RESUMEN
Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.
Asunto(s)
Antiinflamatorios/farmacología , Mannheimia haemolytica/efectos de los fármacos , Oligosacáridos/farmacología , Pasteurella multocida/efectos de los fármacos , Neumonía Enzoótica de los Becerros/tratamiento farmacológico , Animales , Bovinos , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Pulmón/microbiología , Neumonía Enzoótica de los Becerros/microbiologíaRESUMEN
Bacterial lung disease caused by Mannheimia haemolytica inflict significant mortality and morbidity resulting in enormous economic losses to cattle industry. The use of antibiotics is becoming more challenging because of development of anti-microbial resistance. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3), a pattern-recognition receptor is produced at sites of inflammation by many cell types, recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in lungs of normal and M. haemolytica-infected calves and normal and E. coli lipopolysaccharide-treated cattle neutrophils using light and electron microscopic immunochemistry and Western blots. Immunohistology showed the presence of PTX3 in airway epithelial cells, alveolar septa and macrophages in normal and inflamed lungs of calves and the blots showed a significant increase in the expression of PTX3 in lungs from infected calves. Immuno-gold electron microscopy showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages (PIMs). Immunohistochemical staining for PTX3 in peripheral blood neutrophils shows an altered staining pattern in neutrophils stimulated with lipopolysachharide (LPS). However, western blots no significant change in PTX3 amount in LPS-treated neutrophils compared to the controls. These are the first data on the expression of PTX3 in the lungs and the neutrophils of cattle which may add to our understanding of innate immunity in cattle lungs.
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Proteína C-Reactiva/genética , Expresión Génica , Pulmón/microbiología , Mannheimia haemolytica/inmunología , Neutrófilos/inmunología , Neumonía Enzoótica de los Becerros/microbiología , Componente Amiloide P Sérico/genética , Factores de Edad , Animales , Bovinos , Inmunidad Innata , Pulmón/citología , Pulmón/inmunologíaRESUMEN
Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA-LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.
Asunto(s)
Mannheimia haemolytica/metabolismo , Infecciones por Pasteurellaceae/sangre , Neumonía Enzoótica de los Becerros/sangre , Animales , Bovinos , Femenino , Espectroscopía Infrarroja CortaRESUMEN
The objectives of this study were to determine whether plane of nutrition (PON) of milk replacer previously provided to calves, and dosage level of Mannheimia haemolytica (MH), influenced inflammatory responses to a combined viral-bacterial respiratory challenge. Holstein calves (1 d of age; n = 30) were assigned to treatments in a 2 × 3 factorial with pre-weaning PON and MH dose as main effects (n = 5 per treatment). Calves were fed either a low (LPN; n = 15) or a high PON (HPN; n = 15) from birth through weaning. Calves fed LPN were fed 436 g of dry matter (DM) per day of milk replacer until weaning, and HPN calves were fed 797 g of DM per day of milk replacer from d 1 to 10 and 1,080 g of DM per day from d 11 until weaning. Calf starter and water were offered ad libitum. Calves were step-down weaned beginning at d 54 and moved into an enclosed barn at d 70. Indwelling rectal temperature (RT) recording devices and jugular catheters were inserted at d 80. Calves were challenged with 1.5 × 108 plaque-forming units (pfu) per mL of bovine herpesvirus-1 (BHV-1) in each nostril at d 81 and with either 106, 107, or 108 cfu of MH at d 84. Blood samples were collected at varying intervals relative to BHV-1 and MH challenges. Four LPN calves either died or were euthanized soon after the 144-h observation period, whereas all HPN calves survived the entire observation period. As dosage of MH administered increased, acute and systemic inflammatory responses increased. Higher doses of MH resulted in increased leukocyte, neutrophil, and haptoglobin concentrations in infected calves. Data from the current study suggest that the highest dose, 108 cfu, triggered weaned calves' acute disease response, whereas the lower doses, 106 and 107 cfu, caused more moderate inflammation and disease. The effects of PON on inflammation responses to the disease challenge indicated that calves previously fed the LPN diet had more severe pathophysiological responses. Calves fed LPN showed higher peripheral neutrophil and leukocyte counts and serum haptoglobin concentrations following the BHV-1 challenge. Additionally, following the MH challenge, LPN calves had higher peripheral neutrophil counts, neutrophil-to-lymphocyte ratios, and serum tumor necrosis factor-α concentrations. These data demonstrate that higher doses of MH increase the acute inflammatory response and prolong inflammation, and that calves previously fed LPN responded more severely to the combined viral-bacterial respiratory challenge.
