This study was undertaken to compare the MIC (minimum inhibitory concentration) and MPC (mutant prevention concentration) values for oxytetracycline and florfenicol against strains of Pasteurella multocida isolated from cattle and pigs, and for enrofloxacin against strains of Salmonella Typhimurium isolated from horses. Isolates of P. multocida from cattle and pigs, and S. Typhimurium from horses were obtained from specimens or isolates from contributing laboratories. All the equine isolates and 50% of the cattle and pig isolates were from clinically sick animals. All isolates were tested in duplicate with both the MIC and the MPC methods. The MIC method used was the standardized microdilution method performed in microtitre plates. The MPC method used was according to the method described by Blondeau. This method was modified, to make use of smaller plates and lower volumes of antimicrobials, but retaining a final bacterial concentration of 109 colony-forming units per ml. The antimicrobials were dissolved as described in the certificates of analyses. Enrofloxacin and oxytetracycline were dissolved in water, and florfenicol was dissolved in alcohol. For the MPC method, an additional control was added to one quadrant of a four-quadrant 90mm plate/petri dish. The antimicrobials were tested as individual antimicrobials and not as combinations. Both the MIC and MPC methods included ATCC (American Type Culture Collection) strains as control organisms and were evaluated according to the guidelines of the CLSI (Clinical and Laboratory Standards Institute). The MIC50 values for enrofloxacin against Salmonella Typhimurium isolates from horses was 0.25 ìg/ml and the MPC50 values 0.5 ìg/ml. A comparative reference range was not available as enrofloxacin is not registered in South Africa for use in horses, and is used extra-labelly. The results for florfenicol against P. multocida yielded an MIC50 value of 0.5 ìg/ml and an MPC50 value of <2 ìg/ml. The close relationship of these two concentrations is an indication of the effectiveness of florfenicol when used against P. multocida. The PD/PK data with a value of 141.78 for AUC/MIC provided additional support for the efficacy of florfenicol against P. multocida. The PD/PK value of >125, is an effective parameter for treatment of Gram-negative bacteria. The corresponding results for oxytetracycline were above the MIC value but fell within the mutant selection window. The results point to the fact that the use of oxytetracycline against P. multocida may not be effective in preventing the appearance of first step mutant strains when used at current recommended dosages. The PK/PD data, using AUC/MIC, yielded a value of 56. Some of the isolates (55.17%) had an MPC value of 16 ìg/ml. Whereas the MIC method is used routinely in diagnostic laboratories, the MPC method can be employed to generate data that can be applied where antimicrobial treatment of certain bacteria is problematic and standard treatment may lead to the development of resistance. Data obtained from such studies will enable manufacturers of antimicrobial drugs to adapt antimicrobial therapy where practical and feasible to prevent the development of first step mutants. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Über eine lange Zeit galt die Merzung der gesamten Herde und die Repopulation aus von toxinogenen Pasteurella multocida (Pmt) freien Herkünften als der einzig sichere Weg zur Eradikation des Erregers und so zur Verhinderung klinischer Erkrankungen der Progressiven Rhinitis atrophicans (PRa). Die Selektion infizierter Tiere schien nur in seltenen Ausnahmefällen erfolgreich zu verlaufen. In früheren Versuchen (GEIGER et al. 1992, DE JONG et al. 1994, ALT et al. 1996, SCHNÜLL 2001) wurde ein Sanierungserfolg in der Regel in kleinen, manchmal akut infizierten Herden mit weniger als 100 Sauen und / oder durch die Kombination der selektiven Diagnostik mit organisatorischen, medikamentösen und / oder immunprophylaktischen Maßnahmen erreicht. Im Unterschied zu diesen Beispielen wurde hier versucht, die Keime aus einer latent infizierten, geschlossenen Zuchtherde mit etwa 500 produktiven Sauen und Eigenremontierung an nur einem Standort ohne ein Frühabsetzen, Medikation oder Vakzinierung bei laufender Produktion allein durch die Selektion der Keimträger zu eradikieren. Der Bestand, der Mastläufer, Jungsauen für die Eigenremontierung und Zuchteberferkel für eine Besamungseberstation produziert, ist in 4 Stalleinheiten (Belegstall, Wartestall, Abferkel- und Absetzläuferstall, Jungsauenaufzuchtstall) untergebracht. Anlass für den Versuch waren klinische Erscheinungen der PRa bei gelieferten Mastläufern. In der Untersuchungsherde selbst aber traten nie klinische Symptome der Krankheit auf. Der Versuch lief in 4 zeitlichen Phasen ab: 1. Nachweis der Pmt, 2. Reagentenselektion nach gruppenweiser serologischer und bakteriologischer Untersuchung im Enzym linked immunosorbent assay (ELISA), 3. Selektion der Keimträger nach bakteriologischen Gesamtbestandsuntersuchungen durch Nasen- und Rachentupferproben mit der Polymerase-Kettenreaktion (PCR) und 4. Kontrolle und Überwachung des Sanierungserfolgs. Zu Beginn wurden eine serologische Prävalenz von 4,8 % und eine bakteriologische Nachweishäufigkeit von 7,7 % im ELISA ermittelt. Nach einer Erregerreduzierung betrug der Anteil der Pmt-Nachweise mit der PCR anfangs noch 6,2 %. Durch die Selektion infizierter Tiere konnte der Erreger aus der Herde eliminiert werden, was regelmäßige klinische, bakteriologische und serologische Kontrollen bestätigten. Im eigenen Mastbestand wurden keine Symptome der PRa beobachtet. Aus dem Versuch ist zu schlussfolgern: Die Sanierung größerer Zuchtherden auf dem beschriebenen Weg ist möglich. Neben guten Umwelt- und arbeitsorganisatorischen Bedingungen sind dazu ein hoch sensitives Verfahrens zur Erkennung der infizierten Tiere und kurze Untersuchungsperioden notwendig. Zur Diagnostik eignen sich die Entnahme von Nasen- und Rachentupferproben und ihre Untersuchungen in der PCR. Der Sanierungserfolg kann nur nach einer längeren Periode (ca. 2 Jahre) regelmäßiger klinischer, bakteriologischer und serologischer Kontrollen als sicher betrachtet werden.

