جداسازی و شناسائی مولکولی آوی‌باکتریوم پاراگالیناروم جدا شده از مرغان بومی و تخم‌گذار تجاری در ایران

Introduction

Avibacterium paragallinarum is a Gram- negative bacterium and the causative agent of infectious coryza, an important disease of chickens associated with an acute upper respiratory infection, growth retardation, marked drop in egg production (10-40%), reduced hatchability, and increased number of culls. Infectious coryza is normally characterized by the acute onset, very rapid spread in the flock, and by the signs of nasal discharge, facial swelling, lacrimation, anorexia, and diarrhea (Blackall and Soriano‐Vargas, 2020). Horizontal transmission within the flocks occurs through aerosols and direct contact. However, vertical transmission from parents to progeny stocks has yet to be documented. Therefore, the intermittent excretion of Av. paragallinarum from the carrier birds with subclinical infections plays an important role in spreading the disease (Blackall and Soriano‐Vargas, 2020). Infectious coryza has been reported from all around the world including Iran where chickens are raised (Badouei et al., 2014; Nouri et al., 2014; Patil et al., 2017; Crispo et al., 2018; Wahyuni et al., 2018; Nouri et al., 2021).

Antimicrobial agents may be used to treat the diseased flocks in order to reduce the severity and the spread of the disease. However, generally, prevention of infectious coryza mainly relies on good biosecurity practices and vaccination in poultry flocks. Despite these measures, sporadic infectious coryza outbreaks continue to occur and pose significant economic losses to the poultry industry especially in developing countries. Inactivated whole-cell vaccines against infectious coryza are widely used however their protection is only limited to the serovars used to prepare the vaccine (Blackall and Soriano-Vargas, 2020). In Iran, despite extensive vaccination programs against infectious coryza, the disease is frequently observed in the poultry farms. Therefore, unvaccinated poultry flocks and the farms that do not strictly follow the biosecurity principles and/or have not been properly vaccinated, are at risk. Moreover, broilers that are not usually vaccinated are at risk of exposure and infection (Christensen et al., 2002).

Despite the widespread occurrence of the disease in Iran, the genotypic characteristics of Av. paragallinarum in isolates circulating among poultry farms are largely unknown. Therefore, the aims of this study were molecular characterization and determination of antimicrobial susceptibility patterns of Av. paragallinarum isolated from the suspected cases of infectious coryza in backyard and commercial layer chickens in three provinces of Tehran, Alborz, and Qazvin in Iran.

Materials and Methods

Sample Collection

Swab samples were provided from five commercial laying farms (25 samples) and backyard chickens (20 samples) in which suspected chickens to infectious coryza were found during autumn and winter between November 2018 and May 2019. Swabs were taken from eye secretions and mouth cavity of suspected birds and placed in a tube containing pre-prepared NAD-enriched brain-heart infusion (BHI) medium and transferred to the laboratory in less than 24 hours. Only farms with obvious symptoms including unilateral or bilateral facial swelling, subcutaneous sinus swelling, tearing, and rhinorrhea were sampled. Sampled farms were located in Tehran, Alborz, and Qazvin provinces of Iran.

Bacteriological Culture

Each swab sample was streaked onto a sheep blood agar plate, then one or two intersecting lines of Staphylococcus aureus as a nurse bacterium was made on the medium. The inoculated plates were incubated at 37°C and 5% CO₂ for 24 hours. The small colonies with satellite growth as a single colony were carefully searched and pure colonies were isolated by re-culturing onto the sheep blood agar plates as described above. The purified colonies were cultured onto 7% Colombian horse blood agar (QUELAB, Canada). Biochemical reactions were performed to determine catalase and oxidase activities. Three Av. paragallinarum confirmed isolates were lyophilized and frozen in glycerol at -80°C until further use.

