Introduction

Respiratory tract infections are considered as one of the serious problems in intensive poultry production. Diseases cause economic damage to breeding units due to increased mortality, reduced growth, and elevated slaughterhouse condemnation rates (Chin et al., 2013). Various bacterial pathogens have been identified as the etiological agents of respiratory diseases. Ornithobacterium rhinotracheale (ORT) is a recently identified bacterial pathogen associated with respiratory infection in poultry (Thieme et al., 2016) which has been isolated throughout the world (Chin et al., 2013). Currently, ORT infection appears to have become endemic, especially in areas with intensive poultry production and multiple age farms. In Iran, the outbreak of respiratory diseases associated with ORT has also been reported (Mayahi et al., 2016).The sensitivity of ORT to antibiotics is strain-dependent and geography-related (Watteyn et al., 2016). ORT can acquire reduced susceptibility or resistance against antibiotics (Umali et al., 2017). Treatment and control of ORT infections with antibiotics are very difficult due to the varying susceptibility and resistance of this bacterium (Chin et al., 2013). Therefore, the best strategy to prevent Ornitho bacteriosis is believed to be vaccination (Schuijffel et al., 2006). A wide range of vaccines has been developed to control experimental and natural infections associated with ORT (Churria et al., 2012). Sprenger et al. (2000) showed that an inactivated ORT vaccine in poultry and turkeys induced protective immunity in birds. Recently, the Razi Vaccine and Serum Research Institute of Iran has produced an inactivated ORT vaccine. The researchers have demonstrated that the experimental infection of chickens with ORT causes minor lesions, while the concurrent presence of other respiratory pathogens with ORT increases the severity of lesions associated with ORT (Ellakany et al., 2018).Consequently, to more accurately evaluate the ORT vaccine produced by the Razi Vaccine and Serum Research Institute, a simultaneous challenge was performed with ORT and the LaSota strain of Newcastle Disease Virus (NDV).

Materials and Methods

Chickens

In this study, a total of 90 one-day-old specific-pathogen-free (SPF) White Leghorn chickens (Venky^,s Ltd, India) were randomly divided into five equal groups. The chickens were kept in separate cages. The chickens were housed in the Bird Disease Research Building of the Razi Vaccine and Serum Research Institute. Throughout the experiment, the chickens had unrestricted access to sterile water and disinfected feed. No vaccines or medications were given to the chickens until the end of the test.

Bacteria

The ORT used in the present study with the isolation number of R87-7/1387 (JF 810491) had previously been isolated and identified from poultry farms in different provinces of Iran. The bacteria were kept in the Avian Diseases Department of the Razi Vaccine and Serum Research Institute at -70°C. The bacteria were thawed and cultured on Columbia Agar (Oxoid Ltd, Basingstoke, UK) with 5% sheep blood and was incubated for 48 h at 37°C in an atmosphere with 5% CO₂. Next, the bacterial colonies were transferred into the brain heart infusion (Oxoid Ltd, Basingstoke, UK) and were incubated for 24 h at 37°C. The bacteria were washed twice with phosphate-buffered saline. A suspension containing 1×10¹⁰ colony forming units (CFU) per 0.5 mL was prepared.

Experiment Design

At the age of 14 days, the chickens in groups V₁ and V₂ were vaccinated by 0.3 mL of Razi ORT vaccine containing 1×10⁷ whole-cell bacteria in a mineral oil adjuvant subcutaneously into the neck. Simultaneously, the birds in groups C₁, C₂, and C₃ were injected with 0.3 mL of sterile physiological serum subcutaneously. At the age of 42 days, according to Table 1, the birds of group V₁ were challenged with 1×10⁶ EID₅₀/dose of the LaSota strain of NDV by the ocular route and 1×10¹⁰ CFU per 0.5 mL of the ORT through the intratracheal route. The chickens of group V₂ were inoculated only with ORT. The chickens of group C₁ were challenged with both intraocular NDV and intratracheal ORT, while the birds in group C₂ were inoculated only with ORT. Group C₃ was maintained as an unchallenged control group. Each bird in group C₃ was given one drop of intraocular sterile distilled water and 0.5 mL of intratracheal sterile physiological saline.

Table 1. Plan of vaccination and challenge of the experimental groups.

Sampling

In order to determine the antibody titer in the blood serum of chickens against ORT, at the ages of 14 days (before vaccination) and 42 days (before challenge with ORT), in addition to days 2, 4, 6, 8, 10, and 12 after the challenge with ORT (AC), blood samples were taken from chickens of all experimental groups. On days 2, 4, 6, 8, 10, and 12 AC, three chickens of each group were randomly killed by cervical dislocation. The samples from the trachea, lungs, air sacs, liver, and spleen of those birds were collected immediately and were examined by bacteriological and molecular methods.

