Bordetella Bronchiseptica Research Articles

Environmental Origin of the Genus Bordetella

Members of the genus Bordetella include human and animal pathogens that cause a variety of respiratory infections, including whooping cough in humans. Despite the long known ability to switch between a within-animal and an extra-host lifestyle under laboratory growth conditions, no extra-host niches of pathogenic Bordetella species have been defined. To better understand the distribution of Bordetella species in the environment, we probed the NCBI nucleotide database with the 16S ribosomal RNA (16S rRNA) gene sequences from pathogenic Bordetella species. Bacteria of the genus Bordetella were frequently found in soil, water, sediment, and plants. Phylogenetic analyses of their 16S rRNA gene sequences showed that Bordetella recovered from environmental samples are evolutionarily ancestral to animal-associated species. Sequences from environmental samples had a significantly higher genetic diversity, were located closer to the root of the phylogenetic tree and were present in all 10 identified sequence clades, while only four sequence clades possessed animal-associated species. The pathogenic bordetellae appear to have evolved from ancestors in soil and/or water. We show that, despite being animal-adapted pathogens, Bordetella bronchiseptica, and Bordetella hinzii have preserved the ability to grow and proliferate in soil. Our data implicate soil as a probable environmental origin of Bordetella species, including the animal-pathogenic lineages. Soil may further constitute an environmental niche, allowing for persistence and dissemination of the bacterial pathogens. Spread of pathogenic bordetellae from an environmental reservoir such as soil may potentially explain their wide distribution as well as frequent disease outbreaks that start without an obvious infectious source.

Occurrence of Pasteurella multocida among pigs with respiratory disease in China between 2011 and 2015

BackgroundPrior to the 1990s, P. multocida capsular serogroup A was the most prevalent in China, followed by serogroups B and D. Thirty years later, serogroup D became the most prevalent, followed by serogroups A and B. However, the P. multocida capsular serogroups currently circulating in China remain unclear. Therefore, the aim of the present study was to provide an update on P. multocida serogroups isolated from diagnostic samples collected from clinically diseased pigs in Central and Eastern China from 2011 to 2015.ResultsBetween February 2011 and October 2015, 296 isolates of Pasteurella multocida were collected from 3212 pigs with clinical respiratory disease in 12 provinces of China (isolation rate of 9.2%). Of the 296 collected isolates, 146 (49.3%) were P. multocida capsular type A, 141 (47.6%) were capsular type D, and one was capsular type B. Streptococcus suis (94/193; 48.7%), Haemophilus parasuis (76/193; 39.3%), Escherichia coli (53/193; 27.5%), and Bordetella bronchiseptica (26/193; 13.5%) were frequently isolated together with P. multocida. A total of 14 toxigenic P. multocida strains co-isolated with other pathogens from 32 cases of atrophic rhinitis were classified into serogroup D. The virulence of P. multocida capsular type A isolates was higher than that of capsular type D isolates based on LD50 studies in mice.ConclusionsOver the past 5 years, P. multocida capsular type A was the most frequently isolated from diagnostic submissions in Central and Eastern China, followed by serogroups D and B.

Comparison of Two Challenge Models of Atrophic Rhinitis in Piglets

Background: Swine atrophic rhinitis (AR) is a multiple chronic respiratory diseasemainly caused by porcine Bordetella bronchiseptica (Bb) and Toxigenic Pasteurella multocida (T+Pm). There are two challenge models using Bordetella bronchiseptica and Pasteurella multocida. The first model is to treat pigs with Bordetella bronchiseptica followed by Toxigenic Pasteurella multocida infection. Another model is to inoculate pigs with Bordetella bronchiseptica and Pasteurella multocida simultaneously. So far, there is no report about comparison of these two challenge models as for their effectiveness to establish infection. Methods: Thirteen 42 day-old piglets were divided into three groups. Pigs in the first group were challenged with Bordetella bronchiseptica followed by Toxigenic Pasteurella multocida infection (model 1). Pigs in the second group were challenged with Bordetella bronchiseptica and Pasteurella multocida at the same time (model 2). Pigs in the third group worked as sterile controls. Clinical symptoms, turbinate lesions, lung lesions and daily bodyweight gain were used as parameters to evaluate the effectiveness of above challenge models. Results: All challenged piglets showed different degrees of clinical symptoms, turbinate lesions, lung lesions and loss of average daily bodyweight gain. There was no significant difference in clinical symptoms and lung lesions between two challenge models. However, significant differences in turbinate lesion score and average daily gain were observed between these two challenge models. Discussion and Conclusion: Turbinate lesions score of piglets in first group ranged from 4 to 10 and only 1/5 of piglets had a total turbinate score of 10. By contrast, turbinate lesions score of piglets in the second group ranged from 8 to 16 and 4/5 of piglets had a total turbinate score of equal or above to 10. Therefore, all above data indicated that co-infection of Bordetella bronchiseptica and Pasteurella multocida was more suitable for to establish AR infection model.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice.