Antimicrobial Susceptibility Test

The antimicrobial susceptibility of the Av. paragallinarum isolates to a panel of antimicrobial agents was assessed through the agar disk diffusion method on 7% Colombian horse blood agar. The following antimicrobials (concentrations in μg) were tested: cephalexin (30), ceftriaxone (30), ampicillin (10), amoxicillin (30), neomycin (30), streptomycin (10), gentamicin (10), florfenicol (30), oxytetracycline (30), doxycycline (30), linco-spectin (15/200), colistin (10), and trimethoprim-sulfamethoxazole (1.25/23.75). The Escherichia coli ATCC 25922 reference strain was included for quality control. All antibacterial disks were provided from Padtan Teb Co. (Tehran, Iran). Briefly, a bacterial suspension from an overnight culture was adjusted to a turbidity of 0.5 McFarland standards and spread onto 7% Colombian horse blood agar plates. The antimicrobial disks were then placed on the plates and incubated at 37°C and 5% CO₂ for 24 hours. The interpretation of results was carried out according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2018).

Identification of Avibacterium paragallinarum by PCR

The colonies suspected to be Av. paragallinarum were subjected to HPG-2 PCR using primers N1 and R1 resulting to a 500 bp amplified product as shown in Table 1 (Chen et al., 1996; Nouri et al., 2021). No known function has been attributed to HPG-2 amplicon sequence. The extraction of bacterial DNA was performed by phenol-chloroform method as described by Moore & Dowhan (2002). By the use of a NanoDrop^® ND-1000 spectrophotometer (Thermo Fisher, USA), the extracted DNA samples were measured in terms of quantity and purity. The specific primers were synthesized by SinaClon (Tehran, Iran). Amplification reactions were carried out in a 25 μL reaction volume containing 12.5 µL of Mastermix, 0.5 μL of each of the forward and reverse primers, and 6.5 µL of deionized water. Approximately 4 µL of template DNA was added to the mixture. DNA extracts from the cultures of Av. paragallinarum and Ornitobacterium rhinotracheale (ORT) were included in all PCR reaction sets, respectively, as positive and negative controls. Amplification was performed in a thermocycler (Mastercycler, Eppendorf, Germany) as follows: 95°C for 7 min followed by 35 cycles of 94°C for one min, 58°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 7 min. The amplification products were detected by 1% agarose gel electrophoresis in 1 x TAE buffer.

Sequence and Phylogenetic Analysis

The hmtp210 gene of Av. paragallinarum encodes an outer-membrane hemagglutinin (HA) that has an important role in the pathogenicity of this bacterium (Araya-Hidalgo et al., 2017). In all three Av. paragallinarum isolates, the hmtp210 gene was amplified using specific primers (Table 1) and protocol as described previously (Sakamoto et al., 2012). Amplified PCR products were subjected to sequencing with the standard Sanger sequencing method at the Macogen Company (Seoul, South Korea). Blast analysis was performed against the available sequences from Av. paragallinarum strains on the NCBI GenBank database (n=100, accessed on August 2020). Phylogenetic analysis of the hmtp210 genes was done using the Neighbor-Joining and Maximum Composite Likelihood method with 1000 bootstrap replicates following ClustalW alignment option in MEGA-X, version 10.0.5 (Kumar et al., 2018). All ambiguous positions were removed for each sequence pair (pairwise deletion option).

Table 1. Primer sets used for amplification of the DNA fragments in this study

Results

Bacteriological Findings and Antimicrobial Sensitivity Profiles

In total, three Av. paragallinarum isolates were obtained from all the cultured samples. Av. paragallinarum isolated from the commercial and backyard flocks in Iran demonstrated similar antimicrobial sensitivity profiles. All three isolates were resistant to amoxicillin, oxytetracycline, streptomycin, ampicillin, and colistin. Additionally, all the isolates were susceptible to cephalexin, ceftriaxone, florfenicol, gentamicin, lincospectin, neomycin, and doxycycline. Furthermore, all three isolates showed intermediate susceptibility to trimethoprim sulfamethoxazole.