Bacteriological Analysis

For microbiological analysis, samples were taken from the lungs, trachea, air sacs, liver, and spleen of chickens under sterile conditions. The steps of culture and biochemical identification of bacteria were completed according to the method of Ghasemipour et al. (2019) described previously.

Antibody Detection

The titers of antibodies against ORT were measured by enzyme-linked immunosorbent assay (ELISA).For this purpose, the BioChek ORT Antibody test kit (Netherland) was used. The test was carried out according to the manufacturer's instructions.

DNA Extraction

Bacterial genetic material was extracted using the phenol-chloroform method previously described by Ghasemipour et al. (2019).

Primers

Primers used in this study were designed by Van Empel and Hafez (1999). The sequences of primer pairs were OR 16S-F₁ (5´-GAG AAT TAA TTT ACG GAT TAA G-3´) and OR 16S-R₁ (5´-TTC GCT TGG TCT CCG AAG AT-3´). The 16s rRNA gene is a suitable tool for molecular studies (Montes et al., 2018). These primers amplify a 784 bp fragment on the 16s rRNA gene of ORT.

PCR

Molecular tracing of the samples was performed using the PCR method. The details of the technique were previously described by Ghasemipour et al. (2019).

Statistical Analysis

The data were statistically analyzed by the SPSS software version 22 (SPSS Inc., Chicago, Ill., USA). In order to compare the means between and within the groups, a t-test was applied.

Results

Mortality

We did not observe mortality in any of the experimental groups during the experiment.

Serology

The mean of ELISA ORT titer in different sections of the study is presented in Table 2. Antibodies were not present in the blood of chickens in any of the experimental groups before vaccination. After vaccination, the titer of antibody in vaccinated groups was significantly higher than in unvaccinated groups. After challenging the chickens with ORT, the mean of antibodies in the blood of chickens in vaccinated groups augmented. However, the difference between the mean antibody titers before and after challenge with bacteria in vaccinated groups was not significant (Table 2).

Bacterial Identification

The results of bacteriological isolation are presented in Table 3. Bacteria were not isolated from the liver and spleen of any of the chickens in distinct experimental groups in post-challenge sampling. In vaccinated groups, the ORT was not recovered from the lungs, trachea or air sacs of the birds. However, in the unvaccinated groups challenged with ORT (C₁ and C₂), O. rhinotracheale was isolated from lungs, trachea, and air sacs. In group C₁, ORT was isolated from the trachea and air sacs on the second and fourth days after the challenge, while it was isolated from lungs exclusively on the fourth day. In group C₂, the ORT was found in the lungs, trachea, and air sacs on the second day after challenge (AC).

Molecular Detection

To investigate the presence of bacteria after the challenge of chickens in different experimental groups, a PCR test was performed on samples taken from the trachea, lungs, air sacs, liver, and spleen of chickens. Finally, ORT was only detected by PCR in the samples collected from the lungs, trachea, and air sacs of birds on 2 DAC in V₁, V₂, and C₂ groups. In the C₁ group, on days 2 and 4 after challenge with bacteria, ORT was detected in lungs, trachea, and air sacs samples (Table 3).

Table 2. Mean ORT Elisa titer in different phases of the experiment, comparison of mean ORT Elisa titer between groups after challenge, and comparisons of mean ORT Elisa titter before and after challenge within vaccinated groups.

BV: before vaccination; BC: before challenge; AC: after challenge.

Ns: not significant, *: significant difference (P≤0.05),  **: significant difference (P≤0.01).

V₁ (VAC ⁺, ORT ⁺, ND ⁺), V₂ (VAC ⁺, ORT ⁺, ND ^-). C₁ (VAC ^-, ORT ⁺, ND ⁺), C₂ (VAC ^-, ORT ⁺, ND ^-), C₃ (VAC ^-, ORT ^-, ND ^-)

Table 3. Results of culture and PCR of the different organs after challenge.

AC: after challenge

V₁ (VAC ⁺, ORT ⁺, ND ⁺ ), V₂ (VAC⁺, ORT⁺, ND ^- ), C₁ (VAC ^-, ORT ⁺, ND ⁺ ), C₂ (VAC ^-, ORT ⁺, ND ^- ), C₃ (VAC ^-, ORT , ND)

Discussion

Nowadays, ORT is considered a promising pathogen of poultry and a serious threat to control practices in the poultry industry (Churria et al., 2012). In profitable poultry production, vaccination plays an important role in herd health. Commercial and experimental bacterins have been the most commonly used vaccines against ORT since 1998.This study demonstrated the efficacy of the first Iranian native inactivated ORT vaccine in layer chickens under a challenging system.

