Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms

Abstract Background Multi‐causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. Objectives The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi‐causal respiratory disease (MCRD) from 2017 to 2020. Methods Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. Results Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out‐group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. Conclusions The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.


INTRODUCTION
The high prevalence of the multi-causal respiratory disease (MCRD) in broiler flocks has recently caused heavy economic losses in Iran.  (Bashashati & Banani, 2020 listlessness, reduced mobility, decreased feed intake, weight loss, transient nasal discharge, sneezing, and facial oedema. In broiler breeders, the disease affects the birds in the laying period, especially the period of peak production or prior to start of egg production. In addition, it is associated with an increase in mortality, reduced feed intake, and mild respiratory clinical signs. On the other hand, it may decrease egg production, reduce egg size, and lead to poor shell quality in the flocks.

Viral pathogens such as
In laying hens, decreased egg production, increased misshapen eggs, and deaths associated with ORT infection have been reported (Hafez & Chin, 2020). ORT control is primarily performed through strict biosecurity methods to prevent infection from spreading among the flock.
Vaccination of breeders by inactivated vaccine stimulates the production of high levels of antibodies that can pass to their progeny to protect them for up to 4 weeks of age (Warner et al., 2009).
Three main approaches could be used for the diagnosis of MG, MS, and ORT including isolation and identification, serology, and molecular detection (Armour, 2020;Ferguson-Noel & Noormohammadi, 2020;Hafez & Chin, 2020 Under commercial field conditions, co-infection with multiple respiratory pathogens is more common than single respiratory infection. Despite the various studies on single MG, MS, and ORT infections in Iran, these bacteria have not been studied together, and their contribution to the MCRD has not been determined (Banani et al., 2004;Bashashati & Banani, 2020;Bayatzadeh et al., 2014). Due to the high   van Empel and Hafez (1999)

Clinical information, sample collection, and processing
For this study, 350 specimens including tracheal and lung tissues from a total of 70 broiler farms (5000-80,000 birds per flock) were collected in 12 provinces of Iran (i.e., Alborz, Bushehr, Chaharmahal and Bakhtiari, Hamadan, Hormozgan, Isfahan, Kermanshah, Markazi, Mazandaran, Tehran, West Azerbaijan, and Yazd) between 2017 and 2020. The collected samples were not a routine and systematic monitoring, but they were originated from broilers at 3-6 weeks old displaying the overt clinical respiratory signs with high rate of mortality for a minimum of 3 days, which required a further laboratory diagnosis.
Tissue samples (trachea and lungs) from five birds of each flock were pooled together and homogenized in sterile mortar and pestle with brain heart infusion broth to get 10% concentration (w/v). After centrifugation at 1000 × g for 5 min, the supernatant was stored at −70 • C until use.

2.2
Genomic DNA extraction DNA was extracted by the phenol-chloroform method with some modifications (Ghadersohi et al., 1997). Briefly, 1 mL of the supernatant was transferred to a 1.5 mL microtube and centrifuged at 13,000 × g for 30 min. 900 µL of supernatant was discarded, and 100 µL of lysis buffer (50 mM Tris-HCl-50 mM EDTA-100 mM NaCl and 10% SDS) was added. After mixing, 100 µg/mL proteinase K was added to the lysate, and the mixture was incubated at 56 • C for 4 h. The mixture was subjected to three extractions with tris-saturated phenol (pH 8.0), phenol-chloroform-isoamyl alcohol (25:24:1), and chloroform-isoamyl alcohol (24:1) until a clean aqueous phase was obtained. 0.1 volume of sodium acetate buffer (3 M, pH 5.2) was added to the solution, and the DNA was precipitated with 2X sample volume of 100% ethanol. After washing the residue with ice cold 70% ethanol, DNA was diluted in 50 µL of nuclease-free water and kept at −20 • C.

