RESUMEN
Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep.
Asunto(s)
Mannheimia haemolytica , Tipificación de Secuencias Multilocus , Pasteurelosis Neumónica , Enfermedades de las Ovejas , Animales , Mannheimia haemolytica/genética , Mannheimia haemolytica/clasificación , Mannheimia haemolytica/aislamiento & purificación , Mannheimia haemolytica/patogenicidad , Ovinos/microbiología , Enfermedades de las Ovejas/microbiología , India , Pasteurelosis Neumónica/microbiología , Serogrupo , Electroforesis en Gel de Campo Pulsado , Factores de Virulencia/genética , Virulencia/genética , FilogeniaRESUMEN
Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.
Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex , Nasofaringe , Recombinasas , Animales , Bovinos , Nasofaringe/microbiología , Recombinasas/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Secuencias Repetitivas Esparcidas/genética , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/diagnóstico , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Complejo Respiratorio Bovino/microbiología , Conjugación Genética , Sensibilidad y Especificidad , Mannheimia haemolytica/genética , Mannheimia haemolytica/aislamiento & purificación , Pasteurellaceae/genética , Pasteurellaceae/aislamiento & purificaciónRESUMEN
Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.
Asunto(s)
Antibacterianos , Cabras , Mannheimia haemolytica , Pruebas de Sensibilidad Microbiana , Pasteurella multocida , Enfermedades de las Ovejas , Animales , Pasteurella multocida/efectos de los fármacos , Pasteurella multocida/aislamiento & purificación , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/aislamiento & purificación , Etiopía/epidemiología , Ovinos/microbiología , Cabras/microbiología , Antibacterianos/farmacología , Estudios Transversales , Enfermedades de las Ovejas/microbiología , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Cabras/microbiología , Enfermedades de las Cabras/epidemiología , Prevalencia , Factores de Riesgo , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/veterinaria , Infecciones por Pasteurella/epidemiologíaRESUMEN
Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.
Asunto(s)
Abomaso , Mannheimia haemolytica , Necrosis , Infecciones por Pasteurellaceae , Enfermedades de las Ovejas , Animales , Mannheimia haemolytica/aislamiento & purificación , Enfermedades de las Ovejas/patología , Enfermedades de las Ovejas/microbiología , Ovinos , Abomaso/patología , Abomaso/microbiología , Infecciones por Pasteurellaceae/veterinaria , Infecciones por Pasteurellaceae/patología , Infecciones por Pasteurellaceae/microbiología , Necrosis/veterinaria , Necrosis/patología , Necrosis/microbiología , Gastropatías/veterinaria , Gastropatías/patología , Gastropatías/microbiología , Masculino , Femenino , Inmunohistoquímica/veterinariaRESUMEN
A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.
Asunto(s)
Antibacterianos/farmacología , Complejo Respiratorio Bovino/patología , Enfermedades de los Bovinos/patología , Disacáridos/farmacología , Compuestos Heterocíclicos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Pasteurella multocida/efectos de los fármacos , Animales , Antibacterianos/uso terapéutico , Complejo Respiratorio Bovino/tratamiento farmacológico , Complejo Respiratorio Bovino/microbiología , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Enfermedades de los Bovinos/microbiología , Estudios Transversales , ADN Viral/genética , ADN Viral/metabolismo , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Disacáridos/uso terapéutico , Herpesvirus Bovino 1/efectos de los fármacos , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/aislamiento & purificación , Compuestos Heterocíclicos/uso terapéutico , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Nasofaringe/microbiología , Nasofaringe/virología , Pasteurella multocida/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , ARN Viral/genética , ARN Viral/metabolismo , Virus Sincitial Respiratorio Bovino/efectos de los fármacos , Virus Sincitial Respiratorio Bovino/genética , Virus Sincitial Respiratorio Bovino/aislamiento & purificación , Factores de Riesgo , Insuficiencia del TratamientoRESUMEN
This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Colorimetría/veterinaria , Pruebas Diagnósticas de Rutina/veterinaria , Técnicas de Diagnóstico Molecular/veterinaria , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Infecciones por Pasteurellaceae/veterinaria , Pasteurellaceae/aislamiento & purificación , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Colorimetría/instrumentación , Pruebas Diagnósticas de Rutina/instrumentación , Mannheimia haemolytica/aislamiento & purificación , Técnicas de Diagnóstico Molecular/instrumentación , Nariz/microbiología , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Infecciones por Pasteurella/diagnóstico , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/aislamiento & purificación , Infecciones por Pasteurellaceae/diagnóstico , Infecciones por Pasteurellaceae/microbiologíaRESUMEN
Bovine respiratory disease (BRD) is caused by a mixture of viruses and opportunistic bacteria belonging to Pasteurellaceae and Mycoplasma bovis. However, these organisms are also commonly isolated from healthy calves. This study aimed to determine whether the organisms are present in higher numbers in calves sick with acute BRD than in clinically healthy calves, and further to genetically characterize bacteria of the family Pasteurellaceae to understand whether particular types are associated with disease. Forty-six clinically healthy and 46 calves with BRD were sampled by broncheoalveolar lavage (BAL) method in 11 herds geographically spread over Denmark to determine presence and quantity of microorganisms by culture and quantitative real time qPCR. Isolates of Pasteurellaceae were tested for antibiotic resistance and were whole genome sequenced to determine genotypes. Histophilus somni was in particular positively associated with BRD, suggesting particular importance of this organism as likely aetiology of BRD. In addition, quantification of bacteria revealed that higher counts of H. somni as well as of M. haemolytica was also a good indicator of the disease. Pasteurellaceae isolates were susceptible to the commonly used antibiotics in treatment of BRD, and genotypes were shared between isolates from clinically healthy and sick calves.
