Bovine Respiratory Tract Research Articles

Relationship between capsule production and biofilm formation by Mannheimia haemolytica, and establishment of a poly-species biofilm with other Pasteurellaceae

Mannheimia haemolytica is one of the bacterial agents responsible for bovine respiratory disease (BRD). The capability of M. haemolytica to form a biofilm may contribute to the development of chronic BRD infection by making the bacteria more resistant to host innate immunity and antibiotics. To improve therapy and prevent BRD, a greater understanding of the association between M. haemolytica surface components and biofilm formation is needed. M. haemolytica strain 619 (wild-type) made a poorly adherent, low-biomass biofilm. To examine the relationship between capsule and biofilm formation, a capsule-deficient mutant of wild-type M. haemolytica was obtained following mutagenesis with ethyl methanesulfonate to obtain mutant E09. Loss of capsular polysaccharide (CPS) in mutant E09 was supported by transmission electron microscopy and Maneval's staining. Mutant E09 attached to polyvinyl chloride plates more effectively, and produced a significantly denser and more uniform biofilm than the wild-type, as determined by crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy with COMSTAT analysis. The biofilm matrix of E09 contained predominately protein and significantly more eDNA than the wild-type, but not a distinct exopolysaccharide. Furthermore, treatment with DNase I significantly reduced the biofilm content of both the wild-type and E09 mutant. DNA sequencing of E09 showed that a point mutation occurred in the capsule biosynthesis gene wecB. The complementation of wecB in trans in mutant E09 successfully restored CPS production and reduced bacterial attachment/biofilm to levels similar to that of the wild-type. Fluorescence in-situ hybridization microscopy showed that M. haemolytica formed a poly-microbial biofilm with Histophilus somni and Pasteurella multocida. Overall, CPS production by M. haemolytica was inversely correlated with biofilm formation, the integrity of which required eDNA. A poly-microbial biofilm was readily formed between M. haemolytica, H. somni, and P. multocida, suggesting a mutualistic or synergistic interaction that may benefit bacterial colonization of the bovine respiratory tract.

Pasteurella multocida ST20 is widespread in Australian poultry farms and may infect wild waterbirds

The bacterial agent that causes fowl cholera, Pasteurella multocida, was isolated from two deceased wild waterbirds in Victoria, Australia, in 2013. Whole genome sequence analysis placed the isolates into ST20, a subtype described in farmed chickens from Queensland, Australia and more recently in feedlot cattle and in pigs across a broader area of the continent. This study also found ST20 between 2009 and 2022 on three chicken farms and two turkey farms located in four Australian states. The sequences of 25 of these ST20 isolates were compared to 280 P. multocida genomes from 23 countries and to 94 ST20 Illumina datasets from Queensland that have been deposited in public databases. The ST20 isolates formed a single phylogenetic clade and were clustered into four sub-groups with highly similar genomes, possessing either LPS type 1 or type 3 loci. Various repertoires of mobile genetic elements were present in isolates from farmed, but not wild birds, suggesting complex histories of spill-over between avian populations and gene acquisition within farm environments. No major antimicrobial resistance was predicted in any of the ST20 isolates by the genomic analysis. The closest relative of these isolates was a ST394 bovine respiratory tract isolate from Queensland, which differed from ST20 by only one allele and carried beta-lactam and tetracycline resistance genes. These findings underline the importance of understanding the role of wild and commercial birds in the maintenance of fowl cholera, and of implementing regular epidemiological surveillance and biosecurity management programmes in wildlife, as well as free-range poultry farms.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Antiviral activity of bovine type III interferon against bovine viral diarrhea virus is greatly reduced in bovine turbinate cells due to limited expression of IFN lambda receptor 1 (IL-28Rα).

The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor. Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.

The spatial dissimilarities and connections of the microbiota in the upper and lower respiratory tract of beef cattle.

