinal lumen. In addition to intestinal lesions, hepatomegaly (Fig Birds receiving therapeutic S. boulardii (T5) initially exhibited lesions comparable to those of T2 on the 24 th day; however, by the 38 th day, substantial restoration of intestinal architecture, reduced inflammatory infiltration, and marked improvement in hepatic, renal, splenic, and bursal lesions were observed. Residual microscopic lesions persisted in T2 at the end of the experiment, whereas tissues from T3 and T4 appeared near normal and T5 demonstrated partial histological recovery. The villus height-to-crypt depth (VH:CD) ratio differed significantly among treatment groups at both sampling intervals (P<0.05) (Table 3). At 24 th day, the lowest VH:CD ratio was observed in T2 (4.76 ± 0.32), whereas T1 (6.65 ± 0.23) and T3 (6.26 ± 0.41) recorded significantly higher and comparable ratios. T4 (5.27 ± 0.07) and T5 (5.10 ± 0.30) exhibited intermediate values. By 38 th day, T1 (8.04 ± 0.42), T3 (7.86 ± 0.42), and T4 (7.48 ± 0.57) remained statistically comparable, whereas T5 recorded an intermediate ratio (6.69 ± 0.25). T2 continued to record the lowest VH:CD ratio (5.41 ± 0.09).

Ileum

Ileal villus height differed significantly among treatment groups at both sampling intervals (P<0.05) (Table 4). At 24 th day, the lowest villus height was recorded in T2 (668.67 ± 11.85 µm) compared with T1 (852.67 ± 7.86 µm), T3 (834.67 ± 6.57 µm), and T4 (829.33 ± 10.41 µm), whereas T5 recorded an intermediate value (723.33 ± 5.21 µm). By 38 th day, T3 (1176.67 ± 6.96 µm) and T4 (1170.00 ± 4.16 µm) exhibited the greatest villus height, followed by T1 (1136.00 ± 7.57 µm). T2 (1066.00 ± 9.87 µm) remained significantly lower than all other groups, whereas T5 (1103.33 ± 13.48 µm) recorded intermediate values.

Crypt depth also differed significantly among treatment groups at both sampling intervals (P<0.05). At 24 th day, T2 recorded the greatest crypt depth (130.33 ± 2.33 µm), followed by T5 (119.33 ± 3.53 µm) and T4 (111.67 ± 3.76 µm), whereas T1 (100.00 ± 3.06 µm) and T3 (98.33 ± 3.76 µm) recorded the lowest values. At 38 th day, T2 again recorded the greatest crypt depth (207.33 ± 3.53 µm), whereas T4 (172.67 ± 3.53 µm) and T5 (176.67 ± 2.91 µm) showed intermediate values. T1 (162.67 ± 2.91 µm) and T3 (162.33 ± 2.19 µm) remained statistically comparable and exhibited the lowest crypt depths. The VH:CD ratio also differed significantly among treatment groups at both sampling intervals (P<0.05). At 24 th day, T2 recorded the lowest ratio (5.14 ± 0.16), whereas T1 (8.54 ± 0.30) and T3 (8.52 ± 0.39) exhibited the highest and comparable ratios. T4 (7.44 ± 0.16) and T5 (6.07 ± 0.19) recorded intermediate values. By 38 th day, the highest VH:CD ratio was observed in T3 (7.25 ± 0.12), followed by T1 (6.99 ± 0.16) and T4 (6.78 ± 0.14). T5 recorded an intermediate ratio (6.25 ± 0.14), whereas T2 continued to exhibit the lowest VH:CD ratio (5.14 ± 0.08).

Re-isolation and Enumeration of Clostridium perfringens

Experimental Clostridium perfringens was successfully reisolated from the ileo-caecal contents of challenged birds on the 24 th and 38 th day. Typical black sulphite-reducing colonies were observed on Tryptose Sulphite Cycloserine (TSC) agar, while the isolates produced characteristic opalescent zones on egg yolk agar and smooth grey colonies with a double zone of haemolysis on blood agar. Gram staining revealed large Gram-positive rods arranged singly or in short chains, and all isolates were catalase-negative, confirming their identity as C. perfringens (Figure X). Intestinal C. perfringens counts are presented in Table 5. Significant differences in bacterial counts were observed among treatment groups at both sampling intervals (P<0.05). On the 24 th day, T2 recorded the highest bacterial load (6.58 ± 0.49 log₁₀ CFU/g), followed by T5 (6.13 ± 0.14 log₁₀ CFU/g). Lower counts were observed in T4 (5.15 ± 0.09 log₁₀ CFU/g) and T3 (5.08 ± 0.11 log₁₀ CFU/g), whereas T1 recorded the lowest count (3.60 ± 0.08 log₁₀ CFU/g). By the 38 th day, bacterial counts had decreased in all treatment groups; however, T2 continued to record the highest count (6.15 ± 0.17 log₁₀ CFU/g). Among the treated groups, T5 recorded higher counts (5.19 ± 0.12 log₁₀ CFU/g) than T4 (4.14 ± 0.08 log₁₀ CFU/g) and T3 (4.07 ± 0.11 log₁₀ CFU/g), which did not differ significantly from each other. The lowest bacterial count on the 38 th day was recorded in T1 (3.68 ± 0.08 log₁₀ CFU/g).