Asunto(s)
Bacterias/genética , Bacterias/patogenicidad , Complejo Respiratorio Bovino/microbiología , Enfermedades de los Bovinos/virología , Enfermedades Respiratorias/microbiología , Enfermedades Respiratorias/veterinaria , Animales , Antibacterianos/farmacología , Bacterias/clasificación , Bacterias/aislamiento & purificación , Líquido del Lavado Bronquioalveolar/microbiología , Líquido del Lavado Bronquioalveolar/virología , Bovinos , Mannheimia haemolytica/genética , Mannheimia haemolytica/aislamiento & purificación , Mannheimia haemolytica/patogenicidad , Pasteurellaceae/clasificación , Pasteurellaceae/efectos de los fármacos , Pasteurellaceae/genética , Pasteurellaceae/patogenicidad , Enfermedades Respiratorias/virologíaRESUMEN
The relationships between host-pathogen population dynamics in wildlife are poorly understood. An impediment to progress in understanding these relationships is imperfect detection of diagnostic tests used to detect pathogens. If ignored, imperfect detection precludes accurate assessment of pathogen presence and prevalence, foundational parameters for deciphering host-pathogen dynamics and disease etiology. Respiratory disease in bighorn sheep (Ovis canadensis) is a significant impediment to their conservation and restoration, and effective management requires a better understanding of the structure of the pathogen communities. Our primary objective was to develop an easy-to-use and accessible web-based Shiny application that estimates the probability (with associated uncertainty) that a respiratory pathogen is present in a herd and its prevalence given imperfect detection. Our application combines the best-available information on the probabilities of detection for various respiratory pathogen diagnostic protocols with a hierarchical Bayesian model of pathogen prevalence. We demonstrated this application using four examples of diagnostic tests from three herds of bighorn sheep in Montana. For instance, one population with no detections of Mycoplasma ovipneumoniae (PCR assay) still had an 6% probability of the pathogen being present in the herd. Similarly, the apparent prevalence (0.32) of M. ovipneumoniae in another herd was a substantial underestimate of estimated true prevalence (0.46: 95% CI = [0.25, 0.71]). The negative bias of naïve prevalence increased as the probability of detection of testing protocols worsened such that the apparent prevalence of Mannheimia haemolytica (culture assay) in a herd (0.24) was less than one third that of estimated true prevalence (0.78: 95% CI = [0.43, 0.99]). We found a small difference in the estimates of the probability that Mannheimia spp. (culture assay) was present in one herd between the binomial sampling approach (0.24) and the hypergeometric approach (0.22). Ignoring the implications of imperfect detection and sampling variation for assessing pathogen communities in bighorn sheep can result in spurious inference on pathogen presence and prevalence, and potentially poorly informed management decisions. Our Shiny application makes the rigorous assessment of pathogen presence, prevalence and uncertainty straightforward, and we suggest it should be incorporated into a new paradigm of disease monitoring.
