Characterization of Bordetella Bronchiseptica Isolated from Rabbits in Fujian, China CURRENT STATUS: POSTED

Background Bordetella bronchiseptica can infect many animal species, and is a potential zoonotic pathogen that can also infect humans. In rabbits, infection of B. bronchiseptica is associated with respiratory disease, which causes economic losses to the rabbit farming. Fujian Province is a traditional importance rabbit farming area in China. However, no literature about the epidemiology and characteristics of B. bronchiseptica in rabbits in Fujian Province has been reported. Results A total of 219 B. bronchiseptica isolates were recovered from the 833 lung samples of dead rabbits with respiratory disease. The 219 isolates were typed into 11 sequence types (STs) including 5 known STs (ST6, ST10, ST12, ST14 and ST33) and 6 new STs (ST88, ST89, ST90, ST91, ST92 and ST93) by using multi-locus sequence typing (MLST). Surprisingly, all the 219 isolates carried the 5 virulence genes of fhaB , prn , cyaA , dnt and bteA in the PCR screening. Moreover, the isolates resistance to cefixime, ceftizoxime, cefatriaxone and ampicillin were detected, and the resistance rates to the 4 kinds of drug were 33.33, 31.05, 11.87 and 3.20%, respectively. Conclusions In the present study, we showed for the first time that B. bronchiseptica is widespread in rabbits in Fujian Province, and that B. bronchiseptica is an important pathogen associating with respiratory disease in rabbits in Fujian Province. Moreover, it should be alert to the potential occurrence of transmission events between rabbits and humans because the B. bronchiseptica strain of ST12 that can infect humans were also isolated from rabbits in Fujian Province. cefotaxime, ceftizoxime, gentamycin, kanamycin, streptomycin, ofloxacin, ciprofloxacin and norfloxacin were used. The were on the breakpoints for Enterobacteriaceae The as

B. bronchiseptica spreads rapidly in rabbits and the infection is often persistent, causing economic losses to the rabbit farming [9]. Fujian Province, in the southeast of China, is one of the important rabbit farming areas in China [10]. To the best of our knowledge, B. bronchiseptica is a common pathogen isolated from rabbits with respiratory disease in Fujian Province. However, the knowledge of the epidemiology and characteristics of the B. bronchiseptica in rabbits in Fujian province is largely unknown. In this study, B. bronchiseptica strains were recovered from the lung samples of dead rabbits with respiratory disease. The isolates were then characterized by defining the STs, screening the virulence genes and testing the antimicrobial susceptibility.

B. bronchiseptica isolation and identification
Two hundred and nineteen B. bronchiseptica strains were isolated from the 833 lung samples of dead rabbits with respiratory disease. The B. bronchiseptica strains could be recovered from the all 9 rabbit farms and the all 4 rabbit breeds ( Table 1). The infection rates of B. bronchiseptica in the 9 rabbit farms and the 4 rabbit breeds ranged from 13.79 to 38.42% and 15.66 to 50.24%, respectively.

MLST analyses
The results of MLST analyses showed that the 219 B. bronchiseptica strains were typed into 11 STs, with the ST33 (30.14%, 66/219), ST14 (26.94%, 59/219) and ST12 (16.44%, 36/219) were the 3 most predominant STs. Among the 11 STs, six new STs were detected including ST88, ST89, ST90, ST91, ST92 and ST93 (Table 1), and the MLST profiles of the six new STs had been submitted to the PubMLST database. The 7 house-keeping genes of each of the isolate of the 11 STs were concatenated for phylogenetic analysis. A maximum likelihood tree was constructed (No. of bootstrap replications was 1000) by using the MEGA 5.0 software. The 11 STs were mainly grouped into 2 clusters, cluster I and cluster II. Cluster I contained 9 STs including ST6, ST10, ST12, ST14, ST33, ST88, ST89, ST90 and ST92, whereas cluster II only contained 2 STs including ST92 and ST93 (Fig. 1).

Virulence genes detection
Five virulence genes were screened in the 219 B. bronchiseptica isolates. The results showed that all the 5 virulence genes of fhaB, prn, cyaA, dnt and bteA were positive in the all 219 isolates.

