Histopathological and Molecular Detection of Some Respiratory Diseases in Backyard Chickens Around Ahvaz City

1. Introduction
Raising backyard chickens is common in Iran. Local poultry are among the most common domesticated animals worldwide and, due to their close contact with their surroundings, are among the most important sources of infectious diseases, posing a threat to the poultry farming industry. Respiratory problems in poultry farms may occur due to various viral, bacterial, and environmental factors [1]. Infection of backyard chickens with Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) represents one of the most significant health and economic challenges to the poultry sector, particularly in rural and traditional farming systems [2, 3]. Backyard chickens often play an essential role in household food security and income generation; however, limited biosecurity measures and the absence of regular vaccination programs make these birds highly susceptible to infectious viral diseases [4]. The virus that causes Newcastle disease (ND) can infect more than 200 bird species. The virulent strains of NDV are responsible for considerable economic losses in the poultry industry worldwide. In developed countries, outbreaks of virulent Newcastle disease (vND) not only cause substantial economic losses but also imposes high costs on the poultry industry through disease control measures, such as vaccination. In many developing countries, vND is also endemic, thereby representing a major constraint on the growth and sustainability of the poultry industry. ND is a highly contagious viral disease of poultry characterized by severe respiratory, nervous, and gastrointestinal symptoms. In unvaccinated flocks, mortality rates may reach up to 100% [2]. Backyard chickens act as important reservoirs for NDV due to their free-range management and frequent contact with other domestic and wild birds, thereby facilitating virus maintenance and transmission to commercial poultry farms [2, 5].
Avian influenza viruses belong to the family Orthomyxoviridae and are classified into two major pathotypes based on their virulence in poultry, namely highly pathogenic avian influenza (HPAI) viruses and low-pathogenic avian influenza (LPAI) viruses. Avian influenza is a major viral disease affecting poultry worldwide. LPAI viruses such as H9N2 are widely prevalent in backyard poultry and can cause respiratory distress, decreased egg production, and immunosuppression [3, 4]. HPAI strains can lead to severe outbreaks with high mortality rates. Moreover, certain avian influenza viruses pose a potential zoonotic risk, raising concerns for public health [3]. The spread of H9N2 has been associated with factors such as migratory bird movements and the operation of live bird markets in some regions. Since the late 1990s, sporadic human infections with H9N2 have been reported in several Asian countries, prompting concerns regarding its potential public health implications [6]. There is also the possibility of virus transmission to humans, changes in virulence, and participation in genetic recombination with other influenza viruses [7]. 
Infectious bronchitis is characterized as an acute viral infection with high transmissibility, involving the respiratory and urogenital systems of chickens of all ages. The disease has a global distribution, causes high morbidity, low mortality, and significant production losses. IBV infection can result in reduced growth performance, poor feed conversion, kidney damage, and reproductive disorders, especially in young or immunologically naïve birds [5]. The IBV belongs to the family Coronaviridae. More than a dozen serotypes have been identified to date. The genetic diversity and frequent emergence of new IBV variants complicate disease control, particularly in backyard poultry populations with limited vaccination coverage [5].
Backyard chickens are often kept under minimal biosecurity conditions, which can lead to close contact with other birds, contaminated environments, and live bird markets. Consequently, these birds can serve as reservoirs and amplifiers of NDV, AIV, and IBV, increasing the risk of virus spread to industrial poultry operations [4, 8]. Co-infections with multiple respiratory viruses have been shown to exacerbate disease severity, leading to higher morbidity, mortality, and economic losses [8, 9]. Implementation of effective vaccination programs, improvement of biosecurity practices, and education of rural poultry keepers are essential strategies to reduce the circulation of these viruses in backyard chickens. Surveillance and early detection are also crucial to prevent the spread of these viruses to commercial farms and reduce public health risks [2, 4].
Therefore, considering the importance of the subject, the present study aimed to examine the molecular and pathological status of backyard chickens in the Ahvaz region infected with Newcastle, influenza (H9N2), and infectious bronchitis viruses.

2. Materials and Methods
2.1. Sampling
Fifty-four backyard chickens were randomly selected from different areas around Ahvaz City. The age and sex of the birds, clinical signs, and necropsy findings were recorded. Tissue samples of the trachea, lung, kidney, cecum, cecal tonsils, and brain were prepared for molecular and histopathological analysis. 

