Ameliorative effect of methanolic extract of Tridax procumbens leaf powder against experimentally induced Clostridium perfringens infection in broiler chickens

Introduction

Enteric diseases are a major constraint in poultry production, causing significant economic losses due to reduced body weight gain, poor feed conversion ratio, increased mortality, and higher medication costs. Among these, Clostridium perfringens infection is of considerable importance, particularly associated with types A, C and G, with type A being the predominant causative agent in broilers (Sultan et al., 2025; Miller et al., 2010) [44,70] . C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium commonly present in the intestinal tract of healthy birds at low levels (10²-10⁴ CFU/g), but proliferates rapidly under disease-induced conditions to reach pathogenic levels (10⁷-10⁹ CFU/g) (Kiu and Hall, 2018; Shojadoost et al., 2012; Xiu et al., 2020) [37,66,77] . The disease contributes to global economic losses estimated at nearly 2 billion USD annually (Mwangi et al., 2019; Khan et al., 2021; Jamil et al., 2025) [31,35,49] . The extensive use of antibiotics for growth promotion and disease control has led to the emergence of antimicrobial resistance (AMR), posing a serious threat to animal and public health (Prestinaci et al., 2015; Acharya and Wilson, 2019) [7,55] . Although restrictions on antibiotic growth promoters have been implemented in several International Journal of Advanced Biochemistry Research 2026; 10(7): 684-697 regions, their use remains prevalent in developing countries, with a projected 67% increase in antibiotic consumption in animal production systems (Goutard et al., 2017; Van Boeckel et al., 2015) [24,76] . This has necessitated the search for safer and effective alternatives. Medicinal plants have gained increasing attention as potential alternatives due to their antimicrobial, antioxidant, and growth-promoting properties (Abd El-Hack and Alagawany, 2015; Rafeeq et al., 2022) [5,57] . Tridax procumbens L., a member of the Asteraceae family, is widely recognized for its diverse pharmacological activities, including antibacterial, antioxidant, antidiabetic, and hepatoprotective effects (Kethamakka and Deogade, 2014; Bhagwat et al., 2008; Pai et al., 2011; Hemalatha, 2008) [12,25,34,51] . Phytochemical analyses have revealed the presence of bioactive compounds such as flavonoids, alkaloids, glycosides, and terpenoids, which contribute to its antimicrobial efficacy against various pathogenic bacteria (Sarkar et al., 2016) [61] . Considering the aforementioned evidence, the present study was undertaken to induce experimental Clostridium perfringens infection and evaluate the ameliorative potential of methanolic extract of Tridax procumbens leaf powder in broiler chickens.

Materials and Methods

Induction material

Livacox® Q vaccine (M/S Stallen South Asia Pvt. Ltd., Mumbai), containing live attenuated sporulated oocysts of Eimeria tenella, E. acervulina, E. maxima and E. necatrix, was used as a predisposing factor for experimental induction of Clostridium perfringens infection. A lyophilized culture of Clostridium perfringens (MTCC 450) was obtained from the Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India. The culture was revived aseptically in Reinforced Clostridial Medium (RCM) broth and incubated anaerobically at 37 ± 1°C for 24 h. Following incubation, the bacterial suspension was serially diluted in sterile phosphate-buffered saline (PBS) and standardized to approximately 1 × 10⁷ CFU/ml by comparison with McFarland Standard No. 2. The optical density of the inoculum was adjusted to 0.556 at 600 nm to ensure uniform bacterial concentration.

Preparation of methanolic extract

Fresh leaves of Tridax procumbens were shade-dried for 4-5 weeks and ground into a fine powder. Methanolic extraction was carried out by Soxhlet extraction using 100 g of leaf powder and 1000 ml of methanol at 64°C for 24 h. The extract obtained was filtered through Whatman No. 41 filter paper, concentrated and stored at 4°C until further use.

