Detection of Ornithobacterium rhinotracheale antibodies in broiler by ELISA in Mosul city

Abstract


Introduction
Ornithobacterium rhinotracheale (ORT) is a Gramnegative, pleomorphic, rod-shaped bacteria that mainly affect the respiratory system (1,2). ORT has been isolated from bird species worldwide, including chickens, geese, ducks, turkeys, pigeons, and other domestic and wild birds (3)(4)(5). Infection with ORT is frequently associated with growth retardation, reduced and low-quality egg production, and mortality in chickens (4). Many factors can affect ORT to be a primary or secondary pathogen, such as the virulence of the strain, unfavorable environmental factors, the immune status of the flock, and infections with other pathogens (6). The infections by ORT can be spread by horizontal transmission, including direct or indirect contact methods. However, the vertical transmission was also suggested as a rapid means for spreading infection (3). The laboratory diagnosis of ORT infection is mainly performed based on bacterial isolation and identification (7), serological tests (8,9), and molecular detection by polymerase chain reaction (10,11). Nevertheless, bacterial isolation is time-consuming and requires special conditions for successful growth (1,12). On the other hand, Molecular methods require expensive equipment and well-trained personnel. Nevertheless, large genetic variation of species or subspecies within the genus Ornithobacterium renders genetic characterization of this cumbersome bacteria work for routine detection (2). Therefore, primarily ELISA-based tests and serological methods are adequate for rapidly diagnosing ORT infections. Moreover, the ELISA tests can be performed on a large scale, giving an advantage over other tests (8,13). In Iraq, the respiratory infections of broiler flocks have overshadowed their impact and led to significant economic losses for this vital industry. Many recent studies focused mainly on viral causes of infections, including avian influenza virus (AI), Newcastle disease virus (NDV), infectious bronchitis virus (IB) (14)(15)(16), or Mycoplasma infections (17). The first study in Iraq was conducted by Al-Hasan et al. (18), which directed to isolate and identify ORT in broiler farms in the South of Iraq. However, there is no published data related to infection by ORT, especially in poultry in Mosul city, and to the best of our knowledge, this is the first study to detect the presence of ORT in broilers. Accordingly, this study aimed to detect the presence of antibodies against ORT in broilers using ELISA.

Sampling
A total of 16 small broiler farms aged 4-6 weeks were selected randomly from different areas of Mosul city and adjacent areas from April to December 2021. The areas include; Al-Rashidia, Sada and Bawiza, Gogjali, Bashiqa, Bartilla, and Al-Hamdania. All broilers have previous vaccination history with Newcastle disease (ND), infectious bronchitis (IB), and infectious bursal disease (IBD). Blood samples (n=368) were collected from the wing vein of the birds. Samples were submitted to the Department of Microbiology (College of Veterinary Medicine, Mosul University). After collection, serum samples were separated and stored at -20 °C until assayed (19). Also, data collected from these farms include; the type of management of broiler farms (standard or non-standard sanitary and biosecurity practices), antibiotic administration according to sensitivity test results, and presence of a previous history of respiratory infections using a particular questionnaire form.

Serology
A Commercial indirect enzyme-linked immunosorbent assay (ELISA) kit for detecting Ornithobacterium rhinotracheale (ORT) antibodies were purchased from BioChek, UK. Sera were tested according to the manufacturer's instructions. Briefly, 100 µl of diluted sera (1:100), positive and negative controls were added to a coated microplate and incubated for 60 min. Then the plate was washed with 350 µl/well 1X wash buffer solution using an automated microplate washer (BioTek, 50 TS, USA). After that, 100 µl of the conjugate solution was added to each well and incubated for 30 min. Then, the plate was rewashed, as mentioned previously. Finally, 100 µl of substrate solution was added and incubated for 15 min, followed by 100 µl of stop solution. The optical density (OD) was measured at 450 nm using a microplate reader (BioTek 800TS) with Gen5™ software. OD readings were transformed to serum/positive percentage (S/P%) according to a specific equation cited by the manufacturer. The sample considered positive if it gives S/P % >70%, while 60% < S/P % < 70% considered doubtful, and S/P % < 60% considered negative. S/P%=(OD sample-ODNC)/(ODPC-ODNC) (20). Chi-square (χ2) test was performed to compare P values using Statistical Package for Social Sciences (SPSS) software version 22. P<0.05 was considered significant.