In the present challenge trial, contrary to the high rate of mortality reported by Pan et al. (2012), even in the unvaccinated groups that were challenged with ORT and ND, no mortality was observed in chickens. The mortality rate due to ORT is strongly related to the predisposing factors, the virulence of bacteria, and susceptibility of birds. Experimental infections in chickens have shown that the severity of the disease induced by ORT varies between strains (Chin et al., 2013). A difference in pathogenicity between the strains of ORT has also been reported by Van Veen et al.(2000). Moreover, White Leghorn chickens used in this study were found to be less susceptible to ORT infection than broilers and turkeys (Van Veen et al., 2000). Variety in mortality rate may be attributed to the high virulence of bacteria, vaccination, inadequate breeding management conditions, including high bird density per unit area, poor ventilation, and the presence of other pathogens in farms exacerbating any disease that has been brought about. Nevertheless, it seems that the presence of infectious and non-infectious agents can act as an intensifier of ORT pathogenicity. Low bacterial pathogenicity, controlled breeding conditions, and low susceptibility of Leghorn chickens might be due to the lack of mortality in the present investigation.

No antibody titer was detected against ORT in the current study before vaccination in none of the experimental groups, before the challenge in unvaccinated birds, and the birds of the negative control group, which might be due to the use of SPF chickens and the breeding of birds in separate cages and separate rooms. After vaccination and after challenge, the titer of antibody against ORT in challenged vaccinated groups (V₁ and V₂) was significantly higher than challenged unvaccinated groups (C₁ and C₂).

The main reason for vaccinating poultry is to induce high levels of antibody to protect birds in the face of disease challenges. The results of the present study indicated the effective role of the vaccine in inducing antibody titers against ORT, which was consistent with the results of Schuijffel et al. (2006) and Erganis et al. (2010). Several researchers have developed and used bacterins for the control of ORT infection outbreaks in commercial poultry under experimental and recorded conditions (Schuijffel et al., 2006; Erganis et al., 2010). However, an important issue in using bacterial vaccines is that different bacterial serotypes may not have much antigenic similarity with each other leading to inadequate immunity after usage in birds. Serotype A of ORT is the most prevalent serotype in chickens (Chin et al., 2013). Consequently, this serotype was chosen for the inactivated Razi ORT vaccine. This bacterial serotype was isolated from poultry farms with respiratory diseases in different provinces of Iran.

On days 2, 4, 6, 8, 10, and 12 AC, bacteriological and molecular detection were performed on the samples of lungs, air sacs, trachea, liver, and spleen. In none of the experiment groups, ORT was detected by culture and PCR from the liver and spleen, which may be related to the low pathogenicity of the bacterium. This was in agreement with the study by Hegazy et al. (2016) and Umali et al. (2017) who did not isolate ORT from the heart and liver. Researchers have shown that in natural and experimental infections, ORT has been isolated from the trachea and lungs of infected birds (De la Rosa et al., 2018; Gavrilovic et al., 2016; Hauck et al., 2015). In the present investigation, ORT was isolated from respiratory organs in unvaccinated groups on 2 and 4 DAC, while in vaccinated birds ORT was not isolated even on 2 DAC. Lack of bacterial isolation after challenge in vaccinated groups could be attributed to the influential role of the Razi ORT vaccine in preventing infection. In unvaccinated chickens challenged with ORT and NDV, the bacteria were detected by culture and PCR on 2 and 4 DAC. However, in birds challenged exclusively with ORT, the bacteria were isolated on 2 DAC. The NDV appears to have caused a longer bacterial infection in the respiratory organs. Van Empel et al. (1999) demonstrated that ORT is isolated from the trachea and air sacs of poultry only until the second day after exposure to the bacterium. On the other hand, in birds that were first exposed to the virus and then to the bacterium, the bacterium was isolated from the lungs and air sacs of infected birds for up to ten days after a lung infection. Furthermore, the lack of bacterial isolation in the unvaccinated birds challenged with ORT and ND and unvaccinated birds challenged only with ORT on 6, 8, 10, and 12 DAC can be due to the low pathogenicity of the bacterium and the removal of the bacteria by the immune system of the birds. The bacteria were detected in the PCR of the samples of lungs, trachea, and air sacs on 2 DAC. The ORT can normally be isolated by culture only at an early stage of the infection and attempts to recover the organism at a later stage often fail (Chin et al., 2013). The reason for the positive results of PCR on 2 DAC in vaccinated groups despite the negative result of culture could be the high sensitivity of specific primers in the identification of bacterial genetic material. According to the serologic, microbiological, and molecular results presented in this study, the Razi ORT vaccine can effectively protect chickens against infection with serotype A of the bacterium.

Acknowledgments

The authors would like to thank Razi Vaccine & Serum Research Institute of Karaj, Agricultural Research, Education and Extension Organization (AREEO), for providing facilities and financial support. The investigators would like to thank Dr. Abas Nouri for his assistance in vaccination, collecting the samples and cooperation in the laboratory work.

Conflict of Interest

The authors declared no conflict of interest.