Sequence and Phylogenetic Analysis

Two different genotypes of Av. paragallinarum are responsible for infections in the commercial and backyard flocks in Iran. As shown in Figure 1, the results of phylogenetic analysis on the hmtp210 genes showed that the Av. paragallinarum strains isolated from backyard (IR-98-5nat; MN928968) and commercial layer (IR-98-1alay and IR-98-1blay; MN928969 and MN928970, respectively) flocks in this study were closely related to two different reference strains from Germany (strain 2671; KU143740; serovar B-1) and Australia (strain HP60; KU143744; serovar C-4), respectively. Accordingly, the isolates from backyard (IR-98-5nat) and commercial layer (IR-98-1alay and IR-98-1blay) flocks shared between 100% and 99.82% hmtp210 genes nucleotide sequence identity with the German strain 2671 and Australian strain HP60, respectively (Figure 2). Interestingly, both of the isolates from the commercial layer flocks (IR-98-1alay and IR-98-1blay) possessed an insertion of 138 nucleotides which corresponded to nucleotides from 3504 to 3641 from the hmtp210 genes of the Australian strain HP60 (Figure 3). A similar insertion was previously reported for other Australian (HP31; KU167070) and Taiwanese strains (TW07, TW08, and TW13; KJ867498, MT050502 and MT050506, respectively) (Wang et al., 2016; Tan et al., 2020). Collectively, these results emphasized that at least two different genotypes of Av. paragallinarum circulated among Iranian poultry flocks.

Figure 1. Neighbor-Joining tree compared to reference strains for all serovars, MEGA-X v10.0.5. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. This analysis involved 14 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1882 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Figure 2. Pairwise comparison with reference strains for all serovars, CLC Workbench v5.5

Figure 3. Sequence alignment compared to reference strains for all serovars, an overview of insertions/deletions, CLC Workbench v5.5

Discussion

Infectious coryza is an acute respiratory disease of chickens caused by Avibacterium paragallinarum. This disease is very contagious leading to acute symptoms in the upper respiratory tract of birds and becomes a chronic respiratory disease when accompanied by other pathogens (Blackall and Soriano‐Vargas, 2020).

In recent years, due to the increased spread of chronic respiratory disease (CRD), it has become very important to investigate the pathogens in the respiratory system of birds. Unfortunately, due to the misdiagnosis in recognition of the causes of respiratory diseases, infectious coryza is not usually treated properly leading to huge waste of costs for the wrong treatment and possible increased antimicrobial resistance. Moreover, when infectious coryza occurs in poultry flocks of developing countries, owing to the presence of other pathogens and stress factors, more economic losses occur (Blackall and Soriano‐Vargas, 2020).

In this study, three Av. paragallinarum strains were isolated from the poultry sources using the bacteriological culture and then confirmed by PCR using specific primers. Antimicrobial susceptibility patterns of three isolates were determined. The PCR-amplified hmtp210 genes of three isolates were subjected to sequencing and more information on Av. paragallinarum isolates was revealed from Iran.

