Bovine respiratory disease (BRD) is the principal cause of morbidity and mortality among feedlot cattle. Mannheimia haemolytica is consistently implicated in this condition, but treatment options are diminishing with the rise of antimicrobial resistance and intensifying consumer pressure to reduce reliance on conventional therapies. Thus, sustainable alternatives like vaccination are required. In this study, the phenotypic and genotypic diversity of BRD pathogens were examined with the objective to identify vaccine targets using reverse vaccinology, an innovative approach to identify antigens via genomic sequence. Preliminary surveillance confirmed M. haemolytica serotype 2 isolates were predominant in healthy animals (75.5%) while serotypes 1 (70.7%) and 6 (19.5%) were common in diseased animals. Pathogens of BRD, including M. haemolytica, Pasteurella multocida and Histophilus somni were also isolated from North American BRD mortalities, and compared using pulsed-field gel electrophoresis and antimicrobial susceptibility. Concurrently, polymerase chain reaction detection of bacterial and viral agents confirmed that M. haemolytica with bovine viral diarrhea virus were the most prevalent. Whereas isolates from live cattle were found to have a relatively low level of resistance, several pathogens from the mortalities were found to contain integrative conjugative elements (ICE) conferring resistance to seven antimicrobial classes. These ICEs were transferred via conjugation to other bacterial species, emphasizing the need for alternative antimicrobial therapies. Collectively, data from these investigations informed the selection of 11 diverse M. haemolytica strains for whole genome sequencing and comparative analyses. Several bacteriophage associated genes and CRISPR-Cas regulated gene expression systems were identified and are likely contributing to virulence in M. haemolytica. Coding sequences across all genomes were screened using pan-genome analysis, identifying 291 candidates with cell-surface associated signatures. Using a cell-free translation system and enzyme-linked immunosorbent assay the candidates were screened against serum from cattle challenged with serovar 1, 2 or 6 of M. haemolytica, and ranked according to immunogenicity. The top five vaccine candidates included Ssa1, ComE, a solute binding protein, an outer membrane protein, and the periplasmic component of an ABC transporter. With further characterization, these unique antigenic candidates could be developed into a vaccine to effectively reduce the dependence on antimicrobial therapies.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Robert L. Larson / Bradley J. White / There are multiple modalities available to evaluate health or stress status of animals. The objective of my research was to evaluate different modalities including behavior, rectal and nasal temperature, and blood samples to determine the relationship with these outcomes of interest in bovine respiratory disease (BRD) events, environmental conditions, transportation, and Mannheimia haemolytica challenge model. The objective for the final project was to determine whether diagnostic sensitivity or specificity resulted in greater economic value for the industry using simulation models for identification of BRD. There was a positive association with rectal temperature and probability of not finishing the production cycle normally, but this relationship was not linear. Rectal temperature of feedlot calves at first treatment for BRD had limited value as a prognostic indicator of whether those calves would finish the production cycle normally. A positive association between rectal temperature and ambient temperature and temperature-humidity index was determined. Environmental conditions must be considered when rectal temperature is used as a diagnostic tool. At 48 hours after initiation of transportation there were no differences in body weight, rectal temperature, and time spent at various locations in the pen detected between transported and non-transported control heifers. Transportation of heifers during periods of high ambient temperatures caused transient changes in physiologic and behavioral indices of heifers. Calves challenged with Mannheimia haemolytica had more changes in behavior, body weight, and blood biomarkers during high ambient temperatures compared to control calves. Results of this study may guide research in development of objective assessment tools for identification and management of cattle affected with BRD during extreme summer conditions. For both low and high apparent prevalence cohorts, increasing diagnostic specificity resulted in more rapid, positive change in net returns compared to change in increasing sensitivity. Improvement of diagnostic specificity, perhaps through a confirmatory test or pen-level diagnostics, can increase diagnostic value. Mortality risk was the primary driver for net returns. Results from this study are important for determining future research priorities to analyze diagnostic techniques for BRD and provide a novel way for modeling diagnostic tests.