Asunto(s)
Alimentación Animal , Dieta/veterinaria , Herpesvirus Bovino 1 , Rinotraqueítis Infecciosa Bovina/prevención & control , Mannheimia haemolytica , Neumonía Enzoótica de los Becerros/prevención & control , Alimentación Animal/análisis , Animales , Bovinos , Haptoglobinas , Inflamación/prevención & control , Inflamación/veterinaria , Masculino , Leche , Sustitutos de la Leche/administración & dosificación , Neutrófilos , DesteteRESUMEN
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
OBJECTIVE: Bovine respiratory diseases are a common cause of calf loss. This study aimed to analyse associations between an occurrence of enzootic bronchopneumonia (EBP), calf mortality and calving management. MATERIAL AND METHODS: A total of 153 dairy farms participated in the study on a voluntary basis from November 2006 to July 2007. Calf management was inspected on-site during a farm visit and farm managers were required to complete a questionnaire on personal assessment of calving procedures, neonate management and environmental factors. Results were collated and matched with the calf mortality rate of 2006 determined from the HI-Tier database for each farm. Randomly selected serum samples of a mean number of 7 calves at the age 6 months per herd were investigated for antibodies against bovine respiratory syncytial virus (BRSV-AB) and parainfluenzavirus type 3 (PIV3-AB). According to the proportion of calves with BRSV-AB or PIV3-AB (≤ 20 % or > 20 %) farms were divided into 2 groups. RESULTS: Customary timing of the first colostrum feeding as well as the perceived level of importance of EBP to the farm manager, as described in the questionnaire, showed a positive correlation to calf mortality. BRSV-AB occurred more frequently on farms where managers stated that the first colostrum feeding occurred later than 4 hours after birth, that birth monitoring was rarely practiced and that the estimated level of dust in the calf barn was considered high. PIV3-AB was more frequently found at farms practicing tethered calving. CONCLUSION AND CLINICAL RELEVANCE: The results of this study indicate that peri- and postnatal calf management procedures may affect calf mortality and the frequency of occurrence of BRSV-AB or PIV3-AB. The influences of birth monitoring and the time of first colostrum feeding as well as dust exposure should be taken into account in future studies on the frequency of EBP and be included in the veterinary cause analysis of herd EBP-related problems.
Asunto(s)
Anticuerpos Antivirales/sangre , Neumonía Enzoótica de los Becerros , Animales , Animales Recién Nacidos , Bovinos , Calostro , Femenino , Virus de la Parainfluenza 3 Humana/inmunología , Neumonía Enzoótica de los Becerros/epidemiología , Neumonía Enzoótica de los Becerros/mortalidad , Embarazo , Virus Sincitial Respiratorio Bovino/inmunología , Factores de RiesgoRESUMEN
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
Monitoring sickness behavior may improve identification, management, and welfare of sick animals. The objective of this study was to characterize components of sickness behavior in group-housed dairy calves, using an experimental disease challenge model with Mannheimia haemolytica (MH). Holstein bull calves (aged 3-7 weeks; 58.0 ± 12.0 kg of body weight) were group-housed based on age and body weight in sand-bedded pens (6 calves/pen, 6.6 m2/calf) and provided pasteurized waste milk (8 L/d) 2×/d and grain concentrate ad libitum. Within group, calves were randomly assigned to 1 of 2 treatments: (1) inoculation at the tracheal bifurcation with 3 × 109 cfu of MH suspended in 5 mL of sterile phosphate buffered saline (PBS) followed by a 120 mL wash PBS (MH; n = 12, 3/pen), or (2) inoculation with 5 mL + 120 mL of sterile PBS only (control; n = 12, 3/pen). Rectal temperature and health scores were collected from d 0 to +6 of the challenge. A range of behaviors, including feeding patterns and social interactions, were recorded from video from d 0 to +2. The challenge model resulted in calves experiencing a mild disease state: rectal temperatures of MH calves were elevated throughout the challenge compared with control calves, peaking at 12 h postinoculation (39.2 vs. 38.9°C; standard error = 0.14). Many behavioral responses were subject to treatment by day effects, with calves generally becoming less active following inoculation with MH and then returning to baseline. Affected behaviors surrounding feeding included milk feeding time, frequency of competitive displacements, and concentrate feeding time. Lying time was similarly subject to treatment by day effects, and MH calves also spent more time lying on their left side compared with their right (604 vs. 471 min/h; standard error = 32), whereas control calves expressed no laterality. Duration of social lying did not differ, but frequency of social lying bouts decreased in MH calves following inoculation (0.44 vs. 0.75 bouts/h; standard error = 0.04). Social grooming was initiated less by MH calves (0.78 vs. 1.96 min/h; standard error = 0.38), but they tended to receive more social grooming for a greater duration of time (1.59 vs. 1.25 min/h; standard error = 0.13). Overall, we found that infected calves exhibited reduced grooming, feeding, and social interactions, suggesting that these behavioral changes may be useful indicators of early stages of respiratory disease.
