Comparative efficacy of Allium sativum and levofloxacin on histopathological alterations in experimentally induced Escherichia coli infection in broilers

Introduction

The poultry industry, primarily consisting of Gallus gallus domesticus, plays a crucial role in global food production, contributing significantly to the supply of meat and eggs. In India, the poultry sector is of particular importance, ranking third in egg production and sixth in chicken meat production (The Economic Survey 2021-22). This sector has experienced significant growth in recent years, with egg production increasing from 103.80 billion in 2018-19 to 138.38 billion in 2022-23. Similarly, poultry meat production rose from 4.06 million tonnes to 4.78 million tonnes during the same period. This growth underscores the increasing demand for poultry products, both domestically and internationally, highlighting the industry's significance in meeting global food security needs. However, despite these advancements, the poultry industry faces persistent challenges, particularly those related to infectious diseases. Bacterial infections are a major concern as they lead to severe economic losses, resulting from increased mortality rates, reduced productivity, and high treatment costs. Among the bacterial diseases affecting poultry, avian colibacillosis caused by Escherichia coli) stands out as one of the most pressing issues. Avian pathogenic E. coli strains are responsible for various diseases such as respiratory tract infections, pericarditis, airsacculitis, and septicaemia, which contribute to high mortality rates and the condemnation of carcasses (Amin et al., 2017; Panth, 2019) [3,17] . Furthermore, the emergence of antimicrobial-resistant E. coli strains has compounded the difficulty of controlling these infections, posing a significant threat not only to animal health but also to public health through zoonotic transmission (Khan et al., 2023) [12] . The misuse and overuse of antibiotics in poultry farming have exacerbated the problem of antimicrobial resistance, which has become a growing concern within the veterinary and public health sectors (Abd El-Tawab et al., 2016) [1] . The increasing prevalence of antibiotic-resistant bacteria has highlighted the need for alternative therapeutic strategies that can effectively address bacterial infections in poultry without contributing to the global antibiotic resistance crisis. In this context, the present study explores the potential of natural, plant-based alternatives with antimicrobial properties. One such promising alternative is garlic (Allium sativum), a widely used herb known for its rich bioactive composition and broad-spectrum antimicrobial activity. Garlic contains various bioactive compounds, including organosulfur compounds like allicin, which have demonstrated potent antibacterial effects against both Grampositive and Gram-negative bacteria, including E. coli (Goncagul and Ayaz, 2010) [10] . Additionally, garlic has shown anti-inflammatory, antioxidant, antimicrobial, and immunomodulatory effects, making it a viable candidate for enhancing poultry health and improving productivity (Nguyen et al., 2019; Melguizo-Rodríguez et al., 2022) [16,15] . Previous research has highlighted the potential benefits of incorporating medicinal plants like garlic into poultry feed. Garlic supplementation has been found to enhance growth performance, feed conversion efficiency, and immune response, while simultaneously reducing the prevalence of bacterial infections in poultry (Borgohain et al., 2019; Tesfaye, 2021) [4,21] . Furthermore, garlic-based interventions have shown promising results in mitigating oxidative stress and improving the overall health of poultry by maintaining microbial balance in the gut, thereby contributing to better productivity and reduced disease incidence (He Ding et al., 2023) [11] . a broad-spectrum third-generation fluoroquinolone antibiotic, has been extensively used in veterinary medicine for the treatment of bacterial infections, including those caused by E. coli. Levofloxacin is highly effective against Gram-negative bacteria like E. coli and is particularly useful in treating systemic infections in poultry due to its high tissue penetration ability (Chen et al., 2020). Despite its proven efficacy, concerns have arisen regarding the development of antimicrobial resistance in E. coli strains following the extensive and sometimes indiscriminate use of fluoroquinolones in poultry farming. The emergence of fluoroquinolone-resistant E. coli strains represents a significant threat to both animal and public health (Kumari et al., 2020) [13] . Regulatory restrictions on the use of antibiotics in poultry farming have further emphasized the need for alternative treatment options. Given these challenges, a comparative approach involving conventional antibiotics, such as levofloxacin, and natural antimicrobial agents, such as garlic, has become an area of significant interest. While levofloxacin provides rapid therapeutic effects in controlling E. coli infections, plantbased alternatives like garlic have shown potential in offering antibacterial, immunomodulatory, and growth-promoting benefits without the adverse consequences associated with antibiotic resistance (Borgohain et al., 2019) [4] . This study aims to investigate the efficacy of garlic, specifically Allium sativum bulb powder, in controlling E. coli infections in broiler chickens by examining its effects on gross pathological and histopathological changes. The study also compares the effectiveness of garlic with levofloxacin, evaluating the potential of combining or replacing conventional antibiotics with herbal alternatives in poultry production. The central hypothesis of this research is that garlic, due to its antimicrobial and antioxidant properties, can serve as a natural alternative to conventional antibiotics in reducing the morbidity and mortality associated with avian colibacillosis. Through controlled experimentation, this study seeks to provide scientific evidence supporting the use of garlic in poultry production as a sustainable solution to the growing issue of antibiotic resistance and bacterial disease outbreaks in the industry. By exploring the potential of plant-based alternatives like garlic, this research aims to contribute to the development of more sustainable and effective strategies for managing bacterial infections in poultry.