Discussion
In the current study, ORT antibodies were investigated in serum samples of small broiler farms in Mosul city. The results indicated that 32.1% (118/368) of broilers have circulating antibodies against ORT. Other studies also reported the ORT in poultry worldwide, Eid et al. (21) reported that 80% of the tested broiler chickens' flocks were seropositive for ORT, and 46/49 (93.9%) of selected sera found positive for ORT in chickens that were suffering from respiratory infections. Also, Xue et al. (19) reported 44.06% seroprevalence of ORT among the tested chickens. In contrast, Bhuiyan et al. (9) recorded high seropositivity of 43.3% (156/360) of ORT in broilers in Bangladesh.
Similarly, Gullu and Erbas (13) evaluated the presence of ORT in Turkey and found that the seropositivity rate of the disease was 232/420 (55.2%) by ELISA. Finally, Baksi et al. (8) found that 354/476 (74.37%) were seropositive for ORT while testing Broiler breeder farms in India. The high seropositivity of ORT infection is directly correlated to a significant economic impact on the poultry industry since infections with ORT might trigger the co-infection with other pathogens (9,22). Our study revealed that Bashiqa and Al-Hamdania broiler farms recorded significant differences, with the lowest seropositivity at 22/93 (23.7%) and 12/68 (17.6%) values, respectively. Al-Rashidia, Sada, and Bawiza recorded the highest seropositivity at 22/47 (46.8%) and 19/44 (43.2%) values, respectively. This might be due to the type of management adopted by these small broiler farms, and this was confirmed by this study with 81.2% of these farms were reported with poor management systems, including insufficient ventilation with high ammonia levels, poor hygienic conditions such as poor-quality litter and overcrowding (8,19).
On the other hand, only 18.8% of the studied farms followed a standard management system.
Another stress factor that affects the poultry industry is the arbitrary administration of antibiotics. Our study revealed that only 12.5% of the studied farms followed the standard method of antibiotic administration after, i.e., after performing an antibiotic sensitivity test. While 87.5% of the farms did not follow standard methods of antibiotic administration and only relied on arbitrary use of different types of commercial antimicrobial agents that might be of low quality of manufacturing to compensate for lousy management system followed and poor biosecurity regimes. The misuse of massive doses of antibiotics for long periods and insufficient diagnosis could lead to resistant pathogenic strains of bacteria, leaving few choices for treatment shortly after (18,23). A recent study by Al-Hasan et al. (18) reported the highest resistance 100% to antimicrobial agents such as azithromycin, colistin, erythromycin, gentamicin, oxacillin, tilmicosin, and amoxicillin in broiler farms, and this indicates dangerous levels of resistance that could be easily transmitted to human through the food chain and finally could lead to the emergence of resistant bacterial strains (23). Other studies also confirmed these results (3,4,24). Another factor that affects ORT infections is a history of respiratory infections (25). Although all studied broiler farms have a previous history of vaccination with primary viral pathogens such as ND, IB, and IBD, we found that 62.5% of the studied farms have previous respiratory infections with prominent signs such as sneezing, nasal secretions and swelling of the sinuses. This actually might reflect the failure of the vaccine or vaccination process to protect the flock and render them vulnerable to other respiratory pathogens (1,9). Many viral, bacterial, and fungal agents play a crucial role in exacerbating the respiratory problem with the presence of ORT, which leads to the development of co-infection with such pathogens and finally results in extensive economic losses, especially with the absence of adequate vaccination against ORT (22,26,27).

Conclusion
Significant seropositive antibodies against ORT were detected in different small broiler farms in Mosul city, mainly with poor hygienic and management practices, arbitrary use of antibiotics, and the risk of other respiratory pathogens, all rendering the investments in such an important industry vulnerable to severe economic losses.