Polymerase chain reaction
MG, MS, and ORT bacteria were detected in tissue samples using the primers shown in Table 1. These primers were designed to amplify fragments of 531 and 784 bp of 16S rRNA gene for MG and ORT, respectively, and 341-392 bp of vlhA gene for MS (Jeffery et al., 2007;Kiss et al., 1997;van Empel & Hafez, 1999). The same primers were used for molecular analysis of MS and ORT. Complete length of mgc2 gene was amplified using primers MB-mgc2-F and MB-mgc2-R that were previously described (Bashashati & Banani, 2020). In the studied positive flocks, further analysis was performed to detect other respiratory pathogenic viruses (AOaV-1, ACV, and AIV subtype H9N2) using RT-PCR based on the previously released methods (Creelan et al., 2002;Lee et al., 2001;Loa et al., 2006;Qiu et al., 2009). The primer set used for the detection of these viruses is also shown in Table 1. PCR TA B L E 2 Broiler flocks positive for different respiratory pathogens.

Genomic sequencing
The amplicons obtained using PCR were excised from the agarose gel and purified using the GeneJET Gel extraction Kit (Thermo Fisher Scientific). The purified PCR products were cloned in the pJET1.2/blunt vector (Thermo Fisher Scientific) according to the manufacturer's instructions, and these were used to transform E. coli TOP10 competent cells. Bacterial colonies were selected and checked for the integrated desired PCR product by PCR, as well as restriction endonuclease digestion using bglII (Thermo Fisher Scientific). Positive clones were cultured in LB broth containing 100 µL/mL ampicillin, and the plasmid was extracted using the alkaline lysis method (Feliciello & Chinali, 1993). Recombinant plasmids with target genes were sequenced in both directions: vector forward and reverse primers. Sequencing services were provided by Macrogen, Seoul, South Korea.

Nucleotide sequencing and phylogenetic study
Nucleotide sequence editing, analysis, and alignments were performed using the BioEdit program (version 7.1.3.0) (Hall, 1999). Nucleotide sequences were compared with corresponding sequences available in the GenBank database using BLAST analysis (https://blast.ncbi.nlm. nih.gov/Blast.cgi). The phylogenetic trees were constructed using the neighbour-joining method with maximum composite likelihood model in the MEGA software (version X) for mgc2, vlhA, and 16S rRNA genes (Kumar et al., 2018). The topological accuracy of the trees was evaluated by the bootstrap method with 1000 replications.

Sequence comparison and phylogenetic analysis
The recombinant plasmids were purified through alkaline-lysis method and commercially Sanger sequenced. The genetic comparison of nucleotide and amino acid sequences of the mgc2 genes showed that five MG strains were found to share 100% similarity with each other. The homologies of nucleotide and amino acid sequences observed between the studied vlhA genes were 84.7%-100% and 85.1%-100%, respectively. The two studied MS strains possessed 100% similarity at nucleotide and amino acid levels, while one of the isolates from Mazandaran province (MS-MCRD-1) had a unique sequence. Among the studied ORT strains, nucleotide sequences of the 16S rRNA genes showed 100% similarity.

TA B L E 3
Highest similarity of the mgc2, vlhA, and 16S rRNA genes in studied bacteria according to BLAST search.

Bacteria
Most similar to Similarity (%)  showed that studied ORT strains were similar to the previously Iranian published sequences. Phylogenetic analysis revealed that 16S rRNA genes of ORT strains detected in this study were grouped into a cluster with other Iranian ORT isolates reported from 1999 to 2010 (Figure 4).

F I G U R E 1
Phylogenetic relationships of the nucleotide sequences of Mycoplasma gallisepticum (MG) strains based on the partial sequence of the mgc2 gene. The tree was generated by the neighbour-joining method using the MEGA X software. The sequences from this study are indicated by the triangle-shaped symbol.

F I G U R E 2
Phylogenetic relationships of the nucleotide sequences of Mycoplasma gallisepticum (MG) strains based on the complete coding sequence of the mgc2 gene. The tree was generated by the neighbour-joining method using the MEGA X software. The sequences from this study are indicated by the triangle-shaped symbol.

DISCUSSION
Upper respiratory tract infection is among the most common causes of high mortality rates and economic losses in global poultry industry.