Asunto(s)
Animales Salvajes/microbiología , Infecciones por Pasteurellaceae/veterinaria , Neumonía por Mycoplasma/veterinaria , Enfermedades de las Ovejas/epidemiología , Borrego Cimarrón/microbiología , Programas Informáticos , Animales , Teorema de Bayes , Internet , Mannheimia haemolytica/aislamiento & purificación , Montana , Mycoplasma ovipneumoniae/aislamiento & purificación , Infecciones por Pasteurellaceae/epidemiología , Neumonía por Mycoplasma/epidemiología , Prevalencia , Probabilidad , OvinosRESUMEN
BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Enfermedades de los Bovinos/microbiología , Mannheimia haemolytica/genética , Infecciones del Sistema Respiratorio/veterinaria , Secuenciación Completa del Genoma/métodos , Animales , Bovinos , Cromosomas Bacterianos/genética , Genotipo , Mannheimia haemolytica/clasificación , Mannheimia haemolytica/aislamiento & purificación , Mutación , FilogeniaRESUMEN
BACKGROUND: A microbiological diagnosis is essential to better target antimicrobial treatment, control and prevention of respiratory tract infections in cattle. Under field conditions, non-endoscopic broncho-alveolar lavage (nBAL) samples are increasingly collected. To what extent the highly variable turnaround time and storage temperatures between sampling and cultivation affect the isolation rate of bacterial pathogens is unknown. Therefore, the objective of this experimental study was to determine the effect of different storage temperatures (0 °C, 8 °C, 23 °C and 36 °C) and times (0,2,4,6,8,24,48 h) on the isolation rate and concentration of Pasteurellaceae in nBAL samples from clinically affected animals. RESULTS: At a storage temperature temperature of 36 °C isolation rates of Mannheimia haemolytica and Pasteurella multocida were significantly reduced 6 h and 48 h after sampling, respectively. At room temperature (23 °C), a decrease in M. haemolytica and P. multocida isolation rate was noticed, starting at 24 and 48 h after sampling, respectively, but only significant for P. multocida at 48 h. The presence of microbial contamination negatively affected the isolation of P. multocida in clinical nBAL samples, but not of M. haemolytica. CONCLUSION: Optimal M. haemolytica and P. multocida isolation rates from clinical nBAL samples are obtained after storage at 0 °C or 8 °C, provided that the sample is cultivated within 24 h after sampling. The maximum period a sample can be stored without an effect on the M. haemolytica and P. multocida isolation success varies and is dependent on the storage temperature and the degree of microbial contamination.
Asunto(s)
Líquido del Lavado Bronquioalveolar/microbiología , Mannheimia haemolytica/aislamiento & purificación , Pasteurella multocida/aislamiento & purificación , Manejo de Especímenes/veterinaria , Animales , Lavado Broncoalveolar/veterinaria , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/veterinaria , Manejo de Especímenes/métodos , Temperatura , Factores de TiempoRESUMEN
Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INFγ and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.
Asunto(s)
Complejo Respiratorio Bovino/patología , Inmunidad Innata/efectos de los fármacos , Fármacos del Sistema Respiratorio/farmacología , Administración Intranasal , Animales , Complejo Respiratorio Bovino/tratamiento farmacológico , Complejo Respiratorio Bovino/mortalidad , Bovinos , Antígenos de Histocompatibilidad Clase II/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Liposomas/química , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Mannheimia haemolytica/aislamiento & purificación , Mannheimia haemolytica/patogenicidad , Nasofaringe/metabolismo , Nasofaringe/microbiología , Óxido Nítrico/metabolismo , Fagocitosis , Fármacos del Sistema Respiratorio/uso terapéutico , Tasa de Supervivencia , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 9/agonistas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Changes in cattle feeding in the twentieth century led to the "Golden Age of Cattle Feeding" on the US High Plains; this was accompanied by recognition that bovine respiratory disease (BRD) is the leading cause of feedlot morbidity and mortality. Decades of research have illuminated the multiple viruses and bacteria that contribute to BRD, which led to vaccines and antimicrobials to prevent, treat, and control BRD. Despite these discoveries, feedlot BRD morbidities do not appear to have changed substantially over this time. New technologies are being developed that have the potential to improve accuracy of BRD detection.