Materials and Methods

Experimental Design

The chicks in group-T 1 were given neither E. coli nor any treatment and kept as control group. Group-T 2 chicks were given broth of E. coli @ 0.5 ml (1x10 9 CFU/mL) by oral route at the age of 8 th day. The chicks in group-T 3 were given powder of garlic bulbs @ 1.5 gm/ kg of feed by oral route through feed from 8 th day till 15 th day. The chicks in group-T 4 were given broth of E. coli @ 0.5 mL (1x10 9 CFU/mL) by oral route at the age of 8 th day and powder of garlic bulbs @ 1.5 gm/ kg of feed by oral route through feed from 8 th day till 15 th day. Whereas, chicks in group-T 5 were given broth of E. coli @ 0.5 mL(1x10 9 CFU/mL) by oral route at the age of 8 th day and Leofloxacin @ 1 gm/10 kg body weight for 7 days. While birds in group-T 6 were given broth of E. coli @ 0.5 mL(1x10 9 CFU/mL) by oral route at the age of 8 th day, powder of garlic bulbs @ 1.5 gm/kg of feed by oral route through feed from 8 th day till 15 th day and Leofloxacin @ 1 gm/10 kg body weight for 7 days.

IAEC Approval

The birds were reared according to standard management practices and the experimental protocol was approved from Institutional Animal Ethics Committee (IAEC) before start of experiment. All the birds in different groups were kept under close observation during whole experimental period.

Gross lesions

The gross pathological lesions in the visceral organs were recorded from dead and sacrificed birds at 7 th day (15 th day of age), 14 th day (22 nd day of age) and 21 st day (29 th day of age) of experiment.

Microscopic lesions

Pieces of suitable thickness of heart, lungs, trachea, liver, spleen, kidneys, intestine and air sacs were collected from the sacrificed birds of all the groups and those died due to infection and the samples were preserved in 10% neutral formal saline. The tissues were embedded in paraffin and processed as per the standard procedure. The sections were cut at 4 to 5  thickness and were stained with Mayer's haematoxylin and eosin for microscopic examination (Culling, 1974) [7] . The detail microscopic findings in the tissue sections were recorded for each group at 7 th day (15 th day of age), 14 th day (22 nd day of age) and 21 st day (29 th day of age) of experiment.

Results

Clinical Symptoms

Chicks in Group T 1 (Healthy Control) and Group T 3 (Garlic Control) exhibited no abnormal clinical symptoms throughout the experimental period and maintained a healthy physiological state. Similarly, chicks in Group T 6 (E. coli infected, garlic, and levofloxacin treated) remained in good health without significant clinical symptoms for the duration of the trial. In contrast, chicks in Group T 2 (E. coli infected) displayed symptoms as early as 15 hours postinfection, including dullness, depression, ruffled feathers, weakness, huddling, anorexia, sneezing, nasal discharge, respiratory distress, and watery to semi-solid diarrhea with pasty vent. These symptoms are consistent with previous studies by Tandale (2019) [20] , Rathod et al. (2021) [18,19] , Chavan et al. (2022) [5] , and Enan et al. (2023) [9] , corroborating the findings of the present study. Chicks in Group T 4 (E. coli infected and garlic treated) showed minimal clinical symptoms. A few chicks exhibited dullness, depression, and reduced appetite, which lasted up to four days post-infection. Group T 5 (E. coli infected and levofloxacin treated) and Group T 6 experienced temporary dullness and depression lasting 12 to 24 hours postinfection, after which the birds appeared healthy for the remaining trial period. Observations of feed intake, body weight gain, and feed conversion ratio further supported these clinical findings.

