Introduction
Poultry farming is a significant agricultural practice in Nigeria, contributing substantially to the economy and food security. However, the industry faces numerous challenges, including the prevalence of infectious diseases that threaten poultry health and productivity. Central to achieving a disease-free poultry production system is the accurate diagnosis of diseases and the institution of efficient and effective biosecurity measures [1]. One such disease is avian Aspergillosis, a severe and non-contagious respiratory condition primarily caused by the fungus Aspergillus fumigatus [2–4]. The primary cause is A. fumigatus, though other species such as A. niger, A. nidulans, A. flavus, and various Aspergillus species, sometimes in mixed infections, also play significant roles [1,5]. Aspergillus fumigatus predominates in respiratory fungal infections likely due to its smaller, easily airborne spores [6]. Avian aspergillosis affects various domesticated and wild birds, particularly those in captivity, and can lead to substantial economic losses due to high morbidity, mortality, and compromised growth and feed efficiency in affected birds [1,5,7,8]. The disease manifests in two forms: acute and chronic; the acute form is characterized by high morbidity and mortality from inhaling many spores, while the chronic form is characterized by low mortality, and mainly affecting immunocompromised birds. Mortality rates in spontaneous infections range from 4.5% to 90%, with affected birds aged between 3 days and 20 weeks [5]. The immediate risk factor of the disease is stress, and it is typically a case of poor management practices in both commercial and backyard poultry production systems [9]. Contamination of feed and water, lack of proper hygiene, and overcrowding of birds in a pen increase the chance of the growth and spread of fungal spores [7].
In addition to Aspergillosis, poultry farms often contend with secondary bacterial infections that exacerbate the impact of the primary fungal disease. Such secondary infections can further complicate clinical outcomes, making management and treatment more challenging [10]. Escherichia coli is a common opportunistic pathogen that can cause colibacillosis, a significant bacterial infection that affects poultry, leading to high morbidity and mortality rates and increased treatment costs [10–13].
This case report focuses on the laboratory diagnosis of an outbreak of A. fumigatus accompanied by a secondary bacterial infection on a poultry farm in Jos, Plateau State, Nigeria. Jos, the capital of Plateau State, is located in the north-central region of Nigeria (at 9° 53’ 47.4972’’ N latitude and 8° 51’ 29.9916’’ E longitude), characterized by a warm and humid climate that can facilitate the proliferation of fungal spores. The report aims to highlight the diagnostic procedures used, the clinical and pathological findings observed, and the implications for poultry management and disease control in Nigeria.
Understanding the interplay between fungal and bacterial infections in poultry is crucial for developing effective management strategies and mitigating the economic impacts of these diseases. This report underscores the importance of accurate laboratory diagnosis in identifying the causative agents and formulating appropriate intervention measures to control and prevent future outbreaks.
Case Details and Diagnostic Plans
Clinical history
A farmer brought eleven carcasses of 15-week-old Isa Brown pullets to the Avian and Aquatic Clinic at the University of Jos Veterinary Teaching Hospital (VTH). The main concern was the sudden death of approximately 30 birds on the day of the initial visit. The flock originally consisted of 3,200 birds. They were fed Supreme Feed® grower mash and given well water for drinking. According to the vaccination history, the birds had received Newcastle vaccine 8 weeks prior. Upon examination, the birds were diagnosed with Aspergillosis and treated with Polidine® (1.6% iodine) and CuSO4. However, 1 week after the first visit, the farmer returned with six additional carcasses. It was noted that mortality decreased to four birds per days after treatment but began to increase again afterward.
Necropsy
Necropsies were performed on the 17 carcasses (11 from the first visit and 6 from the second visit), and gross postmortem lesions were identified and recorded. At the time of necropsy, the differential diagnoses included aspergillosis, mycoplasmosis, and colibacillosis. Observable gross pathological lesions included slight nasal discharge, congested lungs, and kidneys with white nodules. The nodules were observed within the thorax and peritoneum, also, congested liver, and underdeveloped ovarian follicles were observed and recorded (Fig. 1A and B).
Figure 1.
Photograph (A) showing congested lungs with many whitish nodules; Photograph (B) showing white nodules within the peritoneum and on-air sacs.
Histopathology
Tissue samples from the affected organs (liver, kidney, and spleen) were collected and fixed in 10% neutral buffered formalin. These samples were processed according to the method described by Baker et al. [14]. Briefly, the samples were dehydrated using various concentrations of alcohol, cleared in xylene, and infiltrated with paraffin wax. The samples were then incubated in a vacuum at 60°C, embedded in plastic embedding rings, and sectioned into 5-µm slices using a microtome. Afterward, the samples were deparaffinized with xylene, rehydrated in graded alcohol concentrations, stained with Haematoxylin and Eosin, and examined under a light microscope at 40x magnification. Histopathologic examination of the lungs revealed areas of granulomatous inflammation with a necrotic eosinophilic center surrounded by a zone containing inflammatory cells (giant cells, macrophages, and lymphocytes) (Fig. 2 and 3).
