LABORATORY DIAGNOSIS OF BORDATELLA BRONCHISEPTICA TRACHEOBRONCHITIS IN DOG

In the present paper the laboratory isolation and identification of Bordatella bronchiseptica, the causative agent of canine tracheobronhitis, is described. A dog which suffered persistent cough, loss of appetite and fever was previously unsucceffully treated with antibiotics, which is why nasal swabs were taken and sent for microbiological assessment. The isolation of the causative agent was performed on routinely used standard solid growth mediums. The final identification of the isolate was done with MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) and real-time PCR (polymerase chain reaction) assays. Therapy based on the results of the antibiogram lead to successful recovery. The necessity of cooperation of veterinary clinicians and veterinary microbiologists for timely and reliable identification of the microbe(s) and selection of antimicrobials based on the results of the susceptibility testing is emphasized. The significance of the collaboration between microbiological veterinary laboratories and those dealing with human material is underlined. These can provide precise identification of zoonotic agents.


INTRODUCTION
Bacterial species of the Bordetella genus are inhabitants of the respiratory system in both healthy and diseased animals and humans (Markey et al., 2013).The most infamous among these is B. pertussis, the primary aetiological agent of whooping cough, which can also be caused by B. parapertussis.A similar ailment in dogs is caused by B. bronchiseptica, and results in infectious tracheobronchitis, which may attack any dog breed at any age (Ford, 2006).In laymen it is referred to as kennel or canine cough and among professionals as canine croup.The etymology originates from the idea that it primarily affects dogs dwelling in kennels (but also other places where there are many animals in limited areas), and that it resembles the synonymous childhood diseases.The pathogen is transmitted by direct contact or via Flügge droplets (Vieson et al., 2012), but contaminated fomites may also serve as a source of infection (Datz, 2003).Following an incubation which lasts generally from two days to two weeks, the infected dogs start coughing; in the beginning the cough is a dry, paroxismal cough, but later transforms into productive, with nasal discharge, conjunctivitis and fever (Shelton et al., 1977).The most severe complications develop in young dogs due to the immaturity of their immune systems.Aged animals, those with impaired immunity and pregnant bitches are at higher risk.The disease may lead to tracheal collapse (Oskouizadeh et al., 2011).B. bronchiseptica has been identified in rabbits with bronchopneumonia, causing even septicaemia, in cats, horses, guinea pigs and rats suffering from respiratory ailments, and as an opportunistic agent contributing to atrophic rhinitis in pigs (Pittman, 1984;Datz, 2003).Although relatively rare in humans, it was found in people with endocarditis, peritonitis, meningitis and infected wounds, as well as in immunocompromised persons suffering from respiratory infections (Hadley et al., 2009;Woolfrey and Moody, 1991).
Several virulence factors play role in the pathogenesis of respiratory infections which develop owing to B. bronchisepica.For example, fimbriae (FIM), filamentous haemagglutinin (FHA) and pertactin (PTN) mediate the attachment to specific receptors in the respiratory system (Datz, 2003).Since adhesion is a prerequisite for invasion, flagella also may contribute to the adherence to eukaryotic cells (Savelkoul et al., 1996).B. bronchiseptica (and B. avium) are motile by peritrichous flagella (B.pertussis and B. parapertussis are nonmotile) (Markey et al., 2013).
Not unlike other gram-negative bacteria, the outer membrane of Bordetella species contains a lipopolysaccharide endotoxin (Woolfrey and Moody, 1991).In addition, they produce several toxins: tracheal cytotoxin (TCT), dermonecrotic toxin (DNT), osteotoxin, and adenylate cyclase toxin (ACT) (Markey et al., 2013).TCT disrupts ciliated cells (Cookson et al., 1989), DNT is capable of damaging tissues and suppresses both humoral and cellular immunity (Magyar et al., 2000), ACT inhibits the phagocytic function of neutrophils and macrophages (Datz, 2003) and ACT interferes with the activities of epithelial cells (Woolfrey and Moody, 1991).Pertussis toxin (PTX) is an exoprotein produced only by B. pertussis, although the corresponding genes are found also in B. parapertussis and B. bronchiseptica (Masin et al., 2015).

Isolation and identification of B. bronchiseptica.
From a four-month-old dog with symptoms of respiratory disease samples of nasal discharge were taken with two flexible nasal swabs.One sample was sent to the laboratory of the Scientific Veterinary Institute "Novi Sad" for the isolation of the microbes and the other was delivered to the Institute of Public Health of Vojvodina to be subjected to real time-PCR (polymerase chain reaction) assay.
On arrival, the nasal swab was streaked on to Columbia blood agar base (CM0331, Oxoid, UK) with 5% defibrinated ovine blood and MacConkey agar (CM0007, Oxoid, UK).The plates were incubated at 37 o C in aerobic conditions.After 24h of incubation, the blood agar was covered in very small (0.5-1 mm in diameter), convex, smooth, non-haemolytic colonies, which turned to opaque in the next 24h (Figure 1. A).On the MacConkey agar plate (Figure 1. B) the isolate gave rise to minute, pale colonies.