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Evaluation of an Experimental Vaccine for Atrophic Rhinitis in Piglets

Background: Commercial swine atrophic rhinitis (AR) vaccine contains killed Bordetella bronchiseptica and P. multocida toxin (PMT) from Toxigenic Pasteurella multocida. The recombinant PMT proteins used as vaccine candidates were seldom reported. Methods: In this study, we developed recombinant fragments of PMT to replace P. multocida toxin in AR vaccine to compare the efficacy of this vaccine candidate with the commercial vaccine. Results: The experimental AR vaccine showed similar efficacy after bacterial challenges since there were no significant differences in clinical symptoms, turbinate lesions, lung lesions and daily body gain between the two vaccinated groups. Discussion and conclusion: The replacement of recombinant fragments of PMT to P. multocida toxin in AR vaccine provided same protection as commercial vaccine after bacterial challenges. However, high yield and simple processing of recombinant fragments of PMT from E.coli cells to replace purified P. multocida toxin in AR vaccine will dramatically reduce the cost of vaccines and simplify the antigen preparation.

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.

Survival of bacteria of laboratory animal origin on cage bedding and inactivation by hydrogen peroxide vapour.

This study aims to determine the ability of laboratory animal bacteria to resist desiccation and inactivation by hydrogen peroxide vapour (HPV) on paper bedding pieces. Bedding pieces were saturated with bacterial suspensions in water or 2% (w/v) bovine serum albumin (BSA) in water, and held in a mouse facility. Viable counts showed variable survival rates over time for the bacterial species used ([ Pasteurella] pneumotropica, Muribacter muris, Pseudomonas aeruginosa, Acinetobacter redioresistens, Escherichia coli, Klebsiella oxytoca, Bordetella bronchiseptica, Bordetella hinzii, Enterococcus faecalis, β-haemolytic Streptococcus spp., Staphylococcus aureus and Staphylococcus xylosus). Overall, BSA increased bacterial survival in the bedding pieces. The survival rates of Bacillus safensis were not influenced by BSA but depended on sporulation. When bedding pieces and Petri dishes inoculated with E. coli, P. aeruginosa and S. aureus were subjected to HPV disinfection, all bacterial species on the bedding pieces inoculated with bacterial suspensions in water were readily inactivated. By contrast, S. aureus and P. aeruginosa, but not E. coli cells survived HPV treatment in high numbers when inoculated on bedding pieces as a BSA suspension. Notably, all three bacterial species were readily inactivated by HPV even in the presence of BSA when smeared on smooth surfaces. In conclusion, the suspension medium and the carrier can influence the environmental survival and susceptibility of bacterial species to HPV. Our results may help to develop standard protocols that can be used to ensure the microbiological quality of experimental rodent housing.

Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis.

Isolation of Campylobacter spp. from Client-Owned Dogs and Cats, and Retail Raw Meat Pet Food in the Manawatu, New Zealand.

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food-borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C.jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P<0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C.jejuni 22%, C.lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non-poultry meat (P=0.006). Of the 13 C.jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.