Asunto(s)
Conducta Animal , Enfermedades de los Bovinos/fisiopatología , Conducta Alimentaria , Mannheimia haemolytica/aislamiento & purificación , Neumonía Enzoótica de los Becerros/fisiopatología , Animales , Peso Corporal , Bovinos , Enfermedades de los Bovinos/microbiología , Grano Comestible , Aseo Animal , Masculino , Neumonía Enzoótica de los Becerros/microbiología , Distribución AleatoriaRESUMEN
A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration-time curve (AUC0-∞ )/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC0-∞ /MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK-PD modelling of in vitro bacterial time-kill curves for oxytetracycline in serum established mean AUC0-24 hr /MIC ratios for 3log10 decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48-hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25- and 27-fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively.
Asunto(s)
Antibacterianos/farmacocinética , Mannheimia haemolytica/efectos de los fármacos , Oxitetraciclina/farmacocinética , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/efectos de los fármacos , Neumonía Enzoótica de los Becerros/tratamiento farmacológico , Animales , Antibacterianos/administración & dosificación , Antibacterianos/sangre , Antibacterianos/farmacología , Carga Bacteriana/efectos de los fármacos , Carga Bacteriana/veterinaria , Bovinos , Femenino , Inyecciones Intramusculares/veterinaria , Pruebas de Sensibilidad Microbiana/veterinaria , Oxitetraciclina/administración & dosificación , Oxitetraciclina/sangre , Oxitetraciclina/farmacología , Infecciones por Pasteurella/tratamiento farmacológicoRESUMEN
OBJECTIVE To evaluate changes in behavior and surfactant protein (SP) A and D concentrations in serum and bronchoalveolar lavage fluid (BALF) samples of calves experimentally infected with Mannheimia haemolytica. ANIMALS Twelve 4- to 5-month-old Holstein steers. PROCEDURES Calves were divided into 2 treatment groups and instrumented with a data logger to collect behavioral data. After 10 days of acclimation, calves were experimentally inoculated with 3 × 109 CFUs to 5 × 109 CFUs of M haemolytica suspended in approximately 5 mL of PBS solution (infected calves; n = 6) or 5 mL of PBS solution without M haemolytica (control calves; 6) through a catheter into the right accessory lung lobe. Calves were clinically evaluated twice daily for 7 days after inoculation. Blood and BALF samples were collected from all calves at predetermined times for determination of serum and BALF SP-A and SP-D concentrations. Serum and BALF concentrations of SP-A and SP-D and behavioral data were evaluated over time and between treatment groups. RESULTS Compared with control calves, infected calves spent more time lying in general and more time lying on the right side during the 24 hours and 6 days after inoculation, respectively. Mean rectal temperature for infected calves (41.3°C) was significantly greater than that for control calves (39.2°C) 12 hours after inoculation. Mean respiratory rate for infected calves (52.5 breaths/min) was significantly greater than that for control calves (45.4 breaths/min) throughout the observation period. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated continuous behavioral monitoring may improve detection of calves with respiratory tract disease.
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Enfermedades de los Bovinos/fisiopatología , Mannheimia haemolytica , Neumonía Enzoótica de los Becerros/fisiopatología , Animales , Conducta Animal , Temperatura Corporal , Líquido del Lavado Bronquioalveolar , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológicoRESUMEN
Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the ß subunit of ß2 integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted by Mannheimia (Pasteurella) haemolytica binds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(â5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(â5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.