Experimental birds and design

A total of 100 day-old broiler chicks were randomly divided into five groups (n = 20) and reared under standard management conditions for 38 days. Group T1 served as the healthy control. Birds in groups T2-T5 were predisposed with a ten-fold dose of Livacox® Q vaccine on day 13 and subsequently challenged orally with Clostridium perfringens (1 × 10⁷ CFU/ml; 1 ml/bird) on days 17, 18 and 19 of age. Group T2 served as the infected untreated control. Group T3 received enramycin supplementation at 0.1 g/kg feed from day 1 of the study. Group T4 was administered methanolic extract of Tridax procumbens at 200 mg/kg body weight from day 1 of the study as a prophylactic treatment, whereas Group T5 received the same dose therapeutically following the appearance of clinical signs.

Growth performance and clinical evaluation

Body weight, body weight gain, feed consumption and feed conversion ratio (FCR) were recorded weekly. Birds were observed daily for clinical signs and mortality throughout the experimental period.

Serum biochemical analysis

Blood samples were collected on days 0, 7, 14 and 21 postinfection. Serum was separated and analyzed for total protein, albumin, globulin, albumin-to-globulin ratio, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine using standard diagnostic procedures.

Gross pathology and histopathology

Necropsies were conducted on days 24 and 38 of the experiment. Gross lesions were recorded and tissue samples from the intestine, liver, kidney, spleen and bursa of Fabricius were collected. Samples were fixed in 10% neutral buffered formalin, processed routinely, stained with hematoxylin and eosin, and examined microscopically.

Re-isolation and enumeration of Clostridium perfringens

To confirm experimental infection, Clostridium perfringens was re-isolated from the ileocecal contents of dead and sacrificed birds using Tryptose Sulfite Cycloserine (TSC) agar and Egg Yolk Agar and identified by colony characteristics, Gram staining and catalase testing. For bacterial enumeration, ileocecal contents collected aseptically on days 24 and 38 were serially diluted and cultured on TSC agar in duplicate. Following anaerobic incubation at 37°C for 48 h, bacterial counts were determined and expressed as log₁₀ CFU/g of sample.

Phytochemical analysis

Qualitative phytochemical screening of the methanolic extract was performed using standard procedures. The chemical composition of the methanolic extract of Tridax procumbens leaf powder was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) at the RUSA Centre, R.T.M. Nagpur University, Nagpur. Analysis was performed using a Perkin Elmer GC Clarus 500 GC-MS system equipped with an Elite-1 fused silica capillary column (30 m × 0.25 mm, 1 μm film thickness; 100% dimethyl polysiloxane). Helium (99.999%) was used as the carrier gas at a flow rate of 1 ml/min. A 0.5 μl sample was injected in split mode (10:1). The injector and ion source temperatures were maintained at 250°C and 280°C, respectively. The oven temperature was programmed from 110°C (2 min) to 200°C at 10°C/min, then to 280°C at 5°C/min with a final hold of 9 min. Mass spectra were recorded in electron impact mode (70 eV) over an m/z range of 40-550 Da, with a total run time of 30 min.

Statistical analysis

Data were analyzed using a Completely Randomized Design (CRD) as described by Snedecor and Cochran. Differences among treatment means were considered significant at P<0.05.

Result and Discussion

Clinical observations and mortality Birds in Group T1 remained clinically normal throughout the study. In contrast, birds in Groups T2, T3, T4, and T5 developed clinical signs of Clostridium perfringens infection within 36-48 h post-challenge, including dullness, depression, drooping wings, ruffled feathers, diarrhea, vent pasting, dehydration, polydipsia, and reduced body weight. The severity of clinical signs was highest in the infected untreated group (T2) and progressively decreased in the treatment groups (T3, T4, and T5). Similar observations have been reported by Elbadawy and Aboubakr (2017) [18] , Abadeen et al. (2022) [1] , Abd El-Ghany et al. (2022) [4] , Sorour et al. (2021) [69] , and Eraky and Abd El-Ghany (2022) [21] .

