INTRODUCTIONAvian pathogenic Escherichia coli (APEC) is an extraintestinal pathogenic E. coli (ExPEC) that can cause colibacillosis in chickens, which can impact egg and meat production from poultry. APEC employs a variety of virulence mechanisms and pathogenic traits in poultry, leading to colibacillosis(Kathayat et al., 2021). Among them are adhesins, invasins(Sora et al., 2021), fimbriae or pili, outer membrane proteins, and toxins, including cytotoxins, hemolysins, and endotoxins(Meena et al., 2021). The severity of APEC induced disease depends on the host’s health status, the virulence factors expressed by the specific strain and other predisposing factors. It often manifests as extraintestinal infection, with clinical signs such as air sacculitis, perihepatitis, pericarditis, peritonitis, salpingitis, coligranuloma, omphalitis, cellulitis, swollen head syndrome, and osteomyelitis(Nolan et al., 2020).
Food security and poultry welfare(Jørgensen et al., 2019) are global issues, resulting in huge economic losses in the poultry industry due to high morbidity and mortality rates(Mehat et al., 2021). Colibacillosis inflicts enormous losses on Indonesia’s poultry industry, with an estimated average loss of IDR 14.20 trillion per cycle for broilers and approximately IDR 13.40 trillion per month for layers, amounting to 13.10% of the country’s total poultry assets(Wibisono et al., 2018).
The prevalence rate of colibacillosis is 22.2%, as reported in Indonesia(Panth, 2019). Antibiotic therapy remains a key strategy in managing colibacillosis. However, its overuse and misuse may accelerate antimicrobial resistance (AMR) by producing resistant strains(Naghavi et al., 2022). AMR is defined as a result of changes in microbial responses to antibiotics, leading to treatment failures and relapsing infections(Huemer et al., 2020). At the flock level, poultry disease outbreaks are often inadequately controlled when antimicrobials are used without accurately identifying the etiological agent and susceptibility testing (resulting in unmatched treatment)(Choisy et al., 2019). Factors contributing to AMR encompass inadequate treatment, utilization of unsuitable medications, and self-medication(Ahmed et al., 2024). Therefore, AMR surveillance or monitoring for APEC is essential to guide appropriate control measures and limit the dissemination of AMR.
The prevalence of colibacillosis in broiler and layer chickens caused by APEC has been well documented in various provinces of Indonesia. However, effective control and treatment strategies have not been developed, as there has been no recent data for over ten years. The purpose of this study is to find out the phenotypic traits of APEC bacteria that came from instances of colibacillosis in broiler and layer chickens from commercial farms in the Special Region of Yogyakarta and Central Java. It also wants to confirm the organ pathology shown in these case studies. This study also analyzes antibiotic sensitivity to provide real information about how to choose the best treatment for avian colibacillosis. The results should help improve diagnosis and enable more effective control of colibacillosis in both broiler and laying chickens.
DISCUSSIONThe results of isolation and identification showed that the samples showed culture in EMBA, Gram staining, and biochemical tests that had characteristics consistent with E. coli. On EMBA, E. coli colonies appear with a green metallic sheen and greenish-blue spots(Basavaraju & Gunashree, 2023). In Gram staining, E. coli presents as pink, small rod-shaped structures, consistent with Gram-negative bacteria. The biochemical tests performed included triple sugar iron agar (TSIA), urea, citrate, methyl red, Voges-Proskauer (MRVP), sulfide indole motility (SIM), and indole tests(Bisping & Amtsberg, 1988). These isolates showed typical E. coli results in the indole, methyl red, Voges-Proskauer, citrate (IMViC) test, with SIM.
E. coli pathovars can also be screened using the phenotypical Congo red binding assay(Saha et al., 2020). Twenty-one (51.21%) of the E. coli isolates were considered pathogenic based on Congo red binding, as they developed colonies with a red pigment(Ugwu et al., 2020). Similar observations were made in an earlier study on the Congo red binding positive APEC isolates(Saha et al., 2020). The variation of Congo red dye uptake among E. coli isolates may suggest differences in pathogenicity and virulence potential(Berkhoff & Vinal, 1985).
Hemolysis is often associated with E. coli pathogenicity, especially in strains isolated from blood(Daga et al., 2019). It has been reported that α-hemolysis (partial hemolytic activity) is observed in most pathogenic E. coli isolates(Salam et al., 2024). In this study, pathogenicity testing on whole blood agar media showed that the visible colonies produced incomplete hemolysis, or what is often called α-hemolysis.