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Michael D. Apley / Bovine respiratory disease (BRD) remains a major disease in beef production systems. The administration of antimicrobials for both the control and treatment of acute BRD is common. According to most published accounts, antimicrobial resistance among BRD pathogens is increasing; therefore, judicious antimicrobial usage is vital for continued efficacy. The introduction of a novel antimicrobial class has not occurred for well over a decade, therefore it is paramount to maximize efficacy of the antimicrobials currently available. The challenge is targeting the perfect scenario: maximizing clinical efficacy while minimizing antimicrobial resistance. The host-pathogen-drug interaction is very complex and despite current sophisticated technology, this interaction is still not well understood for many infectious diseases. This dissertation work sought to investigate the effects of the administration of a macrolide for both control and treatment of acute BRD on the prevalence of resistance among isolated Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Whole genome sequencing of M. haemolytica was utilized to investigate the presence/absence of macrolide resistance genes and their relationship to the observed minimum inhibitory concentration. Cattle were sampled (plasma and pulmonary epithelial lining fluid) after administration of gamithromycin for drug concentration analysis. A non-linear mixed effects approach was used to fit a compartmental model to the resulting sparse pharmacokinetic data so that a complete time concentration curve could be simulated. From these curves, the CMAX and AUC were measured and used to calculate standard PKPD indices using the MIC values of the isolated bacteria. Clear associations between the use of gamithromycin for control and treatment of BRD and a statistically significantly increased likelihood of macrolide resistance were not found, possibly due to sample size limitations. The calculation of pharmacokinetic-pharmacodynamic indices found that a longer drug exposure was more closely associated with a successful treatment outcome, but there was not a statistically significant correlation. However, there were few clinical failures in this study giving further credence to the complexity of the in vivo system. There are many factors beyond pharmacokinetics/pharmacodynamics and MICs that contribute to the success of a treatment regimen for cattle suffering from BRD.

No description available.

<p> Pasteurella multocida is a respiratory pathogen that is frequently isolated from cattle suffering  from bovine respiratory disease (BRD), the leading cause of mortality and morbidity on modern day cattle farms. Treatment involves the use of antimicrobials which have been shown to fail for  about 30% of BRD cases, leading to the suspicion that etiologic agents, such as P. multocida, may  be resistant. Phenotypic resistance can be confirmed via laboratory antibiotic susceptibility testing  (AST) but this requires several days to complete. Genotypic resistance could be quickly assessed  via nucleic acid assays based on the presence of known antibiotic resistance genes (ARGs). In  human medicine, resistant genes associated with common antibiotics (i.e., ampicillin and penicillin)  in common pathogens (i.e., Salmonella) are very accurate in predicting phenotypic resistance;  however, ARGs associated with antibiotics used to treat BRD, such as enrofloxacin and  tulathromycin, have shown low genotype-phenotype concordance. Hence, this study aims to  improve P. multocida genotype-phenotype concordance by applying a machine learning (ML)  algorithm to identify novel genomic sequences (biomarkers) that have greater accuracy in  predicting resistance to antibiotics commonly used to treat BRD compared to known ARGs.  Cultures of P. multocida were isolated from cattle with clinical signs of BRD. Antibiotic  susceptibility testing was performed and recorded for each isolate. Genomes were sequenced and  assembled, followed by annotating and identifying ARGs using the comprehensive antibiotic  resistance database (CARD). Assembled genomes were then split into 31-base long segments (31- mers), and these segments along with phenotypic antibiotic susceptibility were used as input data  for the ML algorithm. Important genomic biomarkers for four out of the six tested antibiotics were  found to have greater accuracy when predicting resistance phenotype compared to known ARGs.  The biomarker for enrofloxacin had the highest accuracy of 100% whereas the accuracy for the  12 tulathromycin biomarker was 81% but was still greater than the accuracy given by ARGs of 63%.  