Mortality

During the 29-day experimental period, an overall mortality rate of 2.67% was observed. In Group T 2 , which was orally infected with E. coli culture, three chicks (12.33%) died. Mortality occurred during the first and second weeks of post-infection, with 4% occurring within the first week and 8.33% during the second week. Tandale et al. (2019) [20] reported a 16.66% mortality rate in E. coli-infected chicks, while Abeer et al. (2020) [2] observed a mortality rate of 40%, and Rathod et al. (2021) [18,19] reported 14.25% mortality. These findings align with the current study's results. In Group T 4 (E. coli infected and garlic treated), one chick out of 25 died (4% mortality) during the first week post-infection. The results suggest that dietary garlic supplementation reduced mortality in broiler chicks. No mortality was observed in Groups T 5 (E. coli infected and levofloxacin treated) and T 6 (E. coli infected, garlic, and levofloxacin treated), indicating that the combination of levofloxacin in drinking water and garlic in feed had a protective effect against mortality. Tandale (2019) [20] also reported a protective effect of levofloxacin in E. coliinfected chicks, supporting the findings of the current study.

Gross Finding:

Gross pathological changes were observed to vary significantly among the experimental groups. Groups T 1 (Healthy control) and T 3 (Garlic control) did not exhibit any appreciable gross changes in visceral organs, including the trachea, lungs, air sacs, heart, liver, kidneys, spleen, and intestine, at any post-infection interval. In contrast, group T 2 (E. coli infected) displayed marked pathological changes, such as mild tracheal congestion at 7th and 14th days, severe pulmonary congestion, pneumonia, and necrosis, along with thickened, cloudy air sacs. The liver appeared pale with necrotic surfaces and fibrinous deposits, while the kidneys showed petechial hemorrhages and inflammation. Additionally, splenomegaly with petechial hemorrhages and hemorrhagic enteritis were prominent at earlier intervals. By the 21st day, these lesions had subsided, leaving residual changes such as mild congestion and focal necrosis in the liver and other organs. Treatment groups showed significant mitigation of these lesions. Birds in group T 4 (E. coli + Garlic) had mild lesions, such as tracheal congestion and focal liver necrosis, which resolved by the 21st day. Similarly, birds in group T 5 (E. coli + Levofloxacin) exhibited reduced lesion severity, with only mild congestion and pale liver observed throughout. Remarkably, birds in group T 6 (E. coli + Garlic + Levofloxacin) exhibited minimal or no gross pathological changes in all organs, maintaining near-normal gross anatomy. finding Histopathological examinations confirmed significant lesions in E. coli-infected birds (group T 2 ). Tracheal sections revealed congestion, catarrhal inflammation, and mucosal desquamation until the 14th day, with residual infiltration by the 21st day. Pulmonary lesions included congestion, emphysema, pneumonic changes, fibrinous exudates, and thickened interalveolar septa, which persisted as mild changes at later intervals. Air sacs demonstrated fibrinous airsacculitis and congestion up to the 14th day, resolving by the 21st day. The heart exhibited focal necrosis, hemorrhages, fibrinous pericarditis, and inflammatory infiltration, which diminished over time. Liver sections showed focal necrosis, sinusoidal dilation, fatty degeneration, and inflammatory cell infiltration, while kidneys displayed severe degenerative changes, including hydropic and vacuolar degeneration, hyaline casts, and nephritic changes. The spleen revealed congestion, lymphocyte depopulation, and splenic artery thickening, whereas intestinal lesions involved villous blunting, goblet cell hyperplasia, and inflammatory infiltration. Birds treated with garlic (T 4 ) exhibited partial amelioration, with mild congestion and inflammation in trachea, lungs, liver, and kidneys. Birds treated with levofloxacin (T 5 ) exhibited fewer histopathological changes, such as occasional congestion and minor liver alterations. The combination treatment group (T 6 ) displayed the most significant improvement, with near-normal histoarchitecture in all organs. These findings underscored the synergistic and protective effect of combined garlic and levofloxacin treatment, consistent with previous studies (Deshmukh, 2006; Enan et al., 2023; Kumari et al., 2023) [8,9,14] , and suggested its superior therapeutic efficacy in mitigating E. coli-induced damage. These findings suggest the potential therapeutic efficacy of garlic and levofloxacin, particularly in combination, in alleviating gross and histopathological lesions caused by E. coli infections in broiler birds. Garlic supplementation effectively reduces mortality, clinical symptoms, and histopathological lesions while enhancing overall health. Levofloxacin demonstrates rapid symptom control and significant lesion mitigation. The combination of garlic and levofloxacin provides the most significant improvement, with near-normal gross and histological findings, underscoring their synergistic effect. These results highlight garlic as a viable natural alternative to antibiotics, offering sustainable and effective strategies for managing bacterial infections in poultry while addressing the growing concern of antimicrobial resistance.