Mortality rates of 10% and 5% were recorded in Groups T2 and T4, respectively, while no mortality was observed in the remaining groups. These findings are in agreement with those reported by Elbadawy and Aboubakr (2017) [18] .

Average weekly live body weight:

The mean weekly live body weights of broilers are presented in Table-1. No significant differences were observed among the groups during the first two weeks (P> 0.05). However, from the third week onwards, significant differences were recorded (P<0.05). The infected untreated group (T2) showed significantly lower body weight than the control (T1) and all treatment groups (T3, T4, and T5). Among the treatments, T3 recorded the highest body weight, while T4 and T5 showed comparable values during weeks 4 and 5. The improved body weight in T3, T4, and T5 compared to T2 indicates their beneficial effect in alleviating the growthdepressing effects of Clostridium perfringens infection. The reduced body weight in T2 may be attributed to intestinal damage and impaired nutrient absorption caused by clostridial toxins (Lovland and Kaldhusdal, 1999) [41] . Similar findings have been reported by Abd El-Ghany et al.

(2022) [4] , Elbadawy and Aboubakr (2017) [18] , Salem et al.

(2021) [59] , and Shah et al. (2023) [65] . The growth-promoting effects observed in T3 agree with Abudabos (2012) [6] , while the improvement in T4 and T5 supports the beneficial role of Tridax procumbens, likely due to its bioactive phytoconstituents with antimicrobial and gut healthpromoting properties (Pareek et al., 2009; Krishnaveni, 2018; Ukpong, 2024) [39,52,75] .

Average Weekly Body Weight Gain

The mean weekly body weight gain of broilers is presented in Table-2. No significant differences were observed among the groups during the first two weeks (P> 0.05). However, from the third week onwards, significant differences were recorded (P<0.05). The infected untreated group (T2) exhibited significantly lower body weight gain than the control (T1) and treatment groups (T3, T4, and T5). Among the treatments, T3 showed the highest weight gain and was comparable to T1 during the later weeks, while T4 and T5 also recorded significantly higher gains than T2, with no significant difference between them during weeks 4 and 5.

The reduced weight gain in T2 may be attributed to intestinal damage and impaired nutrient absorption caused by C. perfringens toxins (El-Kady et al., 2012) [19] . Similar findings have been reported by Hussein et al. (2020) [26] , Abadeen et al. (2021b) [3] , Abd El-Ghany et al. (2022) [4] , and Elbadawy and Aboubakr (2017) [18] . The improved growth performance in T3 confirms the beneficial effect of enramycin, while the enhancement observed in T4 and T5 supports the growth-promoting and antimicrobial potential of Tridax procumbens, likely through improved gut health and nutrient utilization ( [16,48,54] .

Average Weekly Feed Conversion Ratio (FCR)

The mean weekly FCR values are presented in Table-3. No significant differences were observed among the groups during the first two weeks (P> 0.05); however, significant differences emerged from the third week onwards (P<0.05). The infected untreated group (T2) recorded a significantly higher FCR than the control (T1) and treatment groups (T3, T4, and T5), indicating poor feed efficiency. Among the treatments, T3 showed FCR values comparable to T1, while T4 and T5 exhibited significantly lower FCR than T2, with no significant difference between them during the later weeks. The elevated FCR in T2 may be attributed to intestinal damage and impaired nutrient utilization caused by C. perfringens infection. Similar findings have been reported by Hussein et al. (2020) [26] , Elbadawy and Aboubakr (2017) [18] , Salem et al. (2021) [59] , and El-Alim et al. (2025) [17] . The improved FCR in T3 confirms the efficacy of enramycin, whereas the better feed efficiency observed in T4 and T5 suggests a beneficial effect of Tridax procumbens, possibly through its antimicrobial and gut health-promoting phytoconstituents (Ukpong, 2024; Odeh et al., 2022) [50,75]