On the other hand, resistance genes for florfenicol and tetracycline showed greater genotype?phenotype concordance, with accuracies of 95% and 91%, respectively. Annotations to important  rulesets determined by ML were associated with clustered regularly interspaced short palindromic  repeats (CRISPR) sequences, ligases that function to recycle murein into the peptidoglycan (PDG) layer, and transferases that control the synthesis and modulation of the lipopolysaccharide (LPS).  External validation revealed that phenotypic resistance could be accurately predicted for  danofloxacin and enrofloxacin using genomic biomarkers determined by ML, and for florfenicol  using the floR gene. This study demonstrated that genomic biomarkers determined by ML can provide an accurate prediction of antibiotic resistance within Pasteurella multocida isolates.  Assays could be developed to target ML-generated biomarkers and known ARGs to predict resistance in sick animals and to limit treatment failures associated with antibiotic resistance in  cattle suffering from BRD. </p>

Bovine respiratory disease (BRD) is a highly dynamic disease complex that results from host, microbial agent, and environmental interactions. Despite nearly a century of targeted research, BRD remains the most economically damaging disease in beef cattle production and appears to be increasing in global incidence. While modern modalities for BRD detection exist, clinical diagnosis and management decisions largely depend upon clinical observations and their associated risk of disease. Though these approaches lack precision, they remain in use for many reasons, including fiscal and time constraints within beef production systems. Advancements in high-throughput sequencing have demonstrated the ability to provide insight into complex biological disorders, leading to the development of predictive biomarkers and individualized therapy. Through the use of observational research methods and previously published data, transcriptome analyses were used to capture biological information related to the host-disease or host-pathogen relationship. These studies independently elaborated findings related to host management of inflammation, ultimately being associated with both acquisition and severity of BRD. Through advances in sequencing technology and data analysis methodology, novel components related to host inflammatory mitigation and antimicrobial defense are described for clinical BRD. Factors related to increased alternative complement activation, decreased specialized proresolving lipid mediator biosynthesis, decreased antimicrobial peptide production, and increased type I interferon stimulation were associated with severe clinical BRD. These findings define molecular networks, mechanisms, and pathways that are associated with BRD outcome, and may serve as a foundation for precision medicine in beef cattle.

Bovine respiratory disease (BRD) is a significant health problem for cattle producers in terms of economic cost and animal welfare. In the United States (US), it is one of the leading causes of sickness and death in beef calves prior to weaning. Although much research has been conducted to develop vaccines for prevention and antibiotics for treatment, the morbidity and mortality of BRD in beef calves prior to weaning has not improved over the years. The identification of risk factors associated with BRD is an area of focus which might ultimately allow producers to minimize morbidity and mortality from BRD. Little research has been performed to understand factors contributing to the risk of BRD in beef calves prior to weaning. BRD affects the beef cattle industry through losses due to mortality, prevention cost, treatment cost, or morbidity effect on productivity. Currently, the economic losses due to BRD for beef calves prior to weaning is not available. Price paid for feeder cattle is a major factor influencing the income of producers. The effect of BRD is a complicated problem since the parameters associated with the cost of BRD in beef cow-calf production are variable and interrelated. To better understand the economic effect of BRD in beef calves prior to weaning, concepts of uncertainty, variability, stochasticity, nonlinearity, and feedback might be involved during the process of assessing risk. The objectives of this dissertation are the following: 1) to test if calf sex, birth weight, and age of dam are associated with BRD of beef calves prior to weaning in different age periods; 2) to identify factors affecting the national market price of beef feeder cattle in the US and how the prices change over time; 3) to investigate the prevention and treatment cost of BRD in beef calves prior to weaning; 4) to estimate the economic cost of BRD in US beef calves prior to weaning; and 5) to understand the effect of BRD occurrence or absence on the national net income of the US beef cow-calf industry.

No description available.

