Pathological and bacteriological investigation of colibacillosis in broiler chickens: A case report

Introduction

Escherichia coli is a Gram negative facultative anaerobic bacterium that colonizes the lower intestine of mammals and birds soon after birth where it coexists in harmony with the host and aids in the synthesis of essential vitamins (Sarowska et al., 2019) [12] . They are motile, catalase-positive, oxidase-negative, citrate-negative, nitrate-reducing and carbohydrate fermenters (Al Humam, 2016) [2] . While the majority of Escherichia coli strains are not harmful, some serotypes can infect poultry and cause disease. E. coli infections in poultry flocks have been documented more frequently in recent years, highlighting the pathogen's expanding importance (Schouler et al., 2012) [13] . The two primary categories of pathogenic E. coli are intestinal pathogenic E. coli (InPEC) and extra-intestinal pathogenic E. coli (ExPEC), which are distinguished by the organs in which they typically exhibit their virulence (Kaper et al., 2004) [7] . Among disease-causing strains, avian pathogenic E. coli (APEC) holds particular importance in the broiler industry because it leads to colibacillosis, a condition responsible for considerable economic losses through bird mortality and reduced performance. The disease can appear in different forms, ranging from yolk sac infections (omphalitis) and septicemia in both chicks and adult birds, to salpingitis in layers. Respiratory involvement, especially airsacculitis, is very common in both broilers and layers, while lesions such as perihepatitis and pericarditis in other internal organs are also frequently observed (Kaper et al., 2004; Nolan et al., 2013) [7,10] . The most susceptible age group for avian colibacillosis in broiler chickens is 4-6 weeks (Dho-Moulin et al., 1999) [6] . Contaminated feed and water are significant sources of the disease because E. coli can persist for longer periods in dry and dusty conditions. Fecal contamination of eggs, where the bacteria can enter through the shell and infect the developing chick, is another important mode of transmission (Khoo et al., 2010; Matin et al., 2017) [8,9] . The present case report describes the occurrence of colibacillosis in broiler chickens, including associated gross pathological lesions and confirmation of bacterial etiology.

Materials and Methods

A thorough and systematic necropsy was performed on carcasses presented to the Department of Veterinary Pathology, School of Veterinary Sciences, Abhilashi University. The gross lesions were closely examined and recorded. The liver sample was aseptically collected in the sterile container to identify and confirm the bacterial etiology and after processing it was inoculated on brain heart infusion (BHI) agar and MacConkey's lactose (MLA) agar, incubated for 24 hours at 37 °C. The discrete lactose-fermenting colonies (pink colonies) were chosen and then sub-cultured onto Eosin Methylene Blue (EMB) agar and incubated for 24 hours at 37 °C. On EMB agar, colonies that displayed the distinctive metallic green sheen were regarded as presumed Escherichia coli. The growth characteristics of bacteria on triple sugar iron (TSI) agar were also observed. Gram staining, catalase test, and oxidase test were also performed.

Results and Discussion

The liver of the affected birds had a distinctive "bread and butter" appearance during necropsy because of a thick layer of fibrinous material that covered the whole organ (Fig. 1). Grossly, the liver was black, swollen, and friable. In addition, the surface of the liver also had focal necrotic regions. Besides, lesions indicative of septicemia were also visible, including perihepatitis and fibrinous pericarditis with an adherent white pericardial sac (Fig. 2). The deposition of fibrinous exudate over the liver and heart surface observed in this case is in accordance with the description by (Apostolakos et al., 2021) [3] , Kumar et al. (2013) [10] and Renu et al. (2012) [11] . The concurrence of our observations with previous reports confirms the consistency of fibrinous inflammatory lesions in colibacillosis. E. coli was successfully identified and confirmed from the liver tissue based on growth characteristics on MacConkey lactose (MLA) agar, eosin and methylene blue (EMB) agar, and triple sugar iron (TSI) agar. On MLA agar, discrete dry pink lactose-fermenting colonies were observed (Fig. 3). On EMB agar, colonies exhibited the characteristic metallic green sheen (Fig. 4). On TSI agar, the bacterial isolate was able to ferment all three sugars (both slant and butt were yellow) with gas production, but no H2S production (Fig. 5). These colonial characteristics of bacteria on MLA, EMB agar, and TSI agar are considered classical hallmarks of E. coli and serve as a reliable means of its presumptive identification (Cheesbrough, 1985) [5] . The isolated bacteria were found Gram negative, catalase positive, and oxidase negative. The observations of this study are in agreement with the findings of Abd El-Mongy et al. (2017) [1] , who also reported lactose-positive pink colonies on MacConkey agar and metallic green sheen on EMB agar while isolating E. coli from poultry and emphasised that these colony characteristics provide a simple and dependable diagnostic tool for the confirmation of avian colibacillosis.