Bordetella bronchisepticaPneumonia Post-Liver Resection in a Patient with Liver Cirrhosis and Cholangiocarcinoma

Background: Although Bordetella bronchiseptica is primarily an animal pathogen, cases of human disease caused by this pathogen have been published recently, most frequently pneumonia in immunocompromised patients. In human disease, transmission through animal vectors may play a key role. Although no standardized sensitivity testing is available for this pathogen in human disease, animal isolates are sensitive to most β-lactam antibiotics. Case Report: A 62-year-old Caucasian male with Child-Pugh class A cirrhosis caused by chronic hepatitis C infection underwent uneventful left lateral segmentectomy for a 3 cm cholangiocarcinoma. Within 48 h, he developed altered mental status, temperature of 39.4°C, leukocytosis (white blood cell count: 13,000/mm3), and dyspnea followed by hypotension requiring vasopressor support and intubation. Computed tomography (CT) scan demonstrated left lower lobe pneumonia. Empiric antibiotic therapy including vancomycin (1 g every 12 h) and piperacillin-tazobactam (3.5 g every 6 h) was initiated and his signs of sepsis resolved within two days. Bordetella bronchiseptica was cultured from sputum. Upon questioning, the patient reported close contact with several pet cats on the days prior to admission. Antibiotics were continued for a total of seven days and he was discharged in good condition doing well at his six-month follow-up. Conclusions: Immunocompromised patients may develop infection with Bordetella bronchiseptica especially if they are in close contact with animals known to be a reservoir of this pathogen. If diagnosed early and treated appropriately, the outcome is favorable.

The First Siphoviridae Family Bacteriophages Infecting Bordetella bronchiseptica Isolated from Environment.

Bordetella bronchiseptica is a well-known etiological agent of kennel cough in dogs and cats and one of the two causative agents of atrophic rhinitis, a serious swine disease. The aim of the study was to isolate B. bronchiseptica bacteriophages from environmental samples for the first time. A total of 29 phages from 65 water samples were isolated using the strain ATCC 10580 as a host. The lytic spectra of the phages were examined at 25 and 37°C, using 12 strains of B. bronchiseptica. All phages were able to plaque on 25.0% to 41.7% of the strains. The selected phages showed similar morphology (Siphoviridae, morphotype B2), but variation of RFLP patterns and efficacy of plating on various strains. The partial genome sequence of phage vB_BbrS_CN1 showed its similarity to phages from genus Yuavirus. Using PCR, it was confirmed that the phages do not originate from the host strain, and environmental origin was additionally confirmed by the analysis of host genome sequence in silico and plating heated and unheated samples in parallel. Accordingly, this is the first isolation of B. bronchiseptica phages from environment and the first isolation and characterization of phages of B. bronchiseptica belonging to family Siphoviridae.

Bordetella bronchiseptica Post-Surgical Meningitis in an Adult

Bordetella bronchiseptica is a well-described pathogen classically causing respiratory infections in household pets. We describe a case of postsurgical meningitis in an adult patient. A 49-year -old man with a recently resected glioblastoma developed altered mental status approximately 2 weeks after surgery and was found to have B bronchiseptica meningitis. B bronchiseptica is a very rare cause of meningitis. It has only been described twice before in the literature, both in pediatric patients. It should be considered in patients with household pets. It can be a particularly virulent organism given its ability to form biofilms. In our case, the patient failed initial treatment with 2 weeks of meropenem and required re-treatment.

Identification and characterization of a FAD-dependent putrescine N-hydroxylase (GorA) from Gordonia rubripertincta CWB2

A putrescine N-hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine>cadaverine>hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His10-tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent Km and kcat of 361.6±0.1μM and 0.266±0.011s−1, respectively, whereas the hydroxylation assay showed GorA with an apparent Km and kcat of 737.1±0.1μM and 0.210±0.001s−1. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.

Functional Identification of Putrescine C- and N-Hydroxylases.

