Introduction
Avian pathogenic Escherichia coli (APEC) causes colibacillosis in chickens. APEC serotypes, as a group, are virulent for birds, which causes avian colibacillosis [1]. All types and age groups of birds are susceptible to the disease, ranging from chicks to adult layers, broilers, and breeders [2]. The organism can invade multiple organs resulting in systemic diseases such as coligranuloma, colisepticemia, omphalitis, synovitis, swollen head syndrome, airsacculitis, and cellulitis [3]. Colibacillosis causes considerable economic losses to the poultry industry due to high morbidity and mortality [4]. Escherichia coli (E. coli) is considered the natural inhabitant of avian gut microflora [5]. Several virulence factors, including adhesins, aerobactin, yersiniabactin, hemolysins, outer membrane protein A, lipopolysaccharide, K1-capsule, and heat stable toxin, have been reported in the propagation of various extra-intestinal diseases in avian species [6].
Risk factors for colibacillosis include poor biosecurity, overcrowding, inadequate ventilation, contaminated water and feed, and stressful conditions compromising the birds’ immune systems [7,8]. Clinical signs vary but often include diarrhea, dehydration, decreased appetite, respiratory distress, swollen wattles, and a drop in egg production [9]. Colibacillosis affects multiple organs in chickens, including the liver, intestines, and respiratory system. Pathological changes include inflammation, necrosis, and congestion in affected tissues [10].
Whereas colibacillosis is primarily an enteric disease in mammals, in poultry, it may be a localized or systemic disease, occurring mostly secondary to impairment of the host defense mechanisms [11]. The acute form of the disease is characterized by septicemia, which results in death. In contrast, its subacute form is accompanied by pericarditis, airsacculitis and perihepatitis, and reproductive tract infections like salpingitis and/or egg peritonitis, resulting in huge mortality [12]. Unlike other intestinal E. coli serotypes, APEC can be diagnosed by typical clinical signs, gross pathology, and microscopic lesions, as well as bacterial culture [13,14]; however, the general mechanism of APEC pathogenesis needs further explanations [15].
Although there are several researches on E. coli infections in different animal species, it will still be evaluated to revalidate existing literature and better understand the mechanism of the diseases. Therefore, this research was designed to explore tissue changes associated with experimental E. coli infection in layer chickens.
Materials and Methods
Ethical approval
Ethical approval was sought from the Ethical Committee on Animal Use and Care of Ahmadu Bello University, Zaria (ABUECAUC), with Approval number ABUECAUC/2017/026.
Location of experimental study
This study was carried out in the Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna State, which is located within the Northern Guinea Savannah Zone of Nigeria, between latitude 70 and 110N, and longitude 70 and 440E. The average rainfall of this zone ranges from 1,000 to 1,250 mm, and the average temperature ranges from 190C to 330C [16].
Experimental animals and design
A total of 10 layer (20 weeks old) chickens that were specifically raised for research purposes and vaccinated against endemic vaccinable diseases in the area, except E. coli infections, were purchased from a reputable farm in Kaduna State, Nigeria. The birds were housed and managed intensively in the poultry research pens of the Department. Before the arrival of the birds, the pens were thoroughly washed with detergent and sprayed with formalin at a concentration of 4 ml/l of water. Throughout the experiment, the birds were fed standard commercial layer mash (Hybrid Feeds®), and water was provided to the birds’ ad libitum.
The bacterial organism
Escherichia coli used in this experiment, APEC serotype O1K1, was obtained from the bacteria bank of the National Veterinary Research Institute, Vom, Plateau State, Nigeria.
Grouping and inoculation of birds with E. coli
The birds were kept for 4 weeks to acclimatize to the new environment and other handling conditions, after which they were divided randomly into two groups (infected and control) of 5 layers each. The control birds were housed in a pen far away from the pen in which the birds of the infected group were housed. The bacteria from a previously prepared slant were reactivated by sub-culturing on eosin and methylene blue. The resulting colonies were then examined for their characteristic features, color, and morphology and tested for gram stain reaction. On the day of infection (Day 0), the bacterial inoculum was prepared using McFarland standards, which were prepared by adding barium chloride to sulphuric acid to obtain a barium precipitate of different turbidity standards. These were used to estimate the number of bacteria in a liquid suspension [17]. In this test, the turbidity of the suspension of bacteria was compared with a turbidity of the appropriate standard. Each of the birds in the infected group was then challenged by inoculating each of the birds in the infected group with 0.5 ml of bacterial aliquot containing 109 colony-forming units of the bacteria intratracheally [10]. After inoculation, the bacteria were recovered from infected birds by following conventional culture, isolation, and identification of bacteria by standard procedures [18].
Clinical observations
From day 0 of infection and throughout the experimental period, birds in the infected group were closely monitored for clinical signs of colibacillosis and recorded accordingly.
Postmortem and histopathological examination
Four birds each from both groups (infected and control) were sacrificed at 10 days post-infection and subjected to post-mortem examination; gross lesions observed were recorded accordingly. All sanitary measures in preventing contamination of the carcass were ensured. The liver, ovarian follicles, kidney, and heart specimens were collected into bottles containing 10% neutral buffered formalin and subsequently processed, using standard techniques, for histopathological examination according to Bancroft and Gamble [19]. Processed slides were examined using a light microscope at different magnifications. Gross lesions were snapped using a digital camera accordingly.