F I G U R E 4
Phylogenetic relationships of the nucleotide sequences of Ornithobacterium rhinotracheale (ORT) strains based on the partial sequence of the 16S rRNA gene. The tree was generated by the neighbour-joining method using the MEGA X software. The sequences from this study are indicated by the triangle-shaped symbol.
In respiratory diseases, the interaction of bacterial and viral agents can be either synergistic or antagonistic and might affect disease progression to varying degrees. These interactions, which can subsequently determine the severity of the disease complex, depend on various factors such as duration of interaction, host immune response, use of biological products (vaccine and drug), and environmental fac-tors (Samy & Naguib, 2018). In addition to MG, MS, and ORT bacteria, other respiratory viruses such as AOaV-1, ACV, and AIV subtype H9N2 were detected in the broiler flocks tested positive for studied bacterial respiratory pathogens. These viruses have been implicated by several authors as primary respiratory pathogens in Iranian commercial poultry farms suggesting that they play a prominent role in the clinical respiratory disease (Bashashati et al., 2021;Haji-Abdolvahab et al., 2019;Molouki et al., 2019). Viral respiratory infections in birds cause tissue damage, accordingly facilitating the invasion of bacteria, resulting in loss of tissue function. Three possible mechanisms are considered for this tissue damage: (a) loss of cilia and ciliated cells as a result of virus replication in the upper respiratory tract, (b) inhibition of mucociliary clearance due to decreased ciliary activity, and (c) bacterial adherence and colonization due to damage to the epithelium (Bakaletz, 1995;El Ahmer et al., 1999;Wilson et al., 1996). On the contrary, a previous bacterial infection is beneficial for the pathogenesis of some viral agents such as low pathogenic AIV because it facilitates the cleavage of HA protein through protease-producing bacteria (Kishida et al., 2004). However, bacterial infection may suppress viral pathogenesis by the modulation of the innate immune response and preventing virus entry to the susceptible cells (Sid et al., 2016). Staphylococcus aureus (Roussan et al., 2015;Samy & Naguib, 2018).
For molecular and phylogenetic analysis of MG, complete sequencing of the mgc2 gene was performed based on a previous study (Bashashati & Banani, 2020). Mgc2 protein in different strains of MG differs in its sequence and number of amino acids. This variation is most noticeable in the proline-rich region in its carboxyl terminus. This protein is responsible for binding to tracheal epithelial cells, and the coding sequence of this protein is often needed to differentiate MG strains (Ferguson-Noel et al., 2005). In the current study, five MG strains were sequenced and compared with the MG sequences retrieved from the GenBank database. Based on complete and partial phylogenetic analysis of the mgc2 gene, the detected MGs were grouped in the cluster of Iranian MG isolates from 2015 to 2016 (Bashashati & Banani, 2020).
In a study conducted in Iran, 10 MG isolates were genetically analyzed based on complete sequencing of mgc2 and pvpA genes. Nucleotide homology of more than 98% was observed between four studied isolates and nine strains obtained from GenBank (Hassanzadeh et al., 2010). Mirzaei et al. indicated a high similarity of the 16S rRNA gene sequences (98%-100%) of ORT isolates from pigeon, quail, and turkey, and other reference sequences were retrieved from GenBank.
The phylogenetic tree showed that the Iranian pigeon ORT isolates formed a separate cluster from other ORTs isolated from broilers (Mirzaie et al., 2011). In the present study, the studied ORT strains were highly similar (about 100%) and clustered with other Iranian isolates.

CONCLUSION
This study has shown that chickens with respiratory clinical signs could harboured multiple chicken respiratory pathogens. Although a low detection rate of MG, MS, and ORT in the multiple respiratory infections was observed, further molecular analyses with larger sample sizes are crucial to determine the epidemiological characteristics of these bacteria and to outline strategies to minimize them in the poultry farms. In addition, whole-genome sequencing or other bacterial typing methods are useful to distinguish the relationship of bacterial genotypes to other strains and trace the source of infection.