Bovine respiratory disease (BRD) causes morbidity and mortality in cattle. The critical roles of the respiratory microbiota in BRD have been widely studied. The nasopharynx was the most popular sampling niche for BRD pathogen studies. The oral cavity and other niches within the respiratory tract, such as nostrils and lung, are less assessed. In this study, oropharyngeal swabs (OS), nasal swabs (NS), nasopharyngeal swabs (NP), and bronchoalveolar lavage (BAL) were collected from calves located in four countries and analyzed for investigation of the dissimilarities and connections of the respiratory microbiota. The results showed that the microbial diversity, structure, and composition in the upper and lower respiratory tract in beef cattle from China, the USA, Canada, and Italy were significantly different. The microbial taxa for each sampling niche were specific and associated with their local physiology and geography. The signature microbiota for OS, NS, NP, and BAL were identified using the LEfSe algorithm. Although the spatial dissimilarities among the respiratory niches existed, the microbial connections were observed in beef cattle regardless of geography. Notably, the nostril and nasopharynx had more similar microbiomes compared to lung communities. The major bacterial immigration patterns in the bovine respiratory tract were estimated and some of them were associated with geography. In addition, the contribution of oral microbiota to the nasal and lung ecosystems was confirmed. Lastly, microbial interactions were characterized to reveal the correlation between the commercial microbiota and BRD-associated pathogens. In conclusion, shared airway microbiota among niches and geography provides the possibility to investigate the common knowledge for bovine respiratory health and diseases. In spite of the dissimilarities of the respiratory microbiota in cattle, the spatial connections among these sampling niches not only allow us to deeply understand the airway ecosystem but also benefit the research and development of probiotics for BRD.

Genetic Organization of Acquired Antimicrobial Resistance Genes and Detection of Resistance-Mediating Mutations in a Gallibacterium anatis Isolate from a Calf Suffering from a Respiratory Tract Infection

Gallibacterium (G.) anatis isolates associated with respiratory diseases in calves and harboring acquired antimicrobial resistance genes have been described in Belgium. The aim of this study was to analyze the genetic organization of acquired resistance genes in the G. anatis isolate IMT49310 from a German calf suffering from a respiratory tract infection. The isolate was submitted to antimicrobial susceptibility testing, and a closed genome was obtained by a hybrid assembly of Illumina MiSeq short-reads and MinION long-reads. Isolate IMT49310 showed elevated MIC values for macrolides, aminoglycosides, florfenicol, tetracyclines, and trimethoprim/sulfamethoxazole. The acquired resistance genes catA1, floR, aadA1, aadB, aphA1, strA, tet(M), tet(B), erm(B), and sul2 were identified within three resistance gene regions in the genome, some of which were associated with IS elements, such as ISVsa5-like or IS15DII. Furthermore, nucleotide exchanges within the QRDRs of gyrA and parC, resulting in amino acid exchanges S83F and D87A in GyrA and S80I in ParC, were identified. Even if the role in the pathogenesis of respiratory tract infections in cattle needs to be further investigated, the identification of a G. anatis isolate with reduced susceptibility to regularly used antimicrobial agents in cases of fatal bovine respiratory tract infections is worrisome, and such isolates might also act as a reservoir for antimicrobial resistance genes.

Effect of maternal separation and transportation stress on the bovine upper respiratory tract microbiome and the immune response to resident opportunistic pathogens