Figure 2.
Photomicrograph of chicken lungs showing granulomatous inflammation with necrotic eosinophilic center (asterisk) surrounded by zone with inflammatory cells (X 40 magnification H&E).
Figure 3.
A. Photomicrograph of chicken lungs showing giant cells (yellow arrows), macrophages (black arrows) and lymphocytes (arrow head). ×40 magnification; B. Photomicrograph of chicken lungs showing fungal hyphae (arrow) and inflammatory cells . ×10 magnification (H&E)
Microbial culture and susceptibility test
The microbiological examination of tissue samples (liver, lungs, spleen, and kidney) was conducted using standard bacteriological and mycological culture techniques. These included the use of Blood agar (Himedia) with 7% horse blood, MacConkey agar (MCA) (Himedia), and Sabouraud Dextrose Agar (SDA) (Himedia), following the phenotypical characterization methods described by Kwoji et al. [15] and Karakurt et al. [16]. Cultures that showed pink lactose-fermenting colonies on MCA and were further identified on Eosin methylene blue agar and purified on Nutrient agar before being subjected to biochemical characterization. These were then Gram stained to examine cellular morphology (pink, slender, short rods) and subjected to biochemical tests (Triple sugar iron, indole test, and citrate test). Isolates displaying a greenish metallic sheen, a positive indole reaction with Kovac’s reagent, acid over acid with gas production on triple sugar iron, and a negative citrate test were presumptively identified as E. coli.
Similarly, seeded SDA plates were sealed with masking tape and incubated at room temperature (25°C ± 2°C) for 48 hours. They were examined macroscopically for colony growth patterns and colour, and microscopically using Lactophenol cotton blue staining. The culture of fungal organisms showed a characteristic colonial morphology of A. fumigatus including velvety growth, downy or powdery, showing different shades of green or most commonly seen blue-green to a grey-green with a narrow white border and tan to pale yellow on the reverse side of the plate (Fig. 6). The color is known to typically darken with age. Isolates were also confirmed using the Lactophenol cotton blue staining technique, revealing a conidiophore, vesicles, metulae, phialides, and conidia (Fig. 4 A and B). Molecular characterization of E. coli and A. fumigatus was not performed due to a lack of funds.
Figure 4.
A: Stained cultures showing characteristic chains of Conidia; B: Microscopic characteristic greenish velvety colony morphology of A. fumigatus on SDA.
Antifungal susceptibility testing was conducted on SDA using the disc diffusion method, with locally prepared discs impregnated with nystatin (300 IU/disc) and copper sulfate (300 μg/disc). Briefly, a few fungal colonies were suspended in normal saline and seeded on SDA to create a uniform lawn. Antifungal agents were then placed on the plates, incubated overnight, and observed for zones of inhibition. The antifungal susceptibility test showed that the A. fumigatus isolate was resistant to copper sulfate, but there was evidence of a zone of inhibition with nystatin (Fig. 5).
Figure 5.
Photograph showing in vitro antifungal susceptibility testing on SDA (Arrow; nystatin at 300 IU/disc and Star; Copper sulfate at 300 μg/disc.).
Additionally, direct antibiotic susceptibility testing was performed as described by She [17] using locally prepared antibiotic discs (Oxytetracycline (250 µg), tylosin (250 µg), colistin (480 IU), enrofloxacin (250 µg), streptomycin (300 µg), gentamicin (250 µg), florfenicol (300 µg), and penstrept (150 IU/300 µg)) on nutrient agar plates seeded with cultures of E. coli. The outcome showed that the E. coli isolate was resistant to oxytetracycline, enrofloxacin, tylosin, and colistin while susceptible to Florfenicol and Penstrept®(penicillin+streptomycin).
Treatment
The confirmatory diagnosis was aspergillosis caused by A. fumigatus complicated by secondary bacterial infection caused by E. coli. Severely affected birds were culled and the remaining flock was treated using 500,000 I.U Oral nystatin at 320 tablets/day for 14 consecutive days (1 tablet per 10 kg body weight) and Florum® (20% florfenicol) at 1mL/2L of drinking water for 5 days. The farmer was advised to change beddings regularly, and wash the pen with disinfectant. A follow-up after 2 weeks of treatment interventions, a significant improvement in birds’ health was observed, and zero mortality was recorded. The farmer could not however put the actual number of mortalities, but give an estimate of about a hundred. Also, he revealed to our team that the birds commenced laying at 17 weeks old. On the second farm visit, when the birds were 19 weeks old, there was no record of mortality and no drop in egg production.