Asunto(s)
Antígenos CD18/genética , Exotoxinas/toxicidad , Mannheimia haemolytica , Neumonía Enzoótica de los Becerros/prevención & control , Sustitución de Aminoácidos , Animales , Antígenos CD18/metabolismo , Bovinos/genética , Línea Celular , Resistencia a la Enfermedad , Feto/metabolismo , Fibroblastos/metabolismo , Edición Génica , Leucocitos/metabolismo , MasculinoRESUMEN
BACKGROUND: Mannheimia haemolytica typically resides in cattle as a commensal member of the upper respiratory tract microbiome. However, some strains can invade their lungs and cause respiratory disease and death, including those with multi-drug resistance. A nucleotide polymorphism typing system was developed for M. haemolytica from the genome sequences of 1133 North American isolates, and used to identify genetic differences between isolates from the lungs and upper respiratory tract of cattle with and without clinical signs of respiratory disease. RESULTS: A total of 26,081 nucleotide polymorphisms were characterized after quality control filtering of 48,403 putative polymorphisms. Phylogenetic analyses of nucleotide polymorphism genotypes split M. haemolytica into two major genotypes (1 and 2) that each were further divided into multiple subtypes. Multiple polymorphisms were identified with alleles that tagged genotypes 1 or 2, and their respective subtypes. Only genotype 2 M. haemolytica associated with the lungs of diseased cattle and the sequence of a particular integrative and conjugative element (ICE). Additionally, isolates belonging to one subtype of genotype 2 (2b), had the majority of antibiotic resistance genes detected in this study, which were assorted into seven combinations that ranged from 1 to 12 resistance genes. CONCLUSIONS: Typing of diverse M. haemolytica by nucleotide polymorphism genotypes successfully identified associations with diseased cattle lungs, ICE sequence, and antibiotic resistance genes. Management of cattle by their carriage of M. haemolytica could be an effective intervention strategy to reduce the prevalence of respiratory disease and supplemental needs for antibiotic treatments in North American herds.
Asunto(s)
Conjugación Genética , Farmacorresistencia Bacteriana , Genoma Bacteriano , Genómica , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/fisiología , Neumonía Enzoótica de los Becerros/microbiología , Animales , Antibacterianos/farmacología , Bovinos , Ligamiento Genético , Genómica/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Mannheimia haemolytica/clasificación , Polimorfismo de Nucleótido SimpleRESUMEN
Mannheimia haemolytica is the most important bacterial pathogen isolated from cases of Bovine Respiratory Disease (BRD). Routine identification of these bacteria is usually performed using phenotypic methods. Our study showed that MALDI-TOF MS is a reliable alternative to these methods. All of the strains analyzed were identified as M. haemolytica. The identification results were compared to those obtained using conventional methods commonly used in microbiological diagnostics, based on detection and analysis of biochemical properties of microorganisms. The degree of agreement between the two methods for identifying M. haemolytica was 100%.
Asunto(s)
Mannheimia haemolytica , Neumonía Enzoótica de los Becerros/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria , Animales , Bovinos , Neumonía Enzoótica de los Becerros/microbiología , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a commercial feedlot. The isolates were obtained from the lungs of 21 calves with fatal pneumonia that were part of a group of 206 total calves. All isolates were serotyped and analyzed by pulsed-field gel electrophoresis (PFGE) and for antibiotic sensitivity patterns. ELISA and immunoblotting assays were performed to compare serum antibody levels to M. haemolytica antigens in calves with fatal pneumonia to those calves that survived the outbreak. Isolates were categorized into 14 different PFGE groups based on 90% similarity. Two Group D isolates (1 and 6), and 3 Group H isolates (14, 15, and 16) were characterized as 100% similar. Antimicrobial susceptibility profiles defined 8 groups based on differences in patterns of resistance between isolates. The two 100% similar isolates from PFGE Group D were both in susceptibility Group 1. All but isolate 14 from PFGE Group H (3, 15, 16, and 19) were in susceptibility Group 4a. Serum antibody levels to M. haemolytica antigens in the dead calves were not different than the antibody levels in the 185 calves that survived the outbreak. Immunoblots of selected isolates from each of the PFGE groups demonstrated only minimal differences in antigenic profiles between strains when reacted with serum from calves that either died from or survived the outbreak. Based on the characteristics of these isolates, multiple strains of M. haemolytica were responsible for fatal pneumonia during this outbreak.
Asunto(s)
Brotes de Enfermedades/veterinaria , Mannheimia haemolytica/genética , Neumonía Enzoótica de los Becerros/microbiología , Animales , Bovinos , Kansas/epidemiología , Mannheimia haemolytica/clasificaciónRESUMEN
OBJECTIVE: To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE: 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES: Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS: Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.