BackgroundThe bovine upper respiratory tract (URT) microbiome includes opportunistic pathogens that cause respiratory disease and stress associated with maternal separation and transportation contributes to the severity of this respiratory disease. Stress is known to alter the gut microbiome but little is known regarding the effect of stress on the URT microbiota. This study used six-month old suckling beef calves to investigate whether maternal separation (weaned), by itself or combined with transportation (weaned + transport), altered the URT microbiome and host immune responses to resident opportunistic pathogens.ResultsTaxonomic and functional composition of the URT microbiome in suckling and weaned beef calves did not change significantly when serially sampled over a one-month period. Subtle temporal changes in the URT microbiome composition were observed in weaned + transport calves. Total bacterial density was lower (p < 0.05) on day 4 post-weaning in both the weaned and weaned + transport groups when compared to suckling calves. In addition, significant (p < 0.05) temporal changes in the density of the opportunistic pathogens, M. haemolytica and P. multocida, were observed independent of treatment but these changes did not correlate with significantly increased (p < 0.05) serum antibody responses to both of these bacteria in the weaned and weaned + transport groups. Serum antibody responses to My. bovis, another opportunistic pathogen, remained unchanged in all treatment groups. Weaning, by itself and in combination with transportation, also had significant (p < 0.05) short- (2 to 8 days post-weaning) and long-term (28 days post-weaning) effects on the expression of adrenergic receptor genes in blood leukocytes when compared to age-matched suckling beef calves.ConclusionsMaternal separation (weaning) and transportation has minor effects on the taxonomic and functional composition of the URT microbiome and temporal changes in the density of opportunistic pathogen residing in the URT did not correlate with significant changes in immune responses to these bacteria. Significant changes in adrenergic receptor expression in blood leukocytes following weaning, with or without transportation, suggests altered neuroimmune regulation should be further investigated as a mechanism by which stress can alter host-microbiome interactions for some opportunistic respiratory pathogens that reside in the URT.

Homologous recombination as a mechanism of genetic changes in bovine parainfluenza-3 virus

Bovine parainfluenza-3 virus (BPIV-3) is one of the main viruses associated with bovine respiratory disease complex (BRDC) worldwide. BPIV-3 infect the bovine respiratory tract causing from subclinical infections to severe pneumonia with significant economic losses in the cattle industry. BPIV-3 is a RNA virus with high genetic variability, nevertheless, the contribution of recombination events to its variability has not been assessed so far. In this study the 25 complete genome sequences (CGS) reported so far and 215 partial sequences of different viral genes of BPIV-3 were analyzed to determine their genotypes and subgenotypes, distribution, and the existence of potential recombination events. Based on the analysis of the HN, M, N, and P genes one hypothetical subgenotype was found (subgenotype A4). Four recombination events between sequences of swine and cattle were detected by RDP4 analysis in conjunction with phylogenetic incongruences in the L gene. In addition, 9 sequences reported from Argentina were found to be miss-classified. These results reveal that homologous recombination events have a relevant role in the evolution of BPIV-3 and highlight the importance of implement advanced molecular characterization to better understand the variability and evolution of BPIV-3 as a component of BRDC.

PRESENCE OF MOLLICUTES AND MYCOPLASMA BOVIS IN NASAL SWABS FROM CALVES AND IN MILK FROM COWS WITH CLINICAL MASTITIS

Mycoplasma bovis is part of the bovine respiratory tract microbiota but is considered an opportunistic pathogen of extreme importance in respiratory diseases of calves. It causes to the herd several diseases such as mastitis, polyarthritis, pneumonia and endometritis. This pathogen is highly contagious and animals with mastitis are potential disseminators of infection to the herd since they release from 106 to 108 CFU per mL milk. Similarly, animals with pneumonia eliminate, through respiratory secretions, high microbial loads of the agent. The present study aimed to perform molecular detection of Mycoplasma bovis in 185 milk samples from cows with clinical mastitis, as well as in 50 nasal swab samples from healthy calves with or without signs of pneumonia and born from cows with mastitis, all belonging to four dairy farms in Paraná State, where cases of mastitis had beendiagnosed. DNA extraction from both secretions was carried out according to the thermolysis method. For polymerase chain reaction (PCR), generic primers were employed to amplify the Mollicutes DNA and positive samples were subjected to PCR with primersspecific for M. bovis. Positivity for M. bovis was 3.78% in milk samples, regardless of the farm, and 20% in nasal swabs.