Antimicrobial susceptibility test
The results of antimicrobial susceptibility test showed that most of the B. bronchiseptica isolates in this study were susceptible or intermediate susceptible to streptomycin, ciprofloxacin, gentamycin, ofloxacin, kanamycin and norfloxacin, and the sensitive rates to the 6 kinds of drug were 100, 99.09, 98.63, 96.80, 96.34 and 21 %, respectively ( Table 2). The isolates resistant to cefixime, ceftizoxime, cefatriaxone and ampicillin were observed, and the resistance rates to these 4 kinds of drug were 33.33, 31.05, 11.87 and 3.20 %, respectively (  "R" represents resistance; "I" represents intermediate; "S" represents susceptible isolated from seal, dog, leopard and horse, but not from rabbits. Notably, strains of ST12 could also be isolated from humans, suggesting the potential zoonotic transmission between rabbits and humans [7]. Expression of the virulence factors facilitates the invasion of B. bronchiseptica in hosts [11].  [12,13]. It should be aware that the PRN-deficient B. pertussis had emerged, and the losses of the prn gene expression in the strains might be driven by using of acellular vaccine containing PRN [14]. The cyaA gene encodes the multifunctional adenylate cyclasehemolysin (AC-Hly) toxin, which possess adenylate cyclase activity, pore-forming activity and cell invasive activity [15]. It was showed that the AC-Hly toxin expressed in the B. bronchiseptica strains isolated from human and rabbit was responsible for the lethality of the intranasally infected mice [7].
The dnt gene encodes the dermononecrotic toxin (DNT), and the toxin is a well-recognized causative factor inducing atrophic rhinitis and bronchopneumonia in pigs [16]. Interestingly, the ability to express DNT is varied among strains. It was showed that the larger amount of DNT was produced in pig isolates than in a rabbit isolate RB50 [17]. The bteA, also known as bopC, encodes the Bordetella type III secretion system effector A (BteA), which is an important effector secreted from the type III secretion system [18,19]. It was showed that BteA could induce the necrotic cell death and inhibit the macrophage phagocytosis [18,19]. Take together, the 219 B. bronchiseptica isolates carrying the 5 virulence genes in this study were the potential pathogen causing severe respiratory infection in rabbits.
Antibiotics play an important role in prevention and treatment of infections caused by B.
bronchiseptica [20][21][22]. However, the widespread use of antibiotics for preventing and treating the bacterial infections leads to the emergence of antibiotic-resistant strains [22][23][24]. It was demonstrated that B. bronchiseptica strains isolated from swine in Germany were resistant to ampicillin, and B. bronchiseptica strains isolated from companion animals in Germany and other European countries showed decreased susceptibility to β-lactam antibiotics and cephalosporines [25].
In consistence with this study, B. bronchiseptica isolates showing resistant to ampicillin and cephalosporines were also observed in this study. It was showed that the production of betalactamases and the reduced membrane permeability to cephalosporins might result in the resistance to ampicillin and cephalosporines, respectively [26]. Therefore, it should be aware that the widespread use of antibiotics to control the infection caused by B. bronchiseptica in rabbits is unsustainable.

Conclusion
The characteristics of the B. bronchiseptica strains isolated from dead rabbits with respiratory disease in Fujian Province were described in this study. The results showed that B. bronchiseptica was an important pathogen causing respiratory disease in rabbits in Fujian Province, and that B.
bronchiseptica stain of ST12 that can infect humans was also isolated from rabbits in Fujian Province.
The results might play important roles in tracking the epidemic strains in rabbits, controlling the B.
bronchiseptica infections in rabbits and preventing the potential rabbit-human transmission events.

Ethics statement
Lung samples were collected from dead rabbits with respiratory disease in according with the In order to isolate B. bronchiseptica, each sample was mixed with sterile phosphate buffer saline and homogenized to make 50% suspension. One hundred microliter of suspension was spread on brain heart infusion (BHI) agar plate (containing 1% glycerol and 10% defibrinated sheep blood) and incubated for 48 to 72 h at 37˚C. Five Gram-negative suspected isolates from each plate were picked up. The identities of the isolates with oxidase and ornithine decarboxylase positives, and glucose fermentation negative were further confirmed by sequencing of the 16S rRNA genes. One isolate from each sample was selected as representative for further characterization.

MLST analyses
The isolates were analyzed by MLST as described in PubMLST website (https://pubmlst.org/). Briefly, 7 house-keeping genes including adk, fumC, glyA, tyrB, icd, pepA and pgm were amplified and sequenced. The seven allelic numbers were given by comparing the seven house-keeping genes of the isolate to the corresponding genes in the PubMLST database. The sequence type (ST) of the isolate was defined according to the seven allelic numbers.

Virulence genes detection
Five well-characterized virulence genes that thought to be involved in interactions with the host were screened in the isolates, including filamentous hemagglutinin (fhaB), pertactin (prn), adenylate cyclase-hemolysin toxin (cyaA), dermononecrotic toxin (dnt) and the Bordetella type-III secretion system effector A (bteA). The primers for amplification of the five virulence genes were designed based on the corresponding genes in the NCBI database (https://www.ncbi.nlm.nih.gov/) ( Antimicrobial susceptibility test 9 The antimicrobial susceptibility test of the isolates were conducted by using disc diffusion method on Veterinary Medicine, and the approved number is FAAS-AHVM2017-0511. We obtained written informed consent from the owners of the animals for using them in this study.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article. Figure 1 Phylogenetic tree of the 11 STs of B. bronchiseptica based on the concatenated 7 housekeeping genes. The black filled triangles indicate the new STs. The black filled circle indicates the B. bronchiseptica strain of ST12 that can be isolated from both rabbits and humans.