Asunto(s)
Antibacterianos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Neumonía Enzoótica de los Becerros/tratamiento farmacológico , Animales , Bovinos , Disacáridos/farmacología , Enrofloxacina , Fluoroquinolonas/farmacología , Compuestos Heterocíclicos/farmacología , Pruebas de Sensibilidad Microbiana/veterinaria , Neumonía Enzoótica de los Becerros/microbiología , Tianfenicol/análogos & derivados , Tianfenicol/farmacología , Tilosina/análogos & derivados , Tilosina/farmacología , Estados UnidosRESUMEN
Mannheimia haemolytica is an important cause of pneumonia in feedlot cattle. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes, such as heme oxygenase 1 (HO-1), within the lung. The expression of Nrf2 and HO-1 was immunohistochemically evaluated in 4 calves 24 h after experimental infection with M. haemolytica. Calves receiving normal saline served as controls. In the infected lungs, cytoplasmic Nrf2 expression was high in macrophages and bronchioles and low in alveolar epithelium, whereas nuclear expression was high in endothelial cells, macrophages, and bronchioles and lowest in alveolar epithelium. Normal lung samples displayed only faint Nrf2 cytoplasmic staining within bronchiolar epithelium. Expression of HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all infected lung samples, whereas normal lungs displayed only weak cytoplasmic staining in bronchiolar epithelial cells. These findings suggest that bronchiolar epithelial cells and macrophages up-regulate Nrf2 expression early in the course of infection, which results in increased expression of HO-1 within these cells.
Mannheimia haemolytica est une cause importante de pneumonie chez les bovins en parc d'engraissement. Le facteur érythroïde-2 nucléaire apparenté au facteur 2 (Nrf2) est un facteur transcriptionnel sensible au potentiel redox responsable de l'induction d'enzymes antioxidants, tel que l'hème oxygénase 1 (HO-1), dans le poumon. L'expression de Nrf2 et HO-1 fut évaluée par épreuve immunohistochimique chez quatre veaux 24 h après une infection expérimentale avec M. haemolytica. Les veaux témoins ont reçu de la saline. Dans les poumons infectés, l'expression cytoplasmique de Nrf2 était élevée dans les macrophages et les bronchioles et faible dans l'épithélium alvéolaire, alors que l'expression nucléaire était élevée dans les cellules endothéliales, macrophages et bronchioles, et à son plus faible dans l'épithélium alvéolaire. Les échantillons de poumons normaux montraient seulement une faible coloration cytoplasmique pour Nrf2 dans l'épithélium des bronchioles. L'expression de HO-1 fut détectée dans le cytoplasme des macrophages et des cellules épithéliales des bronchioles de tous les échantillons de poumons infectés, alors que les échantillons de poumons normaux ne montraient qu'une faible coloration cytoplasmique dans les cellules épithéliales des bronchioles. Ces données suggèrent que les cellules épithéliales des bronchioles et les macrophages régulent à la hausse l'expression de Nrf2 tôt lors de l'infection, ce qui résulte en une expression augmentée d'HO-1 à l'intérieur de ces cellules.(Traduit par Docteur Serge Messier).
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Pulmón/metabolismo , Mannheimia haemolytica/aislamiento & purificación , Factor 2 Relacionado con NF-E2/metabolismo , Neumonía Enzoótica de los Becerros/microbiología , Animales , Bovinos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/inmunología , Hemo-Oxigenasa 1/genética , Inmunohistoquímica/veterinaria , Pulmón/enzimología , Macrófagos Alveolares/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neumonía Enzoótica de los Becerros/inmunologíaRESUMEN
The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time-kill data, based on the sigmoidal Emax equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log10 reduction) and 4log10 reduction in bacterial counts. For these levels, mean AUC(0-24 h) /MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (Cmax )/MIC ratios of 13.9:1 and 25.2:1, area under concentration-time curve (AUC0-∞ )/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).