Results
Gross findings
The gross lesions observed in the E. coli-infected layers were congested lungs (Fig. 1), enlarged liver (Fig. 2), hyperaemic intestine (Fig. 3), enlarged kidney (Fig. 4), and congested ovarian follicles (arrowhead), yellowish fibrinous material (short arrow) congested blood vessels of the oviduct (Fig. 5).
Figure 1.
Photograph showing congested lungs (arrow) from a layer in E. coli-infected group on day 10 pi.
Figure 2.
Photograph showing enlarged liver (arrow in A) from E. coli-infected layer and liver from the control group (arrowhead) on day 10 pi.
Figure 3.
Photograph of intestine showing hyperemia (A) and thickened mucosal membrane from E. coli-infected layer on day 10 pi.
Figure 4.
Photograph showing congested lungs (short arrow) and enlarged kidney (arrow head) from E. coli-infected layer on day 10 pi.
Figure 5.
Photograph showing congested ovarian follicles (arrowhead), yellowish fibrinous material (short arrow), and congested blood vessels of the oviduct (long arrow) in the E. coli-infected layer on day 10 pi.
Histopathology of tissues harvested from E. coli-infected group of layers
The ovarian follicle showed congestion of ovarian blood vessels (Fig. 6). Heart showed necrosis of cardiac myocytes, congested blood vessels, and mononuclear cellular infiltration (Fig. 7). The kidney showed diffused necrosis of renal tubular epithelium, aggregation of mononuclear cells, and congested blood vessels (Fig. 8). The liver showed diffused necrosis of hepatocytes, perivascular aggregation of mononuclear cells, and congestion of the central vein (Fig. 9).
Figure 6.
Photomicrograph of ovary section from E. coli-infected chicken showing (a) congestion of ovarian blood vessel on day 10 pi. H&E X 250
Figure 7.
Photomicrograph of section of heart from E. coli-infected chicken showing congestion (a), mononuclear cellular infiltration (b), necrosis of the cardiac myocytes (c) on day 10 pi. H&E X 250.
Figure 8.
Photomicrograph of section of kidney from E. coli-infected chicken showing diffused necrosis of renal tubular epithelium (a), aggregation of mononuclear cells (b), congested blood vessels (c) on day 10 pi. (H&E × 250).
Figure 9.
Photomicrograph of liver section from E. coli-infected chicken showing diffuse necrosis of hepatocytes (a), perivascular aggregation of mononuclear cells (b), congestion of central vein (c) on day 10 pi. H&E × 400.
Discussion
In the present study, the grossly observed congestion of the serosal blood vessels of the oviduct and fibrin deposition was previously reported following E. coli infection of layer birds [20]. This is indicative of acute condition [1]. It was also reported that lesions associated with ascending E. coli infection from the cloaca consequent of salpingitis [21] could be responsible for egg peritonitis frequently encountered in layer birds [22]. Enlarged liver (hepatomegaly) with necrotic foci was also reported in the liver of broilers and layers in the natural colibacillosis outbreak in Eastern Sudan [23]. In line with our results, Sharif et al. [24], and Sonwane et al. [25] also reported degenerative changes in liver and heart sections of E. coli-infected birds. The gross lesion observed in the lungs is similar to Gangane et al. [26] and Kumari et al. [27] which correspond to congestion, edema, and pneumonic foci. Also, Shah et al. [28] in an experimental study on colibacillosis reported congestion and pneumonic lesions in the lungs. Similarly, congested and hemorrhagic kidneys have been reported [6], which might be due to vascular damage caused by E. coli endotoxin [29].
Histopathological evaluation showed damaged liver, kidney, as well as lungs. Many authors have documented similar histological alterations in the kidney, liver, and heart in E. coli-infected broilers [3,30,31,25]. However, degenerative intestinal changes can be attributed to enterotoxin released by E. coli [3]. The lesions of mycardial congestion, mononuclear cellular infiltration, necrosis of the cardiac myocytes observed is in comformity with Lateif et al. [2]. Similarly, chick-lethal toxin and immunosuppression induced by E. coli infection may cause lymphopenia and necrotic foci in immune organs [32,27]. The observed inflammatory and necrotic lesions in the liver of affected birds might be due to E. coli endotoxin and vascular injury [20]. Similar focal necrosis and cellular infiltration were reported in the liver of birds that came down with colibacillosis [29]. Likewise, congested central veins and sinusoids with cellular infiltration around the portal area were reported by El-Ghany and Madian [33] in the liver of broiler chickens infected with E. coli. Similarly, Dutta et al. [34] reported hemorrhage and degenerative tubular changes in the kidneys of E. coli-infected pigeons. This is because most internal organs are affected when E. coli gets to the vascular system [35] as seen in this study.
Conclusion
This study revealed various lesions of congested lungs, enlarged liver, hyperaemic intestine, enlarged kidney, and congested ovarian follicles, yellowish fibrinous material, congested blood vessels of the oviduct that are indicative of Colibacillosis. The observed changes following experimental E. coli infection were typical of the natural infection and may be attributable to colisepticaemia. Diagnoses based on gross examination can be made accurately on up to 90% of samples. This is the importance of gross description. However, for macroscopic pathology to be of help in establishing a diagnosis; knowledge, precision, and accuracy in gross description are required.
Acknowledgment
The authors are profoundly grateful to all the technical staff of the veterinary clinical pathology and microbiology laboratory of Ahmadu Bello University Zaria.
Conflict of interest
The authors declare that they have no conflict of interest.