2.2. Molecular detection
2.2.1. RNA extraction
After defrosting the tissue samples, 1,000 μL of RNX (SinaClon, Iran) was added to the microtubes containing 50–100 mg of pooled tissue samples from each bird. RNA was extracted according to the company’s instructions [10]. The samples were kept at room temperature for 5 minutes until the cells were lysed and the nucleic acid was released. Thereafter, 200 μL of chloroform was added to each sample, and the samples were shaken gently for 15 seconds and kept at 5 °C for 5 minutes. The samples were then centrifuged for 15 minutes at 4 °C at 12,000 rpm. The supernatant containing the RNA was removed. In the next step, isopropanol of the same volume as the supernatant was added to precipitate the RNA, and the samples were thoroughly mixed and kept on ice for 15 minutes. The tubes were centrifuged again at 4 °C at 12,000 rpm for 15 minutes. After discarding the supernatant, 1 mL of 75% ethanol was added to each tube. The tubes were then centrifuged at 4 °C at 7,500 rpm for 8 minutes. Finally, the supernatant was discarded, and 50 μL of sterile DEPC water was added to each tube. The extracted RNAs were stored at -70 °C until cDNA synthesis. 

2.2.2. cDNA synthesis
To synthesize cDNA from the extracted RNA, a cDNA Synthesis Kit (manufactured by Yekta Tajhiz Azma Company, Iran) and a random hexamer primer were used. 

2.2.3. cDNA amplification
The reaction mixture included 10 μL of 2X Master Mix (with a concentration of 1.5 mM MgCl2; Amplicon, Canada), specific primers for the S gene to detect IBV [11], the HA gene to detect influenza virus subtype H9 [12], and the F gene to detect NDV [13], each at a concentration of 10 picomoles/μL (Table 1), template DNA 3 μL, water 6 μL in a final volume of 20 μL with temperature program.

It is important to note that at every step of the polymerase chain reaction (PCR) reaction, a negative control sample composed of DEPC water was used in place of DNA. Additionally, RNA extracted from the Massachusetts H120 and 4/91 strains of the infectious bronchitis vaccine was used as the positive control. The NDV B1 and La Sota vaccines, as well as allantoic fluid containing the H9N2 virus for influenza, were also utilized as positive controls (Table 2).

The PCR products were analyzed by electrophoresis on a 1% agarose gel for 45 minutes at 100 V, then imaged after staining with a UV-safe dye.

2.3. Histopathology evaluation
Tissue samples were fixed in 10% neutral-buffered formalin. Tissue sections were prepared, and after going through the tissue preparation steps, including dehydration, clarification, and embedding, they were sectioned. Several consecutive sections, 5 μm thick, from each block were stained with hematoxylin and eosin. After staining, the slides were mounted and examined under a light microscope.

3. Results
In this study, among the 54 backyard chickens, 37 were hens and 17 were roosters, aged from 3 months to 2 years. Clinical examination of these birds revealed nonspecific and general signs, including weight loss and ruffled feathers. After euthanasia, necropsy findings showed that most carcasses had hyperemic tracheae containing mucous or sometimes purulent exudate. The lungs and kidneys mainly appeared normal, though in some cases they were slightly congested. No necrotic lesions in the liver or thickening of the intestines were observed.

3.1. Molecular Results 
Forty-six (85%) of the examined birds were infected with at least one of the three viruses, whereas no viruses were detected in 8 birds (Table 3, Figures 1, 2 and 3).

F1

Positive cases occurred in both sexes and across all age (3 months to 2 years).

3.2. Histopathological results 
Microscopic examination of tissue sections from the brain, trachea, lungs, and kidneys, and intestines of various birds revealed different lesions, which are described below:
Brain: Meningoencephalitis was diagnosed in 20 cases (Figure 4).

Kidney: In birds infected with the IBV or co-infected with all three viruses, interstitial lymphocytic nephritis, necrosis of tubular epithelial cells, and glomerulonephritis were observed (Figure 5).

Trachea: In positive samples, tracheitis was observed. The lesions ranged from mild to severe (Figure 6).

Lungs: In affected birds, interstitial lymphocytic pneumonia, bronchopneumonia, and hyperplasia of lymphoid tissue around the bronchi were observed (Figure 7).