Due to the limited number of studies on Av. paragallinarum in Iran, investigation on the presence of Av. paragallinarum in the laying flocks of the country was the main objective of the current study, in which the presence of Av. paragallinarum in backyard and commercial laying flocks was verified. In another study conducted by Badouei et al., (2014), 14 samples of laying hens were cultured. Out of five samples with suspected colonies to Av. paragallinarum, only one sample was verified as Av. paragallinarum using the PCR. Badouei et al. (2014) also did the PCR test on the swab sample of sinus and detected Av. paragallinarum in one backyard farm and two commercial laying flocks. These observations indicate the high sensitivity of PCR in the detection of positive clinical samples. Therefore, according to the results of the direct molecular tests and its comparison to the results of bacterial isolation, it is determined that PCR test on clinical samples could be a reliable alternative for disease detection in poultry. The isolation and identification of Av. paragallinarum from backyard chickens which may be a source of infectious coryza for commercial chicken flocks have also been reported recently in Iran (Nouri et al., 2021). Calderón et al. (2010) in Panama also reported that this disease could reduce egg production up to 45%, as well as an increase in losses in broiler breeder flocks. Muhammad and Sreedevi (2015), using the PCR techbique, detected Av. paragallinarum from outbreaks of infectious coryza in Andhra Pradesh of India (Muhammad and Sreedevi, 2015). From a total of 78 infraorbital sinus and nasal swabs, 56 samples (71.7%) were positive for infectious coryza. They found that the samples collected from chickens at acute stage of the disease or collected before treatment with antibacterial agents demonstrated better results on PCR. In Bangladesh, Khatun et al. (2016) worked on detection of Av. paragallinarum in 10 clinically sick broiler chickens during field outbreaks using the bacteriological culture of nasal and ocular discharges, tracheal swab, tracheal wash, and infraorbital sinus exudates. Based on colonial morphology, Gram staining reaction, sugar fermentation, and biochemical tests, only one isolate was confirmed as Av. paragallinarum among the samples collected from 10 broiler chickens. In the present study, no mortality was observed, indicating the efficacy of vaccines used in the sampled flocks.

        Av. paragallinarum serotypes are typically determined by two related schemes (Blackall and Soriano‐Vargas, 2020). The Page scheme is based on a slide agglutination test that differentiates the isolates into 3 serovars (A, B, and C). While the Kume scheme uses an HI test to classify the isolates into 9 serovars (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4) (Blackall and Soriano‐Vargas, 2020). PCR has been extensively used for the molecular detection and identification of Av. paragallinarum (Chen et al., 1996; Miflin et al., 1999). However, the species-specific PCR is not able to identify serogroups and/or serovars of Av. paragallinarum. Morales-Erasto et al. (2014) evaluated the ability of a multiplex PCR (mPCR) to differentiate the serogroups (A, B, and C) of Av. paragallinarum; however, due to the uncertainty about the accuracy of mPCR for recognition of the serogroups, the mPCR assay was not recommended   to replace the conventional serotyping. In another study, Wang et al. (2016) examined mPCR as well as PCR followed by restriction fragment length polymorphism (RFLP) analysis as alternative approaches to replace the conventional serotyping by the Page scheme, but concluded that neither of the assays were appropriate for serotyping the Av. paragallinarum isolates. Between 2012 and 2013 in Korea, Han et al. (2016) isolated Av. paragallinarum from seven chicken farms and confirmed the isolates by PCR. They identified the isolates as serotype A using the mPCR.

In Iran, few studies have reported the prevalence of different Av. paragallinarum serotypes. Nouri et al. (2014), for the first-time, detected serotype B of Av. paragallinarum using the PCR with group -specific and Page serovar-specific primers that target the hypervariable region of haemagglutinin protein of Av. paragallinarum.

Currently, antimicrobial resistance is a worldwide public health concern (Nhung et al., 2017). Increasing the knowledge on the antimicrobial resistance profile of Av. paragallinarum can prevent the improper use of antimicrobials in poultry flocks since this may result in the selection of resistant strains. There is not much published data on antimicrobial resistance of Av. paragallinarum in the scientific literature. In the present study, we evaluated the antimicrobial susceptibility of three Av. paragallinarum isolates to a panel of antimicrobial agents and found that the antimicrobial susceptibility profile was identical in all three isolates. In other regions, Han et al. (2016) evaluated the antibiotic sensitivity among Av. paragallinarum strains from seven chicken farms and demonstrated that only a few isolates appeared to be susceptible to erythromycin, gentamicin, lincomycin, neomycin, oxytetracycline, spectinomycin, and tylosin. In Bangladesh, Khatun et al. (2016) evaluated the antibacterial susceptibility of one isolated Av. paragallinarum against five agents including ciprofloxacin, azithromycin, gentamicin, ampicillin, and cefalexin and found that the isolate was sensitive to ciprofloxacin, azithromycin, and gentamicin and resistant to ampicillin and cefalexin. Antimicrobial susceptibility was variable among Av. paragallinarum isolates of above studies and there were similarities and differences between antimicrobial susceptibility profile of this study and that of previous works (Han et al., 2016; Khatun et al., 2016; Jeong et al., 2017; Nhung et al., 2017; Heuvelink et al., 2018).