Conclusion

Acknowledgement

AcknowledgmentsAuthors wish to thankful to Associate Dean, College of Veterinary and Animal Sciences, Parbhani, MAFSU, Nagpur (M.S) India for conducting the experiment and providing facilities mandatory for the same.

References

1. Abd El-Tawab AA, Mahboub HD, El-Far AH, AbdelFarag EM, Mahmoud H. Impacts of the misuse of antibiotics in poultry on public health. Veterinary World. 2016;9(9):1038-1042.
2. Abeer AA, Heba M, Hanan E. Evaluation of garlic oil supplementation against avian colibacillosis. Veterinary World. 2020;13(2):396-401.
3. Amin UM, Khan AA, Rahman AA. Pathology of avian colibacillosis caused by Escherichia coli in broilers. Asian Journal of Animal and Veterinary Advances. 2017;12(2):93-102.
4. Borgohain A, Devi MA, Borah P. Garlic as a feed additive in poultry: A review. International Journal of Current Microbiology and Applied Sciences. 2019;8(2):1851-1862.
5. Chavan SN, Awandkar SP, Tandale YR. Pathological changes in Escherichia coli infected broiler birds. Indian Journal of Veterinary Pathology. 2022;46(1):3540.
6. Chen Y, Du X, He J, Zhu Y. Pharmacokinetics and tissue distribution of levofloxacin in chickens. Journal of Veterinary Pharmacology and Therapeutics. 2020;43(6):537-543.
7. Culling CF. Handbook of Histopathological and Histochemical Techniques. 4th ed. London: Butterworth-Heinemann; 1974.
8. Deshmukh V, Kulkarni S, Shinde A. Efficacy of levofloxacin against Escherichia coli in poultry. Veterinary Practitioner. 2006;7(2):108-110.
9. Enan G, Abd El-Moaty DAM, Fadel M, Abbas NA. Garlic and antibiotics against bacterial infections in poultry: Synergistic potential. Animal Feed Science and Technology. 2023;303:115626.
10. Goncagul G, Ayaz E. Antimicrobial effect of garlic (Allium sativum). Recent Patents on Anti-Infective Drug Discovery. 2010;5(2):91-93.
11. He Ding C, Pan L, He C, Zhou Y. Role of garlic in reducing oxidative stress and improving gut health in poultry. Poultry Science. 2023;102(5):102657.
12. Khan M, Rehman T, Hussain M, Khan MT, Naz S. Antimicrobial resistance patterns of Escherichia coli isolated from poultry farms. Veterinary Microbiology. 2023;269:109406.
13. Kumari S, Malik P, Kumar A. Emergence of fluoroquinolone-resistant Escherichia coli in poultry. Journal of Global Antimicrobial Resistance. 2020;21:93-98.
14. Kumari S, Sharma R, Singh P, Patel A, Gupta V. Antimicrobial resistance of levofloxacin and garlic: A comparative study. International Journal of Veterinary Science Research. 2023;11(2):85-92.
15. Melguizo-Rodríguez L, Costela-Ruiz VJ, MartínAmeida T, García-Recio E, Mañas A, Ruiz C. Garlic and its role in health and disease prevention. Phytomedicine. 2022;100:154022.
16. Nguyen T, Nieto JJ, Barber D. Potential of garlic (Allium sativum) in improving poultry performance and immunity. Journal of Applied Animal Research. 2019;47(1):95-102.
17. Panth N. A review on bacterial diseases of poultry in Nepal. International Journal of Applied Sciences and Biotechnology. 2019;7(1):34-39.
18. Rathod P, Ingole R, Ingale V, Kuralkar S, Manwar S, Hajare S, Ingawale M. Effect of experimentally induced Escherichia coli on general performance in broilers. Chemical Science Review and Letters. 2021;10(38):190-194.
19. Rathod SD, Khose KK, Doke SA, Chavan SN. Pathology and therapeutic management of avian colibacillosis. Indian Journal of Animal Research. 2021;55(10):1262-1266.
20. Tandale YR, Rathod SD, Awandkar SP. Pathology of Escherichia coli infection in broiler chicken. Indian Veterinary Journal. 2019;96(3):24-27.
21. Tesfaye A. The potential of medicinal plants as feed additives in poultry: A review. Journal of Veterinary Science and Technology. 2021;12(4):1-6.