4. Discussion
Backyard chickens are usually kept in rural household yards as a source of protein and as part of the family’s income. These birds are typically fed table scraps and kitchen waste, kept in coops at night, and allowed to roam freely during the day. Because of their recognized role in the transmission and maintenance of various viral and bacterial pathogens, backyard birds have been frequently reported as infected hosts in many developing regions. In the present study, analysis of samples obtained from 54 backyard chickens revealed a high circulation of avian influenza virus subtype H9N2, ND, and IBVs within this population. Reports similar to these have been documented by researchers worldwide. Rahman et al. (2021) et al., while detecting influenza, Newcastle, and IBVs in samples collected from 100 backyard chickens, demonstrated the widespread presence of these viruses among these birds. They found that even in cases where these birds had received inactivated influenza and ND vaccines, the two viruses remained detectable. This indicates that these vaccines are unable to control the disease or prevent infection with wild strains in the field [14]. Sharma et al. (2015) screened backyard chicken flocks to determine the occurrence of ND, avian influenza, and chicken infectious anemia viruses in western India and reported that serum samples were positive for antibodies against these agents at varying percentages [15]. The presence of ND, influenza, infectious bronchitis, and EDS viruses in rural poultry of Bangladesh was reported by Biswas et al. (2009) [16]. Shadmanesh and Mokhtari (2013) reported the presence of antibodies against ND and avian influenza viruses, Mycoplasma gallisepticum, and Mycoplasma synoviae bacteria in rural Iranian poultry [17]. Mahzounieh et al. (2006) identified serological evidence of IBV exposure in blood samples collected from rural chickens in Isfahan and reported that 85.3% of the sera tested were positive [18]. In 2021, Pouarbbas et al. conducted a serological study, molecular identification, and phylogenetic analysis of the H9N2 avian influenza virus in domestic poultry in rural areas of Fars Province, Iran. The findings of this study showed that 54% of the serum samples were positive. The results of this study showed that, given the high positive serum titers in a large number of domestic poultry in rural areas, continuous monitoring of these birds for avian influenza infection and the implementation of more effective control programs seem necessary [19]. The high prevalence of IBV, H9N2, and NDV infections in the present study indicates the important role of these viruses in causing respiratory disease in domestic poultry and in the spread of these viruses in broiler flocks around Ahvaz. In 2018, Amer et al. isolated and identified co-infections of H9N2 avian influenza and ND in poultry flocks with respiratory symptoms and mortality in Egypt. Natural co-infections with AIV H9N2 and velogenic ND virus (vNDV) were identified [20]. In the microscopic examination of the lesions in the present study, lymphocytic interstitial nephritis, necrosis of epithelial cells of the renal tubules, glomerulonephritis, meningoencephalitis, and PVC, mild to severe lymphocytic tracheitis, lymphocytic interstitial pneumonia, and bronchopneumonia were observed. As noted in various sources, the microscopic lesions of these three viruses are not pathognomonic or specific, and they cannot be attributed to these infections with 100% certainty. However, because these tissue lesions were not observed in negative cases, they may be attributed to these viruses. The tracheal mucosa of chickens with IBV becomes inflamed. Due to the loss of cilia, rounding and detachment of epithelial cells, and infiltration of heterophils and lymphocytes into the tissue, these changes are observed. Kidney damage caused by the IBV primarily involves interstitial nephritis (inflammation of the tissue between the renal tubules). The virus causes degeneration, vacuolation, and desquamation of the tubular epithelium, and infiltration of heterophils into the interstitial tissue in the acute stages of the disease. Necrotic areas may also be seen, and in chronic cases, signs of attempted tubular epithelial repair are seen. During recovery, the inflammatory cell population changes to lymphocytes and plasma cells, and in some cases, destructive changes may lead to atrophy of various areas. In this study, 35 cases of infectious bronchitis that tested positive by molecular testing showed various microscopic findings: 11 cases showed interstitial nephritis, and 10 showed glomerulonephritis. Also, acute necrosis of the renal tubules was observed in 23 cases, as reported previously, which may be one of the causes of these injuries [21]. Gastrointestinal lesions are less common in mild infections caused by the IBV. In a study by Raj and Jones (1997), no pathological lesions were observed from the crop to the ileum of chickens infected with the enterotropic G strain. However, there are reports of villous atrophy, congestion, and local infiltration of lymphocytes, macrophages, and heterophils in the rectum. In this study, all cases showed a spectrum of enteritis, which is certainly not solely due to viral agents but is more likely due to parasitic agents [22]. ND microscopic lesions are not pathognomonic and depend on many factors. The lymphoid tissues of the intestine, including cecal tonsils and Peyer’s patches, are the primary sites of necrosis and hemorrhage in affected chickens and turkeys [23]. Virulent NDV infection in chickens and turkeys is commonly associated with lymphoid depletion and necrosis affecting the cecal tonsils, spleen, thymus, and bursa. Infection in chickens and turkeys may lead to hemorrhagic, ulcerative, or lymphocytic tracheitis. In this study, 14 cases of ND showed lymphocytic tracheitis. Nine cases were also infected with two other viruses simultaneously. NDV also causes gliosis with perivascular cuffing (PVC) of inflammatory cells around the vessels of the cerebellum and brainstem, which is more common in acute forms. Another study investigated the prevalence of ND in wild pigeons in São Paulo, Brazil, and found that the most significant histopathological changes were mononuclear cell infiltration in the brain, kidneys, ventricles, and spleen [24]. LPAI viruses, such as subtype H9N2, cause the most common lesions in the respiratory tract in broilers; the tracheal mucosa may exhibit edema and congestion, with occasional evidence of hemorrhagic changes. Fibrinous-purulent bronchopneumonia can develop when secondary pathogens such as Pasteurella multocida or Escherichia coli are involved. In several natural cases in laying hens and in chickens infected intravenously, the kidneys were swollen and contained visceral urate deposits [25]. In this study, 18 cases of tracheitis were diagnosed: 14 were H9N2-positive, and 9 were positive for all three agents. LPAI viruses cause a spectrum of pneumonia in poultry, ranging from fibrinous pneumonia to lymphocytic interstitial pneumonia. In acute and severe cases, fibrinous pneumonia with tracheitis is common. In this study, 19 cases of lymphocytic interstitial pneumonia were diagnosed. The predilection for the kidneys is dependent on the type of influenza virus. In fatal cases of LPAI infection, marked depletion of lymphocytes accompanied by necrosis or apoptotic changes has been observed in primary lymphoid organs, including the bursa of Fabricius, thymus, and spleen. In contrast, lymphocytic infiltration is frequently noted in other tissues such as the trachea and nasal mucosa. Detection of viral antigen in lymphocytes is uncommon, whereas strong expression is typically observed in necrotic epithelial tissues of the respiratory system, kidneys, and pancreas. Lymphocytic meningoencephalitis, characterized by focal gliosis, neuronal necrosis, and neurophagy, is commonly observed in severe influenza. In this study, 8 cases of meningoencephalitis were identified, all of which tested positive for all three viruses. However, the occurrence of some microscopic lesions, such as meningoencephalitis, interstitial nephritis, or interstitial pneumonia, may be caused by different viral agents.
Finally, it is concluded that rural poultry in the Ahvaz region are an important reservoir of avian pathogenic viruses and can contribute to their transmission to industrial birds.