The mean serum total protein, albumin, globulin and A/G ratio values are presented in Table-4. No significant differences were observed among the experimental groups before infection (P> 0.05). Following Clostridium perfringens challenge, the infected untreated group (T2) showed significantly lower total protein, albumin and A/G ratio values, along with significantly higher globulin levels compared to the control (T1) and treatment groups (T3, T4 and T5) at all post-infection intervals (P<0.05). Among the treatments, T3 maintained values comparable to T1, while T4 and T5 showed progressive improvement, with T4 generally exhibiting better recovery than T5. The altered protein profile in T2 may be attributed to intestinal damage, impaired nutrient absorption, protein loss, hepatic dysfunction and immune stimulation associated with C. perfringens infection. Similar findings have been reported by Salem et al. (2020) [60] , Abadeen et al. (2021b) [3] , Khodary et al. (2019) [36] , Jamil et al. (2025) [31] and Muneeb et al. (2025b) [47] . The normalization of these parameters in T3 confirms the protective effect of enramycin (Da Silva et al., 2024) [13] , whereas the improvement observed in T4 and T5 suggests that Tridax procumbens (Akintunde et al., 2025) [8] helps maintain protein metabolism and immune homeostasis, possibly through its hepatoprotective, antioxidant and antimicrobial properties.

Serum Cholesterol

Mean serum cholesterol values are presented in Table-5. No significant differences were observed among the groups before infection (P> 0.05). However, following Clostridium perfringens challenge, the infected untreated group (T2) showed significantly lower serum cholesterol levels than the control (T1) and treatment groups (T3, T4, and T5) at all postinfection intervals (P<0.05). Among the treated groups, T3 maintained cholesterol levels closest to T1, while T4 and T5 showed gradual improvement and remained significantly higher than T2. The reduced cholesterol levels in T2 may be attributed to intestinal damage, impaired lipid absorption, and metabolic disturbances caused by C. perfringens infection. Similar findings have been reported by Elkomy et al. (2019) [20] , Qing et al. (2017) [56] , and Muneeb et al.

(2025b) [47] . The improvement observed in T3, T4, and T5 indicates the beneficial effects of enramycin and Tridax procumbens (Ravikumar et al., 2005) [58] in maintaining lipid metabolism and intestinal health during infection. Following Clostridium perfringens challenge, the infected untreated group (T2) showed significantly higher AST levels than the control (T1) and treatment groups (T3, T4, and T5) at all post-infection intervals (P<0.05). Among the treated groups, T3 maintained values closest to T1, while T4 and T5 showed moderate elevations but remained significantly lower than T2. By day 21 post-infection, T3 approached normal levels, whereas T4 and T5 showed partial recovery.

The elevated AST levels in T2 may be attributed to hepatocellular damage and leakage of intracellular enzymes caused by C. perfringens infection. Salem et al. (2020) [60] , Khodary et al. (2019) [36] , Jamil et al. (2025) [31] , and Abadeen et al. (2021b) [3] . The lower AST levels observed in T3, T4, and T5 indicate the protective effects of enramycin and Tridax procumbens on liver function, with the T.procumbens likely exerting hepatoprotective and antioxidant activities (Ravikumar et al., 2005) [58] .

Serum Alanine Aminotransferase (ALT)