The small polyamine putrescine (1,4-diaminobutane) is ubiquitously and abundantly found in all three domains of life. It is a precursor, through N-aminopropylation or N-aminobutylation, for biosynthesis of the longer polyamines spermidine, sym-homospermidine, spermine, and thermospermine and longer and branched chain polyamines. Putrescine is also biochemically modified for purposes of metabolic regulation and catabolism, e.g. N-acetylation and N-glutamylation, and for incorporation into specialized metabolites, e.g. N-methylation, N-citrylation, N-palmitoylation, N-hydroxylation, and N-hydroxycinnamoylation. Only one example is known where putrescine is modified on a methylene carbon: the formation of 2-hydroxyputrescine by an unknown C-hydroxylase. Here, we report the functional identification of a previously undescribed putrescine 2-hydroxylase, a Rieske-type nonheme iron sulfur protein from the β-proteobacteria Bordetella bronchiseptica and Ralstonia solanacearum. Identification of the putrescine 2-hydroxylase will facilitate investigation of the physiological functions of 2-hydroxyputrescine. One known role of 2-hydroxyputrescine has direct biomedical relevance: its role in the biosynthesis of the cyclic hydroxamate siderophore alcaligin, a potential virulence factor of the causative agent of whooping cough, Bordetella pertussis. We also report the functional identification of a putrescine N-hydroxylase from the γ-proteobacterium Shewanella oneidensis, which is homologous to FAD- and NADPH-dependent ornithine and lysine N-monooxygenases involved in siderophore biosynthesis. Heterologous expression of the putrescine N-hydroxylase in E. coli produced free N-hydroxyputrescine, never detected previously in a biological system. Furthermore, the putrescine C- and N-hydroxylases identified here could contribute new functionality to polyamine structural scaffolds, including C-H bond functionalization in synthetic biology strategies.

Comparison of ribotyping and sequence-based typing for discriminating among isolates of Bordetella bronchiseptica

PvuII ribotyping and MLST are each highly discriminatory methods for genotyping Bordetella bronchiseptica, but a direct comparison between these approaches has not been undertaken. The goal of this study was to directly compare the discriminatory power of PvuII ribotyping and MLST, using a single set of geographically and genetically diverse strains, and to determine whether subtyping based on repeat region sequences of the pertactin gene (prn) provides additional resolution. One hundred twenty-two isolates were analyzed, representing 11 mammalian or avian hosts, sourced from the United States, Europe, Israel and Australia. Thirty-two ribotype patterns were identified; one isolate could not be typed. In comparison, all isolates were typeable by MLST and a total of 30 sequence types was identified. An analysis based on Simpson's Index of Diversity (SID) revealed that ribotyping and MLST are nearly equally discriminatory, with SIDs of 0.920 for ribotyping and 0.919 for MLST. Nonetheless, for ten ribotypes and eight MLST sequence types, the alternative method discriminates among isolates that otherwise type identically. Pairing prn repeat region typing with ribotyping yielded 54 genotypes and increased the SID to 0.954. Repeat region typing combined with MLST resulted in 47 genotypes and an SID of 0.944. Given the technical and practical advantages of MLST over ribotyping, and the nominal difference in their SIDs, we conclude MLST is the preferred primary typing tool. We recommend the combination of MLST and prn repeat region typing as a high-resolution, objective and standardized approach valuable for investigating the population structure and epidemiology of B. bronchiseptica.

Retrospective Analysis of Bacterial and Viral Co-Infections in Pneumocystis spp. Positive Lung Samples of Austrian Pigs with Pneumonia.

Aim of this study was the retrospective investigation of viral (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), torque teno sus virus type 1 and 2 (TTSuV1, TTSuV2)) and bacterial (Bordetella bronchiseptica (B. b.), Mycoplasma hyopneumoniae (M. h.), and Pasteurella multocida (P. m.)) co-infections in 110 Pneumocystis spp. positive lung samples of Austrian pigs with pneumonia. Fifty-one % were positive for PCV2, 7% for PRRSV, 22% for TTSuV1, 48% for TTSuV2, 6% for B. b., 29% for M. h., and 21% for P. m. In 38.2% only viral, in 3.6% only bacterial and in 40.0% both, viral and bacterial pathogens were detected. In 29.1% of the cases a co-infection with 1 pathogen, in 28.2% with 2, in 17.3% with 3, and in 7.3% with 4 different infectious agents were observed. The exposure to Pneumocystis significantly decreased the risk of a co-infection with PRRSV in weaning piglets; all other odds ratios were not significant. Four categories of results were compared: I = P. spp. + only viral co-infectants, II = P. spp. + both viral and bacterial co-infectants, III = P. spp. + only bacterial co-infectants, and IV = P. spp. single infection. The evaluation of all samples and the age class of the weaning piglets resulted in a predomination of the categories I and II. In contrast, the suckling piglets showed more samples of category I and IV. In the group of fattening pigs, category II predominated. Suckling piglets can be infected with P. spp. early in life. With increasing age this single infections can be complicated by co-infections with other respiratory diseases.

Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica.

Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica

Quantitative PCR and Cytology of Bronchoalveolar Lavage Fluid in Dogs with Bordetella bronchiseptica Infection.

BackgroundThe use of quantitative PCR (qPCR) for detection of Bordetella bronchiseptica in bronchoalveolar lavage fluid (BALF) and demonstration of bacteria adhering to ciliated epithelial cells in BALF or bronchial brushing fluid (BBF) has not been assessed in a series of affected dogs. Coinfections can worsen the clinical severity in bordetellosis, but the specific association with Mycoplasma cynos has not been evaluated.ObjectivesTo assess the utility of culture, qPCR and cytologic examination of cytospin preparations in the diagnosis of bordetellosis in dogs and the influence of coinfection by M. cynos on disease severity.AnimalsTwenty‐four referred dogs with B. bronchiseptica infection and 10 healthy dogs.MethodsRetrospective case series. qPCR (B. bronchiseptica and M. cynos) and culture results from BALF were recorded. Cytospin preparations from BALF and BBF were reviewed. qPCR on BALF from 10 healthy dogs were used as negative control.ResultsThe BALF culture and qPCR detected B. bronchiseptica in 14/24 and 18/18 dogs, respectively. Coccobacilli were found adhering to ciliated epithelial cells in 20 of the 21 BALF cytologic preparations where epithelial cells were found, and 2/3 BBF cytologic preparations. Quantitative PCR detected a low level of B. bronchiseptica in one healthy dog. The frequency of detection of M. cynos was not significantly different in B. bronchiseptica (9/17 dogs) compared with healthy dogs (2/10 dogs) (P = .09).Conclusion and Clinical ImportanceQuantitative PCR detection of B. bronchiseptica in BALF appears to be a useful diagnostic tool. Cytologic examination of BALF or BBF, when positive, allows a rapid and reliable diagnosis.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BackgroundActinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae.ResultsFor the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure.ConclusionsIn conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0742-3) contains supplementary material, which is available to authorized users.

Molecular surveillance of traditional and emerging pathogens associated with canine infectious respiratory disease

A molecular survey for traditional and emerging pathogens associated with canine infectious respiratory disease (CIRD) was conducted in Italy between 2011 and 2013 on a total of 138 dogs, including 78 early acute clinically ill CIRD animals, 22 non-clinical but exposed to clinically ill CIRD dogs and 38 CIRD convalescent dogs. The results showed that canine parainfluenza virus (CPIV) was the most commonly detected CIRD pathogen, followed by canine respiratory coronavirus (CRCoV), Bordetella bronchiseptica, Mycoplasma cynos, Mycoplasma canis and canine pneumovirus (CnPnV). Some classical CIRD agents, such as canine adenoviruses, canine distemper virus and canid herpesvirus 1, were not detected at all, as were not other emerging respiratory viruses (canine influenza virus, canine hepacivirus) and bacteria (Streptococcus equi subsp. zooepidemicus). Most severe forms of respiratory disease were observed in the presence of CPIV, CRCoV and M. cynos alone or in combination with other pathogens, whereas single CnPnV or M. canis infections were detected in dogs with no or very mild respiratory signs. Interestingly, only the association of M. cynos (alone or in combination with either CRCoV or M. canis) with severe clinical forms was statistically significant. The study, while confirming CPIV as the main responsible for CIRD occurrence, highlights the increasing role of recently discovered viruses, such as CRCoV and CnPnV, for which effective vaccines are not available in the market.