Discussion

The present study successfully established an experimental necrotic enteritis (NE) model following oral challenge with Clostridium perfringens, as demonstrated by characteristic clinical signs, gross and histopathological lesions, increased intestinal bacterial counts, altered intestinal morphology, impaired growth performance, and changes in serum biochemical parameters. Among the treatment groups, prophylactic supplementation with Saccharomyces boulardii consistently produced greater improvement than therapeutic supplementation and showed efficacy comparable to enramycin for most evaluated parameters. Experimental C. perfringens infection markedly impaired growth performance, as evidenced by reduced feed intake, body weight, and body weight gain together with poorer feed conversion ratio. These changes were accompanied by significantly higher intestinal C. perfringens counts, indicating successful bacterial colonization. Similar reductions in productive performance following experimental NE have been reported by Jayaraman et al. (2013) [26] , Shojadoost et al. (2012) [3] , and Wu et al. (2018) [27] . Birds receiving enramycin and prophylactic S. boulardii exhibited lower intestinal bacterial counts together with improved productive performance, whereas therapeutic supplementation produced comparatively moderate improvement. Similar preventive effects of probiotic supplementation have been reported by Nari (2020) [28] and Caly et al. (2015) [21] . Gross and histopathological examination of the infected untreated group revealed classical lesions of NE, including intestinal distension, mucosal congestion, hemorrhages, fibrinonecrotic pseudomembrane formation, villus necrosis, epithelial desquamation, inflammatory cell infiltration, and lesions in the liver, kidneys, spleen, and bursa of Fabricius. These observations are consistent with previous descriptions of experimental NE by Shojadoost et al. (2012) [3] , Keyburn et al. (2008) [7] , and Mwangi et al. (2019) [29] . The pathological findings were supported by intestinal morphometry, where infected untreated birds exhibited reduced villus height, increased crypt depth, and a lower villus height-to-crypt depth (VH:CD) ratio in both the jejunum and ileum. In contrast, enramycin and prophylactic S. boulardii preserved intestinal architecture, with morphometric measurements approaching those of the negative control. Similar improvements in intestinal morphology following probiotic supplementation have been reported by Jayaraman et al. (2013) [26] , Wu et al. (2018) [27] , and Nari (2020) [28] . The comparatively lower VH:CD ratio in the therapeutic group further indicates that supplementation after disease establishment was less effective than preventive administration. Serum biochemical alterations further reflected the systemic effects of NE. The infected untreated birds exhibited reduced serum total protein, globulin, and cholesterol concentrations together with increased ALT, AST, and creatinine activities, whereas enramycin-and prophylactic S. boulardii-treated birds maintained biochemical values closer to those of the negative control. Comparable biochemical alterations during experimental C. perfringens infection have been reported by Song et al. (2017) [30] , Jayaraman et al. (2013) [26] , and Wu et al. (2018) [27] . The comparatively higher serum protein and globulin concentrations together with lower serum transaminase activities observed in the treated groups corresponded with the reduced intestinal and hepatic lesions observed in the present study. Overall, the findings demonstrate that prophylactic supplementation with S. boulardii effectively reduced intestinal colonization by C. perfringens, preserved intestinal architecture, improved productive performance, and minimized biochemical alterations associated with experimental necrotic enteritis. Its protective efficacy was comparable to enramycin for most evaluated parameters, supporting its potential as a non-antibiotic strategy for the prevention of necrotic enteritis in broiler chickens.

Conclusion

The present study demonstrated that experimental challenge with Clostridium perfringens successfully induced necrotic enteritis in broiler chickens, resulting in impaired growth performance, alterations in serum biochemical parameters, severe gross and histopathological intestinal lesions, disruption of intestinal morphometry, and increased intestinal bacterial colonization. Dietary supplementation with Saccharomyces boulardii effectively mitigated these adverse effects, with prophylactic administration consistently producing greater protective effects than therapeutic supplementation. Birds receiving prophylactic S. boulardii exhibited improved growth performance, preservation of intestinal architecture, lower intestinal C. perfringens counts, reduced lesion severity, and maintenance of serum biochemical parameters comparable to those observed in the enramycin-treated group. Overall, the findings demonstrate that prophylactic S. boulardii supplementation is an effective non-antibiotic strategy for reducing the severity of necrotic enteritis and maintaining intestinal health and productivity in broiler chickens. These results support its potential application as a sustainable alternative to antibiotic growth promoters in commercial poultry production.

Acknowledgement

Acknowledgments:The authors thank the Department of Veterinary Pathology at the College of Veterinary and Animal Sciences in Parbhani, Maharashtra Animal and Fishery Sciences University, Nagpur, for offering the facilities needed to carry out this research. The authors report no conflicts of interest related to this work.

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