Mean serum ALT values are presented in Table-7. No significant differences were observed among the groups before infection (P> 0.05). Following Clostridium perfringens challenge, the infected untreated group (T2) showed significantly higher ALT levels than the control (T1) and treatment groups (T3, T4, and T5) at all post-infection intervals (P<0.05). Among the treated groups, T3 and T4 maintained values closer to T1, while T5 showed comparatively higher levels but remained significantly lower than T2. By day 21 post-infection, T3 and T4 approached normal values, whereas T5 showed partial recovery. The elevated ALT levels in T2 may be attributed to hepatocellular damage induced by C. perfringens toxins. Similar findings have been reported by Ibrahim et al. (2017) [28] , Salem et al. (2020) [60] , Khodary et al. (2019) [36] , and Jamil et al. (2025) [31] . The lower ALT levels observed in T3, T4, and T5 indicate the hepatoprotective effects of enramycin and Tridax procumbens, with T. procumbens supporting recovery of liver function during infection (Ravikumar et al., 2005) [58] . showed significantly higher creatinine levels than the control (T1) and treatment groups (T3, T4, and T5) at all postinfection intervals (P<0.05), indicating renal dysfunction. Among the treated groups, T3 maintained values closest to T1, while T4 and T5 showed moderate elevations but remained significantly lower than T2. By day 21 postinfection, all treated groups exhibited partial recovery, with T3 showing the greatest improvement. The elevated creatinine levels in T2 may be attributed to renal impairment caused by systemic toxemia and tissue damage associated with C. perfringens infection. Similar findings have been reported by Abadeen et al. (2021b) [3] , Khodary et al. (2019) [36] , Ibrahim et al. (2017) [28] , and Muneeb et al. (2025b) [47] .

In contrast, the reduced creatinine levels in T3, T4, and T5 indicate that enramycin and Tridax procumbens helped preserve renal function, with Tridax procumbens likely contributing renoprotective effects (Ikewuchi et al., 2021) [29] .

Re-isolation and confirmation of Clostridium perfringens

In the present study, Clostridium perfringens was successfully re-isolated and confirmed from the ileocecal contents of dead and sacrificed birds based on cultural, biochemical, and microscopic characteristics. On TSC agar, the organism produced characteristic black colonies, while growth on Egg Yolk Agar showed creamy-white to greyish colonies surrounded by an opaque zone indicative of lecithinase activity. The isolate exhibited a catalase-negative reaction and appeared as Gram-positive, thick rod-shaped bacilli arranged singly or in short chains on Gram staining (Figure -1). These findings confirmed the presence of C. perfringens and are consistent with the observations of Malmarugan et al. (2012) [42] , Thomas et al. (2014) [72] , Dar et al. (2017) [15] , Abadeen et al. (2021a) [2] , Eraky and Abd El-Ghany (2022) [4] , and Auob et al. (2025) [10] . [38] , Fuke (2020) [23] and Muneeb et al. (2025a) [46] , who observed reduced intestinal C. perfringens counts in enramycin treated birds. The reduction in bacterial load in T4 and T5 suggests the antibacterial activity of Tridax procumbens, supporting earlier findings by Sarkar et al. (2016) [61] and Muthusamy et al. (2013) [48] . Gross pathological examination was conducted on the 24 th and 38 th days of the experimental period. Birds from the T1 group did not exhibit noticeable gross lesions, and all organs appeared normal during both slaughter evaluations. Birds in the T2 group showed pronounced gross pathological changes (Figure -3), predominantly affecting the mid-small intestine, particularly the jejunum and ileum. The intestinal mucosa was thickened and covered with a diphtheritic membrane, while the duodenum exhibited comparatively milder lesions.

The intestines also showed ballooning with minimal to moderate hemorrhages. Additionally, hepatomegaly with pale liver, mildly swollen and congested kidneys and minimal splenomegaly with dark discoloration were observed. Birds from T3, T4, and T5 groups exhibited milder hepatic and intestinal lesions compared to those of the T2 group. The gross pathological lesions observed in Clostridium perfringens-infected birds in the present study corroborate the findings of Islam et al. (2009) [30] , Malmarugan et al. (2010) [43] , Asaduzzaman et al. (2011) [9] ,

Ferdoush et al. (2013) [22] , To et al. (2017) [74] , and Abadeen et al. (2022) [1] .

Histopathological Observations Intestine

Histopathological evaluation of the duodenum, jejunum, and ileum was performed on days 24 and 38 of study period in all experimental groups.