Asunto(s)
Complejo Respiratorio Bovino/etiología , Animales , Complejo Respiratorio Bovino/diagnóstico , Complejo Respiratorio Bovino/microbiología , Bovinos , Industria Lechera/métodos , Femenino , Mannheimia haemolytica/aislamiento & purificaciónRESUMEN
Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors associated with epidemic respiratory disease in calves. A cross-sectional study was conducted, involving 128 outbreaks (29 dairy, 58 dairy-mixed, and 41 beef) in Belgium (2016-2018). A semiquantitative PCR for 7 respiratory pathogens was done on a pooled nonendoscopic bronchoalveolar lavage sample for each herd. Potential risk factors were collected by questionnaire and derived from the national cattle registration databank. Most outbreaks occurred between October and March, and single and multiple viral infections were detected in 58.6% (75/128) and 13.3% (17/128), respectively. Bovine coronavirus (BCV) was the most frequently isolated virus (38.4%), followed by bovine respiratory syncytial virus (bRSV; 29.4%) and parainfluenzavirus type 3 (PI-3; 8.1%). Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were detected in 33.3, 41.2, 89.1, and 36.4% of the herds, respectively. Specific risk factors for BCV detection were detection of M. haemolytica [odds ratio (OR) = 2.8 (95% confidence interval = 1.1-7.5)], increasing herd size [OR = 1.3 (1.0-1.8) for each increase with 100 animals] and detection of BCV by antigen ELISA on feces in calves in the last year [OR = 3.6 (1.2-11.1)]. A seasonal effect was shown for bRSV only {more in winter compared with autumn [OR = 10.3 (2.8-37.5)]}. Other factors associated with bRSV were PI-3 detection [OR = 13.4 (2.1-86.0)], prevalence of calves with respiratory disease [OR = 1.02 (1.00-1.04) per 1% increase], and number of days with respiratory signs before sampling [OR = 0.99 (0.98-0.99) per day increase]. Next to its association with BCV, M. haemolytica was more frequently detected in herds with 5 to 10 animals per pen [OR = 8.0 (1.4-46.9)] compared with <5 animals, and in herds with sawdust as bedding [OR = 18.3 (1.8-191.6)]. Also, for H. somni, housing on sawdust was a risk factor [OR = 5.2 (1.2-23.0)]. Purchase of cattle [OR = 2.9 (1.0-8.0)] and housing of recently purchased animals in the same airspace [OR = 5.0 (1.5-16.5)] were risk factors for M. bovis. This study identified pathogen-specific risk factors that might be useful for the development of customized control and prevention and for the design of decision support tools to justify antimicrobial use by predicting the most likely pathogen before sampling results are available.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Coronavirus Bovino/aislamiento & purificación , Brotes de Enfermedades/veterinaria , Infecciones del Sistema Respiratorio/veterinaria , Animales , Bélgica/epidemiología , Lavado Broncoalveolar/veterinaria , Bovinos , Enfermedades de los Bovinos/microbiología , Estudios Transversales , Heces/microbiología , Femenino , Masculino , Mannheimia haemolytica/aislamiento & purificación , Mycoplasma bovis/aislamiento & purificación , Virus de la Parainfluenza 3 Bovina/aislamiento & purificación , Pasteurella multocida/aislamiento & purificación , Pasteurellaceae/aislamiento & purificación , Virus Sincitial Respiratorio Bovino/aislamiento & purificación , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiología , Factores de Riesgo , Especificidad de la Especie , Encuestas y CuestionariosRESUMEN
Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Enfermedades de los Bovinos/diagnóstico , Infecciones por Moraxellaceae/veterinaria , Infecciones por Pasteurella/veterinaria , Infecciones por Pasteurellaceae/veterinaria , Infecciones del Sistema Respiratorio/veterinaria , Animales , Líquido del Lavado Bronquioalveolar/microbiología , Bovinos , Enfermedades de los Bovinos/microbiología , Humanos , Mannheimia haemolytica/clasificación , Mannheimia haemolytica/aislamiento & purificación , Moraxella/clasificación , Moraxella/aislamiento & purificación , Infecciones por Moraxellaceae/diagnóstico , Infecciones por Moraxellaceae/microbiología , Infecciones por Pasteurella/diagnóstico , Infecciones por Pasteurella/microbiología , Pasteurella multocida/clasificación , Pasteurella multocida/aislamiento & purificación , Pasteurellaceae/clasificación , Pasteurellaceae/aislamiento & purificación , Infecciones por Pasteurellaceae/diagnóstico , Infecciones por Pasteurellaceae/microbiología , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Bacterial pneumonia causes significant economic loss to the beef industry and occurs at times of stress and viral infection. Administering antibiotics to at-risk calves is often used to prevent the disease, but alternatives to mass treatment with antibiotics are needed. Tracheal antimicrobial peptide (TAP), a ß-defensin naturally produced by bovine airways, has bactericidal activity against the pathogens that cause pneumonia in cattle. However, TAP expression is suppressed by glucocorticoid (stress) and viral infection. We hypothesized that delivering TAP to the respiratory tract would prevent development of pneumonia in calves infected with Mannheimia haemolytica. Clean-catch calves (i.e. obtained prior to contact with the dam) were challenged by aerosol with M. haemolytica, and TAP or water was delivered to the respiratory tract at 0.3, 2 and 6 hours post-infection. TAP treatment did not protect against development of disease. Calves treated with TAP had similar bacterial loads in the nasal cavity and lung compared to calves treated with water. Similarly, TAP treatment did not affect the development of clinical signs, elevated rectal temperatures, or increased levels of blood neutrophils, haptoglobin and fibrinogen that occurred after bacterial challenge. Postmortem gross and histologic lung lesions were also similar in the two groups. To determine why there was a lack of protective effect, we tested the effect of substances in respiratory lining fluid on the bactericidal activity of TAP. Physiologic concentrations of sodium chloride inhibited TAP bactericidal activity in vitro, as did serum at concentrations of 0.62 to 2.5%, but concentrated bronchoalveolar lavage fluid had no consistent effect. These findings suggest that TAP does not have in vivo bactericidal activity against M. haemolytica because of interference by physiological sodium chloride levels and by serum. Thus, administration of TAP may not be effective for prevention of M. haemolytica pneumonia.