Bovine respiratory coronavirus enhances bacterial adherence by upregulating expression of cellular receptors on bovine respiratory epithelial cells

Bovine coronavirus (BCoV) is one of the agents causing bovine respiratory disease complex (BRDC), with single infection tending to be mild to moderate; the probability of developing pneumonia in BRDC may be affected by viral and bacterial combinations. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection enhances adherence of Pasteurella multocida (PM) to cells derived from the bovine lower respiratory tract but that BRSV infection in cells derived from the upper respiratory tract reduces PM adherence. In this study, we sought to clarify whether the modulation of bacterial adherence to cells derived from the bovine upper and lower respiratory tract is shared by other BRDC-related viruses by infecting bovine epithelial cells from the trachea, bronchus and lung with BCoV and/or PM. The results showed that cells derived from both the upper and lower respiratory tract were susceptible to BCoV infection. Furthermore, all cells infected with BCoV exhibited increased PM adherence via upregulation of two major bacterial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PAF-R), suggesting that compared with BRSV infection, BCoV infection differentially modulates bacterial adherence. In summary, we identified distinct interaction between bovine respiratory viruses and bacterial infections.

Mannheimia pernigra sp. nov., isolated from bovine respiratory tract.

Over a period of 1 year, 270 isolates identified as Taxon 39 of Bisgaard were obtained from the nasopharynx of veal calves at 11 epidemiologically independent Swiss fattening farms. Two isolates from each farm and the Australian Taxon 39 reference strain BNO311 were further characterized by genetic and phenotypic methods. Phylogenetic analysis of 16S rRNA and recN gene sequences placed the isolates in a single, distinct cluster within the genus Mannheimia. As to the rpoB gene, most isolates clustered together, but four strains formed a separate cluster close to Mannheimia varigena. Genome sequence analysis of isolates from both rpoB clusters confirmed their species status, with an average nucleotide identity (ANI) >98.9 % between isolates and <84 % to the closest species, M. varigena. Based upon whole genome sequences, the G+C content was determined as 39.1 mol%. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates clearly separated from the other Mannheimia species, making this the method of choice for identification. In addition, numerous biochemical markers based on classical as well as commercial identification schemes were determined, allowing separation from other Mannheimia species and identification of the new taxon. Major fatty acids for strain 17CN0883T are C14 : 0, C16 : 0, C16 : 1 ω7c and C18 : 1 ω7c. Major respiratory quinones are ubiquinone-7 and ubiquinone-8. We propose the name Mannheimia pernigra sp. nov. for former Taxon 39 of Bisgaard. The type strain is 17CN0883T (=CCUG 74657T=DSM 111153T) isolated from a veal calf in Switzerland.

Überblick über die Ultraschallunter­suchung der Lunge beim Kalb

This paper reviews the technique used for thoracic -ultrasonography of the bovine lower respiratory tract and the ultrasonographic findings in calves with -bronchopneumonia. Studies that compare the results of auscultation with those of ultrasonography and postmortem examination are included as are studies that examine the relationship between ultrasonographic findings and bacterial lung infection and prognosis. Lesions associated with bronchopneumonia, pleuropneumonia, pneumothorax and lung abscesses are -easily imaged ultrasonographically and characterised in calves.

Bovine Respiratory Syncytial Virus Enhances the Adherence of Pasteurella multocida to Bovine Lower Respiratory Tract Epithelial Cells by Upregulating the Platelet-Activating Factor Receptor.

Coinfection by bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM) frequently has been observed in cattle that develop severe pneumonia. We recently reported that BRSV infection significantly increased PM adherence to bovine lower respiratory tract epithelial cells. However, the molecular mechanisms of enhanced PM adherence are not completely understood. To investigate whether BRSV infection regulates any cellular adherence receptors on bovine bronchus- and lung-epithelial cells, we performed proteomic and functional analyses. The proteomic analysis showed that BRSV infection increased the accumulation of the platelet-activating factor receptor (PAFR) in both cell types. Molecular experiments, including specific blockade, knockdown, and overexpression of PAFR, indicated that PM adherence to these cell types depended on PAFR expression. These findings highlight the role, in cattle with severe pneumonia, of the synergistic effect of coinfection by BRSV and PM in the lower respiratory tract.

Replication of Influenza D Viruses of Bovine and Swine Origin in Ovine Respiratory Explants and Their Attachment to the Respiratory Tract of Bovine, Sheep, Goat, Horse, and Swine.