Discussion
Avian Aspergillosis poses a significant threats to the poultry industry, often resulting from inadequate management practices such as stress, immunosuppression, poor litter management, and the use of locally formulated feed. In this case report, despite the farmer using commercially formulated feed, the ubiquitous nature of Aspergillus species allows their conidia to easily contaminate feed, water, and surfaces within poultry houses. Similar findings were observed by Ameji et al. [7], where feed contamination by Aspergillus conidia was documented. Although various Aspergillus species can infect birds and mammals, A. fumigatus is the primary pathogen, as confirmed in this case. Aspergillus fumigatus was isolated from tissue samples of infected birds, consistent with previous reports of Aspergillosis in newly hatched chicks in Jos, North-central Nigeria [1], and poultry farms in Ado-Ekiti, South-western Nigeria [4].
Aspergillus spores are highly resilient, capable of withstanding harsh environmental conditions and proliferating rapidly under favorable humidity and temperature conditions [18], aligning with the climate in Jos, Plateau State. Necropsy of affected birds revealed granulomatous lesions on the lungs and peritoneum, characteristic of Aspergillosis. These findings are consistent with previous studies by Ameji et al. [7], Oladele et al. [1], and Shapaan and Girh [9]. Histopathologic examination corroborated the gross pathological findings, with fungal hyphae observed within lung lesions, indicating inhalation as the primary transmission route for avian Aspergillosis [19].
Treatment of the remaining birds included oral nystatin (500,000 I.U/tablet) at a dose of 1 tablet per 10 kg body weight (320 tablets/day) for 2 weeks, dissolved in drinking water. Nystatin is a potent antifungal agent that interferes with glucose metabolism by altering cell permeability without significantly affecting hexokinase [2]. To address the secondary bacterial infection, birds were also treated with florfenicol, which is effective against many Gram-negative and Gram-positive bacteria and more potent than chloramphenicol. Florfenicol disrupts bacterial protein synthesis by binding to the 50 S subunits of bacterial ribosomes and is generally considered bacteriostatic [20]. These treatments proved effective, with the farmer observing a significant reduction in mortality and the resumption of egg laying. It is important to understand that secondary bacterial infections can significantly exacerbate the severity and complexity of Aspergillosis in poultry. When birds are afflicted by A. fumigatus, their immune systems are already compromised, making them more susceptible to additional pathogens.
The farmer was also advised to regularly sanitize the birds’ drinking water using Isochlor® (sodium dichloroisocyanurate) at 1 tablet per 1,000L of water and to maintain strict biosecurity measures, including proper feed storage, limiting farm access, regular cleaning and disinfection, flock health monitoring, separation of sick birds, and good personnel hygiene protocols. Prevention and control of Aspergillosis require comprehensive biosecurity, vaccination, and good management practices.
Despite the success in treatment, it is crucial to note that avian Aspergillosis is challenging to manage once established. Therefore, prevention remains the most effective strategy. Using antifungal susceptibility tests via the disc diffusion method on SDA can help veterinarians avoid antimicrobial resistance, drug wastage, and high mortality rates in flocks. A notable limitation of this study is the absence of molecular characterization of the identified pathogens, which would have accurately identified the circulating strains of the pathogens in the flock. Molecular assays are robust, sensitive and specific. This case report highlights the need for improved diagnostic methods and management practices to mitigate the impact of avian Aspergillosis on the poultry industry.
Conclusion
Aspergillosis and colibacillosis are two important diseases of the poultry industry caused by Aspergillus species and E. coli. Diagnosis can be achieved through clinical signs, postmortem lesions, histopathological examination, standard mycological and bacterial culture, and enhancing hygiene and sanitation practices at both the farm and hatchery levels, along with implementing proper disinfection protocols.
Acknowledgment
The authors appreciate the unwavering support from the management of the Pathology Faculty at the Postgraduate College of Veterinary Surgeons of Nigeria (CVSN) NVRI, Vom Study Centre. We are equally grateful to the Head of the VTH CVSN Clinic Training Centre, University of Jos for providing an enabling environment during the course of our clinical training at the Veterinary Microbiology Laboratory of the VTH. This case report did not receive any funding.
Conflict of interest
The authors of this case report declared no competing interests concerning its publication.
Authors contribution
A.A. Bitrus, A. R. Jambalang, O. O. Oladele, N.O. Ameji, and Y. J. Isa’ac were involved in the laboratory diagnosis and clinical management for this case report. G.Y. Gurumyen handled the processing and interpretation of the histopathologic slides. A. A. Bitrus prepared the initial draft of the manuscript. All authors have reviewed and approved the final manuscript.