O complexo respiratório bovino é um dos principais problemas encontrados em bovinos tanto confinados quanto àqueles criados a pasto, levando a importantes perdas econômicas devido aos altos índices de morbidade e mortalidade. O objetivo geral deste trabalho foi determinar a prevalência de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Parainfluenza Bovino tipo-3 (PIb- 3) e Influenza vírus D e a prevalência de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (BoHV1) e o Vírus Respiratório Sincicial Bovino (VRSB) em bezerros sadios e com broncopneumonia criados nos assentamentos de Caiuá, Presidente Epitácio e Mirante de Paranapanema - SP. Além disso, objetivase avaliar a associação destes micro-organismos com a presença da broncopneumonia e dos sinais e sintomas apresentados pelos animais durante a avaliação física. Estudou-se 141 bezerros, machos e fêmeas de até um ano de idade, os quais, após exame físico, foram classificados em sadios e com pneumonia. Foram coletadas amostras de lavado traqueobrônquico e sangue total para obtenção do soro. As amostras foram utilizadas para isolamento e identificação de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida e M. haemolytica, detecção molecular de Parainfluenza Bovino tipo-3 (PIb-3) e Influenza vírus D (IVD) e detecção de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (HVBo-1) e o Vírus Respiratório Sincicial Bovino (VRSB). Não houve isolamento de P. multocida e M. haemolytica. Dentre as bactérias aeróbias isoladas, observou-se maior frequência de isolamento de Bacillus sp., Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras. M. díspar foi a única espécie de micoplasma identificada com os oligonucleotídeos iniciadores utilizados. Faz-se necessário a busca por outras espécies de micoplasmas que podem estar relacionadas aos casos de broncopneumonia em bovinos. Não houve detecção de Parainfluenza Bovino tipo 3 e Influenza vírus D nas amostras de lavado traqueobrônquico. Os três municípios estudados apresentaram anticorpos para os vírus estudados. Observou-se prevalência de BoHV-1, VDVB e VRSB de 31,7%, 24,6 e 38,8%, respectivamente. Não houve associação entre o status de saúde dos bezerros e os achados microbiológicos e sorológicos. Foi observada associação entre enterobactérias e a variável &ldquo;desidratação leve&rdquo; (p&#61;0,033/ OR&#61; 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável &ldquo;secreção nasal serosa&rdquo; (p=0,03). Concluiu-se que a broncopneumonia é uma enfermidade multifatorial. Faz-se, necessário, portanto, a associação dos achados de exames físico, microbiológicos e características do ambiente e manejo / Bovine respiratory disease complex is one of the major problems observed in feedlot and grazing cattle, causing economic losses due to high morbidity and mortality rates. The aim of this study was to determine the prevalence of M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 (bPI-3) and Influenza vírus D (IVD) and the prevalence of antibodies against Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB) in healthy and pneumonic calves raised in settlements located in Caiuá, Presidente Epitácio e Mirante de Paranapanema São Paulo State. Moreover, we aimed to evaluate the association between those microorganisms and antibodies and the presence of bronchopneumonia and the symptoms observed during the physical examination. We studied 141 males and females calves that were classified as healthy and pneumonic calves. We collected tracheobronchial lavage samples and total blood to obtain serum samples. Isolation and biochemical/ molecular identification were performed in order to detect M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 and Influenzavírus D. Serological survey was performed to detect antibodies aginst Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB). There was not any isolation of P. multocida and M. haemolytica. Bacillus sp., Staphylococcus intermedius and Non-fermenter Gram-negative bacteria were higher prevalent compared to the other aerobic bacteria isolated. M. díspar was the only mycoplasma species identified by the primers used. It is necessary the identification of other mycoplasma species that could be related to the cases of bronchopneumonia in cattle. Bovine Parainfluenza type-3 and Influenza vírus D were not identified. Antibodies were detected in all municipalities. The prevalence of BoHV, BVDV e VRSB was 31.7%, 24.6 and 38.8%, respectively. The association between health status and microbial/ serological findings was not observed. Enterobacterias were associated to &ldquo;mild dehydration&rdquo; (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and &ldquo;serous nasal discharge&rdquo; (p=0.03) was also detected. In conclusion, S. intermedius was associated to &ldquo;nasal air flow&rdquo; (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results