Duodenum: Sections from group T1 showed normal histoarchitecture on both days. Birds from groups T2 and T5 exhibited minimal to mild vascular congestion, epithelial desquamation, inflammatory cell infiltration, and acute inflammatory exudate in the lumen, with occasional exfoliation of the mucosal lining. Similar but less intense lesions were observed in groups T3 and T4.

Jejunum: Jejunal sections from group T1 remained within normal histological limits throughout the study. Birds in group T2 exhibited mild to moderate congestion, epithelial desquamation, villous denudation, inflammatory cell infiltration, goblet cell hyperplasia, enterocyte proliferation, villous fusion, epithelial exfoliation, and focal necrosis. Thickening of the epithelium and lamina propria was also observed. Groups T3 and T4 showed comparatively milder lesions characterized by minimal to mild congestion, enterocyte hyperplasia, inflammatory cell infiltration, and occasional goblet cell hyperplasia. Group T5 exhibited lesions similar to T2 on day 24; however, lesions were milder on day 38 and characterized by mild congestion, enterocyte proliferation, inflammatory cell infiltration, goblet cell hyperplasia, and occasional villous disorganization.

Ileum: Ileal sections from group T1 showed normal histological architecture on both days. On day 24, groups T2 and T5 exhibited moderate to severe lymphocytic infiltration and acute inflammatory changes, whereas groups T3 and T4 showed only minimal enteric alterations. By day 38, group T2 revealed goblet cell hyperplasia with persistent inflammatory changes, while groups T3 and T4 remained largely within normal limits. Group T5 showed occasional congestion and lymphoid hyperplasia. The intestinal microscopic lesions observed in the present study were consistent with the findings of Islam et al.

(2009) [30] , Thomas et al. (2014) [72] , Sivaseelan et al. (2013) [68] , and Salem et al. (2021) [59] . Daneshmand et al. (2022) [14] reported that Clostridium perfringens induces intestinal damage through toxin-mediated mechanisms, resulting in epithelial necrosis, villous blunting, fusion, denudation, and inflammatory cell infiltration. Da Silva et al. (2024) [13] observed that enramycin preserved intestinal architecture by suppressing bacterial proliferation and toxin production. The lesion severity was noticeably lower in groups T4 and T5 than in T2, which may be attributed to the antimicrobial and antibacterial properties of Tridax procumbens (Mundada and Shivhare, 2010; Pai et al., 2011) [45,51] and the presence of bioactive compounds such as phytol, neophytadiene, and hexadecanoic acid methyl ester.

Liver

Histomorphological examination of liver sections from birds of group T1 on the 24 th and 38 th days did not reveal any significant histopathological alterations. On the 24 th day, liver sections from birds of groups T2 and T5 exhibited mild to moderate congestion, vacuolar degeneration, widening of sinusoidal spaces, disorganization of hepatic cords, focal mononuclear cell infiltration, occasional coagulative necrosis, bile duct hyperplasia, cytoplasmic rarefaction, and fatty changes. Similar but less intense lesions were observed in group T2 on the 38 th day. Birds of groups T3 and T4 showed minimal to mild congestion, focal degenerative and fatty changes, and mononuclear cell infiltration at both intervals. On the 38 th day, group T5 birds exhibited mild congestion, sinusoidal dilatation, focal degenerative changes, and mononuclear cell infiltration. The hepatic microscopic alterations observed in the present investigation were comparable to those reported by Abadeen et al. (2022) [1] , Sivaseelan et al. (2013) [68] , Suryakanth et al. (2019) [71] , and Sawale et al. (2010) [62] . Salem et al. (2021) [59] reported that Clostridium perfringens causes hepatic damage through toxin-mediated enterotoxemia, resulting in hepatocellular degeneration, focal necrosis, sinusoidal congestion, inflammatory cell infiltration, and disorganization of hepatic architecture. Da Silva et al. (2024) [13] observed preservation of liver architecture in enramycin-treated birds through suppression of C. perfringens multiplication and toxin production. Birds treated with methanolic extract of Tridax procumbens (T4 and T5) exhibited markedly milder hepatic lesions than group T2, which may be associated with the hepatoprotective activity of T. procumbens (Ravikumar et al., 2005) [58] . [1] and Behera et al.