Bovine is considered the main reservoir of influenza D virus (IDV), however, low levels of seropositivity in other farmed species suggest a wide range of potential hosts. Nevertheless, it is not clear whether this scenario is the result of rare spillover events upon contact with bovines, or a lack of adaptation of IDV to these hosts. Among these species, sheep represents a crucial component of the rural economy in many developing countries, but little is known about its role in the ecology of the disease. To evaluate the susceptibility of sheep to IDV viruses of different origin, we used ovine respiratory tissues as an ex vivo model and investigated the infective phenotype of two IDV strains isolated from either bovine (IDV-BOV) or swine (IDV-SW). For translatability purposes, we included a parainfluenza type 3 virus, as positive control, given its known respiratory tropism in sheep. We performed a timed evaluation of the viral infectivity, cell tropism and the associated histopathology, by means of tissue culture infectious dose assays on supernatants and histological/immunohistochemical analyses on explanted tissues, respectively. To further investigate differences in the phenotype of these two strains and to identify the potential targets of replication in the most commonly land-based farmed mammalian species, we carried out virus binding assays on histological sections of the respiratory tract of bovine, caprine, ovine, horse and swine. Our results demonstrated that IDV successfully replicates in nasal, tracheal and lung ovine tissues, suggesting a moderate susceptibility of this species to IDV infection. Interestingly, despite the high genetic identity of these strains, IDV- BOV consistently replicated to higher titers than IDV-SW in all respiratory tracts, suggesting IDV viruses might display considerable levels of variability in their phenotype when crossing the species barrier. Virus binding assays confirmed a superior affinity of the IDV viruses for the bovine upper respiratory tract, and a preference for the pharyngeal epithelium of small ruminants, indicating possible targets to improve the sensitivity of virological sampling for diagnostic and post-mortem purposes. Further pathogenesis and cross-species transmission studies will be necessary to elucidate the ecology of IDV and eventually allow the design of cost-effective surveillance strategies.

KA-1002, a Novel Lysophosphatidic Acid Signaling Antagonist, Alleviates Bovine Tracheal Cell Disruption and Inflammation.

Simple SummaryBovine respiratory diseases are a key factor reducing the productivity of cows in an industrial livestock environment. We have identified a novel lysophosphatidic acid signaling antagonist, KA-1002, which alleviates lysophosphatidic acid-mediated bovine tracheal cell disruptive tissue injury and inflammation. KA-1002 could be considered a novel therapeutic reagent candidate for maintaining respiratory health in reared cattle.The industrial livestock environment can cause stress and weakened immunity in cattle, leading to microbial infections which reduce productivity. As such, there is a need for an effective therapeutic agent that can alleviate uncontrolled destructive respiratory inflammation. We found that lysophosphatidic acid (LPA), a potent endogenous stress-induced inflammatory agent, causes respiratory tissue damage and triggers inflammation in bovine bronchial cells. LPA also inflames pulmonary bovine blood vessel cells to produce inflammatory cytokines. These findings strongly suggest that LPA is a highly important endogenous material exacerbating bovine respiratory diseases. We further identified a novel LPA-signaling antagonist, KA-1002, and showed that it alleviated LPA-mediated bovine tracheal cell disruption and inflammation. Therefore, KA-1002 could potentially serve as a novel therapeutic agent to maintain physiologically healthy and balanced conditions in bovine respiratory tracts.

Oxidative stress pathway gene transcription after bovine respiratory syncytial virus infection in vitro and ex vivo

Studies in mouse and lamb models indicate important roles of reactive oxygen species (ROS) in the pathology and immune response to respiratory syncytial virus (RSV). The role of ROS in bovine RSV (BRSV) infection of calves remains unclear. BRSV naturally infects calves, leading to similar disease course, micro- and macro-lesions, and symptomology as is observed in RSV infection of human neonates. Furthermore, humans, lambs, and calves, but not mice, have an active lung oxidative system involving lactoperoxidase (LPO) and the dual oxidases (DUOX) 1 and 2. To gain insight into the role of ROS in the BRSV-infected lung, we examined gene expression in infected bovine cells using qPCR. A panel of 19 primers was used to assay ex vivo and in vitro BRSV-infected cells. The panel targeted genes involved in both production and regulation of ROS. BRSV infection significantly increased transcription of five genes in bovine respiratory tract cells in vitro and ex vivo. PTGS2 expression more than doubled in both sample types. Four transcripts varied significantly in lung lesions, but not non-lesion samples, compared with uninfected lung. This is the first report of the transcriptional profile of ROS-related genes in the airway after BRSV infection in the natural host.