A. B.
Figure 1.Colonies of isolate on blood (A) and MacConkey agar (B) after 48h incubation at 37°C.
The isolate did not ferment carbohydrates (glucose, sucrose and arabinose), but was positive for catalase, urease and oxidase production, and citrate utilization.When the slides were stained with Gram, small Gram-negative coccobacilli were revealed with light microscopy.Based on these characters, the isolate was identified as B. bronchiseptica, and was sent to the Institute of Public Health for confirmation by MALDI TOF (matrix-assisted laser desorption/ ionization -time of flight).
The isolate was prepared using the standard Bruker's direct transfer sample preparation procedure for MALDI-TOF MS.A single bacterial colony was spot-ted directly onto a MALDI target plate (Bruker Daltonics, Germany), allowed to dry and overlaid with 1.0 μL of matrix solution (Bruker Matrix HCCA; α-Cyano-4-hydroxycinnamic acid).MALDI-TOF mass spectrometry was performed on Microflex LT/SH Biotyper system (Bruker Daltonics, Germany) under the control of flexControl software ver.The susceptibility of B. bronchiseptica isolates to antibiotics was assessed with the standard disc-diffusion test on Müller-Hinton agar medium and presented in Table 1.No breakpoints have been set for any of the antimicrobials tested for B. bronchiseptica susceptibility (Morrissey et al., 2016), which is why it is impossible to catagorize the isolates even based on their MIC values (Kadlec et al., 2006).

COMMENT
Besides parainfluenza virus and canine adenovirus type 2, B. bronchiseptica is the most frequent causative agent of canine respiratory diseases (Datz, 2003;Vieson et al., 2012).Although bordetella infection in the dog is usually mild and results in a self-limiting disease, it can be fatal for young animals.In this case, a four-month-old dog of mixed breed was treated empirically, with the combination of antibiotics: penicillin and streptomycin.Both were administered i.m., on five consecutive days: benzyl penicillin 20,000 IU/kg and streptomycin 150 mg/kg.The laboratory testing was required due to the absence of the response to the therapy, but not before two weeks had passed from the onset of the symptoms.The results of in vitro investigation of the nasal swabs confirmed the clinical suspicion of antibiotic resistance, which is understandable.Streptomycin seems generally ineffective against B. bronchiseptica in vitro (Woolfrey and Moody, 1991).Despite the wide use of penicillin, ampicillin and amoxicillin for canine respiratory infections, they have been proven ineffective against B. bronchiseptica, except when the latter is combined with clavulanate (Lappin et al., 2017).Resistance to penicillin has been reported in canine isolates (Markey et al., 2013).In addition, penicillin does not penetrate well into bronchial secretions, which impairs its efficacy.The susceptibility of B. bronchiseptica is intrinsically low to some β-lactams (e.g.penicillins and first-generation cephalosporines) owing to the production of β-lactamase and/ or low membrane permeability to cephalosporines (Prüller et al., 2015;Morrissey et al., 2016).By contrast, aminoglycosides appear to be highly effective against B. bronchiseptica: in severe infections when animals do not respond to parenteral therapy, aerosolized gentamicin may be helpful (Vieson et al., 2012).The most commonly used antibiotics are amoxicillin/clavulanic acid and cephalexin (Vieson et al., 2012).Tetracyclines are also highly efficacious in treating bordetellosis.
To conclude, antibiotics should be selected based on culture and sensitivity tests.Definitive diagnosis of B. bronchiseptica infection in dogs should be confirmed by microbiological findings in nasal or pharyngeal swabs.History and clinical signs can only imply that it is infectious tracheobronchitis caused by B. bronchiseptica we are dealing with.Frequently, it is necessary to cooperate with public health diagnostic laboratories, which are provided with more sophisticated equipment and are capable of performing more precise diagnostic procedures, as it was in this case.In addition, this collaboration can result in better insight into the epidemiology of zoonoses.The initiation of therapy should be guided by the clinical signs: in critically ill animals, empiric therapy should start on the collection of swabs, but in those more stable, antimicrobial therapy should be postponed until the results of the antibiogram arrive, which takes two to three days.Treatment should last about two weeks, or seven days beyond the resolution of health problems (Leekha et al., 2011).When deciding on antibiotic therapy, veterinary surgeons must always have in mind the threatening possibility of resistance development and by giving adequate therapy not contribute to its emergence and spread.

Table 1 .
Antibiotic susceptibility of B. bronchiseptica isolate