Asunto(s)
Amoxicilina/uso terapéutico , Antibacterianos/uso terapéutico , Enfermedades de los Bovinos/tratamiento farmacológico , Mannheimia haemolytica/efectos de los fármacos , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/efectos de los fármacos , Neumonía Enzoótica de los Becerros/tratamiento farmacológico , Amoxicilina/administración & dosificación , Amoxicilina/farmacocinética , Animales , Animales Recién Nacidos , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Bovinos , Enfermedades de los Bovinos/microbiología , Preparaciones de Acción Retardada , Femenino , Inyecciones Intramusculares/veterinaria , Pruebas de Sensibilidad Microbiana/veterinaria , Infecciones por Pasteurella/tratamiento farmacológico , Infecciones por Pasteurella/microbiología , Neumonía Enzoótica de los Becerros/microbiologíaAsunto(s)
Antibacterianos/administración & dosificación , Enfermedades de los Bovinos/prevención & control , Infecciones del Sistema Respiratorio/veterinaria , Administración Oral , Alimentación Animal , Animales , Bronconeumonía/epidemiología , Bronconeumonía/prevención & control , Bronconeumonía/veterinaria , Bovinos , Enfermedades de los Bovinos/epidemiología , Farmacorresistencia Bacteriana , Hierro/administración & dosificación , Neumonía Enzoótica de los Becerros/epidemiología , Neumonía Enzoótica de los Becerros/prevención & control , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/prevención & control , Suiza/epidemiologíaRESUMEN
BACKGROUND: Serum haptoglobin (Hp) and haptoglobin matrix metalloproteinase 9 complexes (Hp-MMP 9) have been identified as biomarkers with diagnostic potential in cattle with conditions resulting in an acute inflammatory response. The purpose of this study was to evaluate potential diagnostic applications of serum Hp and Hp-MMP 9 concentrations in calves with BRD and establish a timeline for their detection in calves experimentally challenged with Bibersteinia trehalosi and Mannheimia haemolytica. Thirty-five cross bred dairy calves were inoculated via tracheal catheterization with either a PCR confirmed leukotoxin negative B. trehalosi isolate, a PCR confirmed leukotoxin positive B. trehalosi isolate, a Mannheimia haemolytica isolate, a combination of leukotoxin negative B. trehalosi and M. haemolytica, or a negative control. Serum samples were collected throughout the study. Calves were euthanized and necropsy performed on day 10 post inoculation. RESULTS: M. haemolytica inoculated calves had increased lung involvement. Serum Hp and Hp- MMP 9 concentrations were elevated compared to the other treatment groups. Increases in serum Hp and Hp-MMP 9 concentrations for the M. haemolytica group were significantly different from other study groups on day 7 of the study. B. trehalosi inoculated calves did not have increased lung involvement compared to control calves, but the leukotoxin positive B. trehalosi group demonstrated increased serum Hp-MMP 9 concentrations from day 3 to the end of the study compared to the pre-inoculation concentrations. CONCLUSION: Serum Hp-MMP 9 concentration is a useful diagnostic tool for detecting early pulmonary inflammation in calves challenged with B. trehalosi and M. haemolytica. Serum Hp-MMP 9 may also be a useful tool in detecting subclinical pulmonary inflammation in challenged calves.
Asunto(s)
Enfermedades de los Bovinos/sangre , Haptoglobinas/análisis , Metaloproteinasa 9 de la Matriz/sangre , Neumonía Enzoótica de los Becerros/sangre , Animales , Biomarcadores/sangre , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pulmón/microbiología , Mannheimia haemolytica/inmunología , Neumonía Enzoótica de los Becerros/diagnóstico , Neumonía Enzoótica de los Becerros/microbiologíaRESUMEN
Respiratory disease of young dairy calves is a significant cause of morbidity, mortality, economic loss, and animal welfare concern but there is no gold standard diagnostic test for antemortem diagnosis. Clinical signs typically used to make a diagnosis of respiratory disease of calves are fever, cough, ocular or nasal discharge, abnormal breathing, and auscultation of abnormal lung sounds. Unfortunately, routine screening of calves for respiratory disease on the farm is rarely performed and until more comprehensive, practical and affordable respiratory disease-screening tools such as accelerometers, pedometers, appetite monitors, feed consumption detection systems, remote temperature recording devices, radiant heat detectors, electronic stethoscopes, and thoracic ultrasound are validated, timely diagnosis of respiratory disease can be facilitated using a standardized scoring system. We have developed a scoring system that attributes severity scores to each of four clinical parameters; rectal temperature, cough, nasal discharge, ocular discharge or ear position. A total respiratory score of five points or higher (provided that at least two abnormal parameters are observed) can be used to distinguish affected from unaffected calves. This can be applied as a screening tool twice-weekly to identify pre-weaned calves with respiratory disease thereby facilitating early detection. Coupled with effective treatment protocols, this scoring system will reduce post-weaning pneumonia, chronic pneumonia, and otitis media.