Bacterial topography of the bovine respiratory tract

It is generally accepted that the nasopharynx is the primary source of bovine respiratory disease (BRD) pathogens for the lower respiratory tract (LRT) of cattle. Indeed, it has long been held that the nasopharynx plays a causative role in the development of BRD. However, it has been shown that there is poor agreement between the microbiota of the nasopharynx and the LRT in cattle. Conflicting results from recent studies suggest that BRD pathogens in the nasopharynx may not always significantly differ between healthy cattle and those diagnosed with BRD. These observations make sense in light of recent research in human medicine, which suggests that bacterial pathogens that cause lung infections don’t originate from the nasopharynx and that the nasopharyngeal microbiota contribute very little to composition of lung microbiota. There was surprisingly more similarity between the mouth/oropharyngeal microbiotas and the lung microbiota. In the pursuit of finding alternative strategies for controlling BRD at the feedlot such as probiotics or local antimicrobials, it is crucial to understand which upper respiratory tract (URT) microbiotas contribute the most to the LRT microbiotas so that we can modulate the correct bacterial communities. Therefore, the objective of this study is to map the bacterial microbiotas present along the entire beef cattle respiratory tract and determine the relative contributions of these communities to the microbiota of the lung.

Co-infection of epithelial cells established from the upper and lower bovine respiratory tract with bovine respiratory syncytial virus and bacteria

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.

Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract

Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation.

Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves

BackgroundThe microbiota of the bovine upper respiratory tract has been recently characterized, but no data for the lower respiratory tract are available. A major health problem in bovine medicine is infectious bronchopneumonia, the most common respiratory syndrome affecting cattle. With this study, we used 16S rRNA gene sequencing to characterize and compare the microbial community composition of the upper and lower respiratory tracts in calves.ResultsThe microbiota of the upper (nasal swab [NS]) and the lower (trans-tracheal aspiration [TTA]) respiratory tracts of 19 post-weaned Piedmontese calves with (8/19) and without (11/19) clinical signs of respiratory disease, coming from six different farms, was characterized by 16S rRNA gene metabarcoding. A total of 29 phyla (29 in NS, 21 in TTA) and 305 genera (289 in NS, 182 in TTA) were identified. Mycoplasma (60.8%) was the most abundant genus identified in both the NS (27.3%) and TTA (76.7%) samples, followed by Moraxella (16.6%) in the NS and Pasteurella (7.3%) in the TTA samples. Pasteurella multocida (7.3% of total operational taxonomic units [OTUs]) was the most abundant species in the TTA and Psychrobacter sanguinis (1.1% of total OTUs) in the NS samples. Statistically significant differences between the NS and the TTA samples were found for both alpha (Shannon index, observed species, Chao1 index, and Simpson index; P = 0.001) and beta (Adonis; P = 0.001) diversity. Comparison of the NS and TTA samples by farm origin and clinical signs revealed no statistical difference (P > 0.05), except for farm origin for the NS samples when compared by the unweighted UniFrac metric (P = 0.05).ConclusionsUsing 16S rRNA gene sequencing, we characterized the microbiota of the upper and lower respiratory tracts of calves, both healthy individuals and those with clinical signs of respiratory disease. Our results suggest that environmental factors may influence the composition of the upper airway microbiota in cattle. While the two microbial communities (upper and lower airways) differed in microbial composition, they shared several OTUs, suggesting that the lung microbiota may be a self-sustaining, more homogeneous ecosystem, influenced by the upper respiratory tract microbiota.