Haemagglutinin is a 210-kDa protein (HMTp210) of Av. paragallinarum that is known to have an important role in the bacterial virulence and the protective immunity against the bacterium (Wang et al., 2014). Purified and recombinant HMTp210 antigens are shown to protect the chickens against experimental challenge with Av. paragallinarum (Sakamoto et al., 2013). In addition, a mutant strain lacking the HMTp210 antigen was less virulent and exhibited no haemagglutinin activity and failed to provoke haemagglutinin inhibition antibodies in infected chickens. This mutant showed reduced ability in some other biological functions as evaluated by Wang et al. (2014). The presence of a hypervariable region in the HMTp210 proteins in some isolates of Page serovars A and C was reported (Wu et al., 2011). Hypervariable regions of hmtp210 gene of serovars A and C strains are known to be the most antigenic region of the HMTp210 protein. This highly antigenic hypervariable region within the hmtp210 gene has been proposed as a candidate for recombinant vaccine production (Wu et al., 2011). However, there is a paucity of information about the genetic diversity, variability, and complexity of the hmtp210 hypervariable region. Recently, Araya-Hidalgo et al. (2017) analyzed the hmtp210 hypervariable region in 16 clinical isolates from Costa Rica by sequencing and compared those 16 clinical isolates with four vaccine strains and other hmtp210 sequences available in GenBank. Except for one isolate, all other isolates demonstrated high similarity with 2 of 4 reference vaccine strains (Araya-Hidalgo et al., 2017). In a recent study, Xu et al. (2019) characterized 28 Av. paragallinarum field isolates by the sequence analysis of the hemagglutinin gene and found that most field strains (25/28) were placed in the same cluster in the phylogenetic tree. Molecular characterization of the three isolates of this study was performed through sequencing of the hmtp210 genes. Analysis of the hmtp210 genes revealed two different lineages of the Av. paragallinarum in commercial and backyard flocks closely related to the bacterial isolates from serovars B-1 and C-4, respectively. The isolation of two different lineages of Av. paragallinarum from the backyard and commercial layer flocks suggested a limited role for backyard flocks in spreading the disease to commercial poultry flocks in Iran. Due to a shortage of sequence data on A. paragallinarum strains from various parts of the world, the above studies may serve as preliminary investigations for understanding the genetic variability and diversity of the hypervariable region of hmtp210.

Conclusion

In this study, Av. paragallinarum isolates recovered from layer flocks were genotypically closely related to serovar C strains while the isolate recovered from backyard chickens was closely related to serovar B strains. Our findings showed that despite vaccine administration to Iranian poultry flocks, infectious coryza may still occur in poultry flocks. The impact of poultry production types, including their proximity to backyard flocks, on the incidence of infectious coryza, needs more investigation. The prevalence of different serotypes in various parts of the country should be studied. Due to the possible antimicrobial susceptibility variability among the Av. paragallinarum isolates, antimicrobial susceptibility should be evaluated whenever these bacteria are isolated. Future studies on a larger pool of Av. paragallinarum isolates from different countries are required to obtain complete genetic data on the hypervariable region of hmtp210 of A. paragallinarum. This additional information is necessary to develop novel diagnostics, vaccines, and therapeutics to better control the disease and reduce the risk of infection with multi-drug resistant Av. paragallinarum isolates in poultry flocks.

Acknowledgments

This research was funded by grant numbers “7508007-6-31” from the Research Council of the University of Tehran and “12-18-18-9452-94003” from Razi Vaccine and Serum Research Institute.

Conflict of Interest

The authors declare they have no conflict of interest associated with this work.