Kidney

(2025) [11] . Birds treated with methanolic extract of Tridax procumbens (T4 and T5) exhibited less severe renal lesions than group T2, suggesting the nephroprotective potential of T. procumbens as reported by Ikewuchi et al. (2021) [29] .

Spleen

Histopathological evaluation of spleen sections from group T1 birds on the 24th and 38th days revealed normal histological architecture without noticeable abnormalities. On the 24th day, birds from group T2 exhibited lymphoid depletion and splenic congestion, whereas birds from groups T3, T4, and T5 showed only minimal lesions. By the 38th day, group T2 revealed marked congestion with thickening of the splenic artery. Birds from group T5 also showed splenic congestion, while groups T3 and T4 exhibited comparatively milder changes.The splenic histopathological alterations observed in the present investigation corresponded closely with the findings of Immerseel et al. (2004). Birds belonging to groups T4 and T5 exhibited less severe splenic lesions than group T2, which may be associated with the immunomodulatory effects of Tridax procumbens (Tiwari et al., 2004) [73] .

Bursa of Fabricius

Histopathological examination of the bursa of Fabricius from birds of group T1 on the 24th and 38th days did not reveal appreciable histopathological abnormalities. On the 24th day, birds from group T2 exhibited mild lymphocytic depletion within the bursal follicles, whereas birds from group T5 showed congestion of the bursal lining epithelium. In contrast, groups T3 and T4 maintained an apparently normal histological architecture. By the 38th day, groups T2 and T5 showed reduced lesion severity, while groups T3 [73] . [33] , Jhample et al. (2015) [32] , and Kushwaha et al. (2019) [40] . Similarly, Sawant and Godghate (2013) [63] detected phenols, flavonoids, tannins, saponins, and alkaloids in various leaf extracts of T. procumbens, with steroids additionally observed in the chloroform-water extract.

Phytochemical analysis of methanol extracts of

Quantitative Phytochemical analysis Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

GC-MS analysis of the methanolic extract of Tridax procumbens leaf powder generated 13 peaks, and all detected compounds accounting for 100% of the total peak area were identified using the NIST14 spectral library. The major constituent was D-alanine (RT [53,64,67] . Their occurrence in the extract may contribute to the antimicrobial activity of T. procumbens. Similar compounds were also identified by Singh et al. (2025) [67] in the methanolic wholeplant extract of T. procumbens, supporting the present findings.

Conclusion

Experimental infection with Clostridium perfringens successfully induced enteritis in broiler chickens, resulting in impaired growth performance, biochemical alterations, increased intestinal bacterial load, and characteristic gross and histopathological lesions. Among the treatment groups, enramycin showed the highest antibacterial efficacy, while the methanolic extract of Tridax procumbens leaf powder significantly ameliorated the adverse effects of infection, with prophylactic administration proving more effective than therapeutic use. The protective effects of the extract are likely attributable to its rich phytochemical composition, including phytol, phenols, flavonoids, tannins, saponins, and alkaloids. Overall, the findings demonstrate that the methanolic extract of Tridax procumbens leaf powder is a promising natural alternative for the prevention and management of C. perfringens infection in broiler chickens owing to its antimicrobial, ameliorative, hepatoprotective, and renoprotective properties.

Acknowledgement

AcknowledgementThe authors express their sincere gratitude to the Associate Dean, College of Veterinary and Animal Sciences, Parbhani, for providing necessary facilities, constant support, and guidance during the course of this study. Their encouragement and valuable suggestions were instrumental in the successful completion of this research work.

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