Colibacillosis cases can be identified through necropsy, which typically reveals pronounced lesions of generalized polyserositis accompanied by various combinations of pericarditis, perihepatitis, air sacculitis, and peritonitis. Typical postmortem observations include the presence of fibrin, leftover egg yolk, or milky fluid within the peritoneal cavity and on the surfaces of several organs. In cases of peritonitis, a caseous exudate can accumulate in the body cavity, resembling clumped egg yolk, a condition referred to as yolk peritonitis(Nolan et al., 2013). Yolk accumulation in the peritoneal cavity can lead to yolk peritonitis in laying hens. In broilers, colibacillosis frequently occurs in conjunction with perihepatitis, pericarditis, and air sacculitis, with E. coli isolated from the affected organs(Apostolakos et al., 2021);(Li et al., 2024). These results align with the postmortem findings in this study.
Anatomical pathological analysis conducted in this study revealed pulmonary hemorrhage and the presence of a caseous mass. Lungs exhibiting pathological alterations due to bacterial infection showed inflammation, which was characterized by a red-brown, meat-like coloration and increased tissue density(Meha et al., 2016). In APEC-infected chickens, Abalaka et al. (2017)(Abalaka et al., 2017) also described perivascular and interstitial edema, pulmonary hemorrhage, granulomatous inflammation, and giant cells in the lungs. In infected broilers, histopathological examinations consistently showed marked pulmonary lesions characterized by heterophile infiltration and accumulation of eosinophilic exudates, as well as intravascular fibrin thrombi(Ghahramani et al., 2024). The endotoxins released during APEC infection may enhance vascular permeability and contribute to pulmonary edema, congestion, and hemorrhage, while inflammatory stimulation of vascular injury aggravates respiratory distress(Kathayat et al., 2021).
The air sacs became thickened due to hypertrophy of the connective tissue and infiltration of heterophilic inflammatory cells. Abdelhamid et al. (2024)(Abdelhamid et al., 2024) described lung congestion and pneumonic lesions in their study of colibacillosis. Bhalerao et al. (2013)(Bhalerao et al., 2013) reported similar lesions, including congestion, edema, and foci of pneumonia. Pathological examination in this investigation revealed opacification of the air sacs, indicating air sacculitis. Birds with colibacillosis exhibit air sacculitis at all ages due to fibrin mass formation. It was most commonly observed in birds older than three weeks(Bhalerao et al., 2013);(Taunde et al., 2021). Histopathology showed thickening and proliferation of connective tissue in the air sacs with heterophilic inflammatory cells.
Anatomopathology in this study showed thickened layers of pericardium with fibrin coating, as seen here. These changes cause the pericardium and epicardium to adhere through fibrinous connective tissue in their respective layers, a sign of pericarditis. Nolan et al. (2020)(Nolan et al., 2020) said pericarditis is colisepticemia, starting with tissue response edema or fluid accumulation in the pericardial sac and exudate, then fibrinous exudate. In chronic infections, the exudate becomes thickened and caseous. In long-standing cases, the pericardium becomes adherent to the epicardium. Microscopy showed infiltration of inflammatory cells (pericarditis and myocarditis) in the pericardium and interstitial tissues and muscle atrophy (muscle vacuolation). These findings are consistent with Nolan et al. (2020)(Nolan et al., 2020).
According to Taunde et al. (2021)(Taunde et al., 2021), the present study observed anatomical, pathological, and histopathological characteristics of the liver, describing a case of colibacillosis with fibrin exudate on the surface of Glisson’s capsule. This is consistent with Abdelhamid et al. (2024)(Abdelhamid et al., 2024) and Ghahramani et al. (2024)(Ghahramani et al., 2024), who noted that the liver capsule can be thickened and surrounded by fibrinous exudates, with heterophilic infiltration; further degenerative changes, such as fatty change, were also observed.
Colibacillosis in chickens causes renal congestion, with the kidney appearing pale and containing hard, pale areas of necrosis, as well as being congested and inflamed with infiltration by inflammatory cells (Abalaka et al., 2017). Chickens with colibacillosis showed kidney lesions such as hemorrhage and discoloration, as noted by Taunde et al. (2021)(Taunde et al., 2021), which is in line with the results of this study. The ovaries of the broilers in this study were underdeveloped, exhibiting atrophied follicles, irregular development, a soft texture, and a color change from pale yellow to white. These changes are in line with Ozaki et al. (2018)(Ozaki et al., 2018), who described post-mortem findings of oophoritis and salpingitis in chickens with colibacillosis. In this study, the visible lesions were similar to those of colibacillosis, characterized by mostly inactive, yellowish, or pale follicular structures.
Excessive and improper use of poultry antimicrobials has driven the emergence of AMR, posing a significant public health threat(Abreu et al., 2023). Chickens are the most closely kept birds among humans across the poultry value chain, with an accompanying risk of a serious public health challenge(Ugwu et al., 2020). In this survey, amoxicillin (including enrofloxacin and trimethoprim-sulfadiazine) was used by farmers for the oral treatment of colibacillosis. While AMR can occur naturally, inappropriate use of antimicrobials in humans and animals has contributed to its increased emergence. Before 2018, antibiotic growth promoters were banned in Indonesia due to the Indonesian Regulation No.14/PERMENTAN/ PK.350/5/2017-Ministry of Agriculture regarding the classification of veterinary drugs.