Asunto(s)
Antiinfecciosos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Enfermedades de los Bovinos/tratamiento farmacológico , Mannheimia haemolytica/patogenicidad , Infecciones por Pasteurellaceae/tratamiento farmacológico , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Líquido del Lavado Bronquioalveolar/microbiología , Bovinos , Fibrinógeno/análisis , Haptoglobinas/análisis , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/aislamiento & purificación , Oxidación-Reducción , Infecciones por Pasteurellaceae/veterinaria , Cloruro de Sodio/farmacologíaRESUMEN
We investigated three bovine respiratory pathobionts in healthy cattle using qPCR optimised and validated to quantify Histophilus somni, Mannheimia haemolytica and Pasteurella multocida over a wide dynamic range. A longitudinal study was conducted to investigate the carriage and density of these bacteria in the nasal passages of healthy beef calves (N = 60) housed over winter in an experimental farm setting. The three pathobiont species exhibited remarkably different carriage rates and density profiles. At housing, high carriage rates were observed for P. multocida (95%), and H. somni (75%), while fewer calves were positive for M. haemolytica (13%). Carriage rates for all three bacterial species declined over the 75-day study, but not all individuals became colonised despite sharing of environment and airspace. Colonisation patterns ranged from continuous to intermittent and were different among pathobiont species. Interval-censored exponential survival models estimated the median duration of H. somni and P. multocida carriage at 14.8 (CI95%: 10.6-20.9) and 55.5 (CI95%: 43.3-71.3) days respectively, and found higher density P. multocida carriage was associated with slower clearance (p = 0.036). This work offers insights into the dynamics of pathobiont carriage and provides a potential platform for further data collection and modelling studies.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Mannheimia haemolytica/crecimiento & desarrollo , Pasteurella multocida/crecimiento & desarrollo , Infecciones por Pasteurellaceae/microbiología , Infecciones por Pasteurellaceae/veterinaria , Pasteurellaceae/crecimiento & desarrollo , Animales , Carga Bacteriana , Portador Sano , Bovinos , Enfermedades de los Bovinos/diagnóstico , ADN Bacteriano/genética , Estudios Longitudinales , Masculino , Mannheimia haemolytica/clasificación , Mannheimia haemolytica/aislamiento & purificación , Cavidad Nasal/microbiología , Pasteurella multocida/clasificación , Pasteurella multocida/aislamiento & purificación , Pasteurellaceae/clasificación , Pasteurellaceae/genética , Pasteurellaceae/aislamiento & purificación , Infecciones por Pasteurellaceae/diagnóstico , Reacción en Cadena de la PolimerasaRESUMEN
Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in beef cattle. Recent evidence suggests that commensal bacteria of the bovine nasopharynx have an important role in maintaining respiratory health by providing colonization resistance against pathogens. The objective of this study was to screen and select bacterial therapeutic candidates from the nasopharynxes of feedlot cattle to mitigate the BRD pathogen Mannheimia haemolytica In a stepwise approach, bacteria (n = 300) isolated from the nasopharynxes of 100 healthy feedlot cattle were identified and initially screened (n = 178 isolates from 12 different genera) for growth inhibition of M. haemolytica Subsequently, selected isolates were evaluated for the ability to adhere to bovine turbinate (BT) cells (n = 47), compete against M. haemolytica for BT cell adherence (n = 15), and modulate gene expression in BT cells (n = 10). Lactobacillus strains had the strongest inhibition of M. haemolytica, with 88% of the isolates (n =33) having inhibition zones ranging from 17 to 23 mm. Adherence to BT cells ranged from 3.4 to 8.0 log10 CFU per 105 BT cells. All the isolates tested in competition assays reduced M. haemolytica adherence to BT cells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulated expression of interleukin 8 (IL-8) and IL-6 (P < 0.05). After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 Lactobacillus strains from 4 different species were selected as the best candidates for further development as intranasal bacterial therapeutics to mitigate M. haemolytica infection in feedlot cattle.IMPORTANCE Bovine respiratory disease (BRD) is a significant animal health issue impacting the beef industry. Current BRD prevention strategies rely