O complexo respiratório bovino é um dos principais problemas encontrados em bovinos tanto confinados quanto àqueles criados a pasto, levando a importantes perdas econômicas devido aos altos índices de morbidade e mortalidade. O objetivo geral deste trabalho foi determinar a prevalência de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Parainfluenza Bovino tipo-3 (PIb- 3) e Influenza vírus D e a prevalência de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (BoHV1) e o Vírus Respiratório Sincicial Bovino (VRSB) em bezerros sadios e com broncopneumonia criados nos assentamentos de Caiuá, Presidente Epitácio e Mirante de Paranapanema - SP. Além disso, objetivase avaliar a associação destes micro-organismos com a presença da broncopneumonia e dos sinais e sintomas apresentados pelos animais durante a avaliação física. Estudou-se 141 bezerros, machos e fêmeas de até um ano de idade, os quais, após exame físico, foram classificados em sadios e com pneumonia. Foram coletadas amostras de lavado traqueobrônquico e sangue total para obtenção do soro. As amostras foram utilizadas para isolamento e identificação de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida e M. haemolytica, detecção molecular de Parainfluenza Bovino tipo-3 (PIb-3) e Influenza vírus D (IVD) e detecção de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (HVBo-1) e o Vírus Respiratório Sincicial Bovino (VRSB). Não houve isolamento de P. multocida e M. haemolytica. Dentre as bactérias aeróbias isoladas, observou-se maior frequência de isolamento de Bacillus sp., Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras. M. díspar foi a única espécie de micoplasma identificada com os oligonucleotídeos iniciadores utilizados. Faz-se necessário a busca por outras espécies de micoplasmas que podem estar relacionadas aos casos de broncopneumonia em bovinos. Não houve detecção de Parainfluenza Bovino tipo 3 e Influenza vírus D nas amostras de lavado traqueobrônquico. Os três municípios estudados apresentaram anticorpos para os vírus estudados. Observou-se prevalência de BoHV-1, VDVB e VRSB de 31,7%, 24,6 e 38,8%, respectivamente. Não houve associação entre o status de saúde dos bezerros e os achados microbiológicos e sorológicos. Foi observada associação entre enterobactérias e a variável &ldquo;desidratação leve&rdquo; (p&#61;0,033/ OR&#61; 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável &ldquo;secreção nasal serosa&rdquo; (p=0,03). Concluiu-se que a broncopneumonia é uma enfermidade multifatorial. Faz-se, necessário, portanto, a associação dos achados de exames físico, microbiológicos e características do ambiente e manejo / Bovine respiratory disease complex is one of the major problems observed in feedlot and grazing cattle, causing economic losses due to high morbidity and mortality rates. The aim of this study was to determine the prevalence of M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 (bPI-3) and Influenza vírus D (IVD) and the prevalence of antibodies against Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB) in healthy and pneumonic calves raised in settlements located in Caiuá, Presidente Epitácio e Mirante de Paranapanema São Paulo State. Moreover, we aimed to evaluate the association between those microorganisms and antibodies and the presence of bronchopneumonia and the symptoms observed during the physical examination. We studied 141 males and females calves that were classified as healthy and pneumonic calves. We collected tracheobronchial lavage samples and total blood to obtain serum samples. Isolation and biochemical/ molecular identification were performed in order to detect M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 and Influenzavírus D. Serological survey was performed to detect antibodies aginst Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB). There was not any isolation of P. multocida and M. haemolytica. Bacillus sp., Staphylococcus intermedius and Non-fermenter Gram-negative bacteria were higher prevalent compared to the other aerobic bacteria isolated. M. díspar was the only mycoplasma species identified by the primers used. It is necessary the identification of other mycoplasma species that could be related to the cases of bronchopneumonia in cattle. Bovine Parainfluenza type-3 and Influenza vírus D were not identified. Antibodies were detected in all municipalities. The prevalence of BoHV, BVDV e VRSB was 31.7%, 24.6 and 38.8%, respectively. The association between health status and microbial/ serological findings was not observed. Enterobacterias were associated to &ldquo;mild dehydration&rdquo; (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and &ldquo;serous nasal discharge&rdquo; (p=0.03) was also detected. In conclusion, S. intermedius was associated to &ldquo;nasal air flow&rdquo; (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results