This study revealed a high resistance to amoxicillin (66.67%; 14/21), a β-lactam antibiotic. This resistance rate is lower than that reported in China (81.7%)(Afayibo et al., 2022) and Ukraine (78.4%)(Nechypurenko et al., 2024). A significant prevalence of APEC resistance to clinically relevant antibiotics, notably β-lactams, poses a serious threat to public health(Osman et al., 2018) Antibiotic sensitivity tests also indicated a 57.14% (12/21) of this samples resistance to enrofloxacin and resistance to nalidixic acid was 47.62% (10/21) which is consistent with the resistance of 45% reported by Sgariglia et al. (2019)(Sgariglia et al., 2019). Fluoroquinolones inhibit bacterial gyrase and topoisomerase IV enzymes(Grabowski et al., 2022). Enrofloxacin is commonly used in broiler farms to treat colibacillosis(Temmerman et al., 2021).
Aminoglycosides such as streptomycin, gentamicin, and neomycin are all classified as aminoglycosides. The streptomycin resistance rate in this study was 52.38% (11/21), which is lower than the findings reported by Afayibo et al. (2022)(Afayibo et al., 2022). The resistance rate to gentamicin was 42.86% (9/21), which is similar to the findings of Hardiati et al. (2021)(Hardiati et al., 2021). Aminoglycosides are effective against Gram-negative species by binding to 30S ribosomal subunit, so bacteria can’t produce protein. They can treat a wide spectrum of diseases(Dezanet et al., 2020). Sulfonamides, often combined with diaminopyrimidine compounds like trimethoprim, exert a synergistic antibacterial effect by inhibiting different stages of bacterial folic acid synthesis. This synergy reduces the dosages required for both agents, broadens antibacterial spectra, and decreases resistance(Stastny et al., 2024). The high resistance rate to trimethoprim-sulfamethoxazole observed in this study, 52.38% (11/21), was lower than that reported in previous studies conducted in Algeria and Jordan(Ibrahim et al., 2019);(Aberkane et al., 2023).
The study’s results showed that resistance to tetracycline antibiotics, specifically tetracycline (42.86%, 9/21) and oxytetracycline (47.62%, 10/21), was relatively low but not significantly different between the two drugs. Jahantigh et al. (2020)(Jahantigh et al., 2020) reported higher resistances in APEC compared to our study, although not as high as those reported by Nechypurenko et al. (2024)(Nechypurenko et al., 2024). Tetracyclines are commonly used as cheap antibiotics in the poultry industry. Resistance can arise from excessive administration for prophylactic, therapeutic, or growth performance purposes(Jahantigh et al., 2020).
Denissen et al. (2022)(Denissen et al., 2022) explain that multidrug resistance (MDR) manifests when an individual acquires resistance to a minimum of three distinct classes of antibiotics. In this study, we found that 71.43% (15/21) of the tested APEC strains were MDR. This finding aligns with previous investigations in Iran(Jahantigh et al., 2020). E. coli significantly contributes to the issue due to its rapid transmission among humans and animals via the fecal-oral route (Galindo-Méndez, 2020). The antibiotic resistance genes can be spread through horizontal gene transfer from other bacterial strains or species(Larsson & Flach, 2022). Imprinted DNA can also persist in the environment long after resistant strains have died(Jian et al., 2021).
Guidelines emphasize administering antibiotics based on accurate diagnosis, choosing the most appropriate antibacterial agent, limiting last-resort antibiotics, and strictly following label instructions(Ungemach et al., 2006). Concerns arise that repeated low-dose antibiotic exposure in animals contributes to AMR. This is problematic in poultry production. The unrestricted availability of antibiotics to livestock farmers without prescriptions promotes their excessive use(Islam et al., 2024). Prudent antibiotic use requires optimized dosage regimens, including correct dosage, interval, initiation, and duration of therapy. Strategies that maximize antimicrobial pharmacokinetics and pharmacodynamics can help minimize AMR selection in pathogens, commensal bacteria, and the environment(Guardabassi et al., 2018). For the chicken’s health and to reduce side effects, it is essential to have an accurate dosage schedule that includes the dose, the time between doses, and the length of treatment. This is especially important for antibiotics, which are necessary to keep AMR in check. It is essential to get the right amount of antibiotics at the right time. Antimicrobials must attain effective concentrations at the infection site and persist for adequate durations to facilitate recovery(Caneschi et al., 2023).