mainly on metaphylactic use of antimicrobials when cattle enter feedlots. However, a recent increase in BRD-associated bacterial pathogens that are resistant to metaphylactic antimicrobials highlights a pressing need for the development of novel mitigation strategies. Based upon previous research showing the importance of respiratory commensal bacteria in protecting against bronchopneumonia, this study aimed to develop bacterial therapeutics that could be used to mitigate the BRD pathogen Mannheimia haemolytica Bacteria isolated from the respiratory tracts of healthy cattle were characterized for their inhibitory, adhesive, and immunomodulatory properties. In total, 6 strains were identified as having the best properties for use as intranasal therapeutics to inhibit M. haemolytica If successful in vivo, these strains offer an alternative to metaphylactic antimicrobial use in feedlot cattle for mitigating BRD.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/terapia , Mannheimia haemolytica/patogenicidad , Neumonía Enzoótica de los Becerros/microbiología , Neumonía Enzoótica de los Becerros/terapia , Infecciones del Sistema Respiratorio/terapia , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Bronconeumonía/microbiología , Bronconeumonía/terapia , Bovinos , Enfermedades de los Bovinos/inmunología , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Inmunidad Innata , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Lactobacillus/efectos de los fármacos , Lactobacillus/fisiología , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/crecimiento & desarrollo , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Nasofaringe/microbiología , Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
Bovine Respiratory Disease (BRD) is a major threat to animal health and welfare in the cattle industry. Strains of Mannheimia haemolytica (Mh) that are resistant to multiple classes of antimicrobials are becoming a major concern in the beef industry, as the frequency of isolation of these strains has been increasing. Mobile genetic elements, such as integrative conjugative elements (ICE), are frequently implicated in this rapid increase in multi-drug resistance. The objectives of the current study were to determine the genetic relationship between the isolates collected at arrival before metaphylaxis and at revaccination after metaphylaxis, to identify which resistance genes might be present in these isolates, and to determine if they were carried on an ICE. Twenty calves culture positive for Mh at arrival and revaccination were identified, and a total of 48 isolates with unique susceptibility profiles (26 from arrival, and 22 from revaccination) were submitted for whole-genome sequencing (WGS). A phylogenetic tree was constructed, showing the arrival isolates falling into four clades, and all revaccination isolates within one clade. All revaccination isolates, and one arrival isolate, were positive for the presence of an ICE. Three different ICEs with resistance gene modules were identified. The resistance genes aphA1, strA, strB, sul2, floR, erm42, tetH/R, aadB, aadA25, blaOXA-2, msrE, mphE were all located within an ICE. The gene bla-ROB1 was also present in the isolates, but was not located within an ICE.
Asunto(s)
Antibacterianos/farmacología , Bovinos/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/genética , Pasteurelosis Neumónica/microbiología , Animales , Antibacterianos/uso terapéutico , Disacáridos/uso terapéutico , Variación Genética , Genoma Bacteriano , Compuestos Heterocíclicos/uso terapéutico , Inmunización Secundaria , Secuencias Repetitivas Esparcidas , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Pasteurelosis Neumónica/tratamiento farmacológico , Filogenia , Vacunación , Secuenciación Completa del GenomaRESUMEN
Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including ß-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.
Asunto(s)
Farmacorresistencia Bacteriana , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/genética , Secuenciación de Nanoporos/métodos , Neumonía Enzoótica de los Becerros/microbiología , Animales , Antibacterianos/farmacología , Bovinos , Genoma Bacteriano , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Neumonía Enzoótica de los Becerros/diagnóstico , Análisis de Secuencia de ADN , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area. RESULTS: Bacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin. CONCLUSION: The study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.