​​​​​​​5. Conclusion
The present study demonstrates a high molecular prevalence of avian influenza virus subtype H9N2, NDV, and IBV in backyard chickens around Ahvaz City, with a considerable rate of mixed infections. The detection of single and co-infections, together with characteristic histopathological lesions in different tissues, indicates active circulation of these viruses in backyard poultry populations. These findings highlight the epidemiological significance of backyard chickens as important reservoirs for avian respiratory viruses and their potential role in transmitting infections to industrial poultry farms. Therefore, continuous surveillance, implementation of appropriate biosecurity measures, and inclusion of backyard flocks in control and prevention programs are essential to reduce the risk of virus spread and economic losses in the poultry industry.

Acknowledgements
The authors acknowledge the staff members of the Department of Avian Medicine and Parasitology, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Compliance with ethical guidelines
Ethical approval for this research was granted by the Research Ethics Committee of Shahid Chamran University of Ahvaz, Ahvaz, Iran (Code: IR.SCU.REC.1403.113).

Funding
This study was financially supported by the Department of Avian Medicine’s statutory funds, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Authors' contributions
Conceptualization and study design: Zahra Boroomand; Experiments: Ali Ghanei; Data analysis and interpretation: Zahra Boroomand and Anahita Rezaie; Writing the original draft: Setayesh Farokhian; Review and editing: Zahra Boroomand and Setayesh Farokhian.

Conflict of interest
The authors declared no conflict of interest.

Data availability
All data generated or analyzed during this study are included in the current article.

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