Conclusion

The present investigation confirms colibacillosis as the cause of mortality in broiler chickens based on characteristic gross lesions along with successful bacteriological identification of E. coli. The presence of fibrinous perihepatitis, "bread and butter" liver, and fibrinous pericarditis, coupled with the cultural identification of lactose-positive pink colonies on MacConkey agar, metallic green sheen colonies on EMB agar, and characteristic reaction on TSI agar was highly suggestive of the disease.

References

1. Abd El-Mongy M, Bayome AM, Abd-El-Moneam GM, Moawad AA. Prevalence, bacteriology, pathogenesis and isolation of Escherichia coli in broilers. Kafrelsheikh Veterinary Medical Journal. 2017;15(2):16.
2. Al Humam NA. Special biochemical profiles of Escherichia coli strains isolated from humans and camels by the VITEK 2 automated system in Al-Ahsa, Saudi Arabia. African Journal of Microbiology Research. 2016;10(22):783-790.
3. Apostolakos I, Laconi A, Mughini-Gras L, Yapicier ÖŞ, Piccirillo A. Occurrence of colibacillosis in broilers and its relationship with avian pathogenic Escherichia coli (APEC) population structure and molecular characteristics. Frontiers in Veterinary Science. 2021;8:737720.
4. Cheesbrough M. Medical laboratory manual for tropical countries. Vol. II. Microbiology. London: Butterworths; 1985. p. 400-480.
5. Dho-Moulin M, Fairbrother JM. Avian pathogenic Escherichia coli (APEC). Veterinary Research. 1999;30(2-3):299-316.
6. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nature Reviews Microbiology. 2004;2(2):123-140.
7. Khoo LL, Hasnah Y, Rosnah Y, Saiful N, Maswati MA, Ramlan M. The prevalence of avian pathogenic Escherichia coli (APEC) in peninsular Malaysia. Malaysian Journal of Veterinary Research. 2010;1(1):27-31.
8. Matin MA, Islam MA, Khatun MM. Prevalence of colibacillosis in chickens in greater Mymensingh district of Bangladesh. Veterinary World. 2017;10(1):29.
9. Nolan LK, Barnes HJ, Vaillancourt JP, Abdul-Aziz T, Logue CM. Colibacillosis. In: Swayne DE, editor. Diseases of Poultry. 13th ed. Ames: Wiley-Blackwell; 2013. p. 751-805.
10. Renu AP, Mishra S, Londhe M, Lathar D, Sharma A. Etio-pathological studies on poultry mortality with reference to Escherichia coli infections. Indian Journal of Poultry Science. 2012;47(2):222-226.
11. Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, FrejMadrzak M, Ksiazczyk M, Bugla-Ploskonska G, Choroszy-Krol I. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathogens. 2019;11(1):10.
12. Schouler C, Schaeffer B, Brée A, Mora A, Dahbi G, Biet F, Oswald E, Mainil J, Blanco J, Moulin-Schouleur M. Diagnostic strategy for identifying avian pathogenic Escherichia coli based on four patterns of virulence genes. Journal of Clinical Microbiology. 2012;50(5):1673-1678.