Bovine Respiratory Disease Complex is a multifactorial disease affecting cattle worldwide resulting in high mortality and morbidity rates in the cattle farming industry. This complex is caused by multiple viral and bacterial pathogens such as Bovine Herpesvirus-1, Bovine Respiratory Syncytial Virus, Mannheimia haemolytica, and Pasteurella multocida; two of the main contributors to the initiation of this disease are Bovine Herpesvirus-1 and the bacteria, Mannheimia haemolytica. Together, these microbes co-infect immunocompromised cattle during times of increased stress and induce a severe pneumonic response along with other health complications. Research has been primarily focused on these microorganisms individually or their effect on the host, however there is a need to study them together due to the increased mortality rate associated with co-infections. In this study, we used Bovine Herpesvirus-1, Mannheimia haemolytica, Pasteurella multocida, and Bovine Respiratory Syncytial Virus to co-infect bovine tissue cultures to determine how they affect each other.

<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>

To identify antigens unique to live Pasteurella multocida P1059, 10 week old specific pathogen-free (SPF) chickens were vaccinated three times with one of the following: viable cells from P. multocida P1059, 3865, 3866, or cells from formalin-killed strain PI059 or formalin-killed strain P1059 that were opsonized with antiserum directed against killed strain PI059 prior to immunization. Vaccinated birds were challenged with 1.5 x 10⁷ CFU of live strain P1059. Eight, 71, 86, and 50% of the birds that received live strains P1059, 3865, 3866 and killed strain P1059 (respectively), exhibited clinical signs of fowl cholera. Antisera directed against live strain PI059 recognized 23 proteins ranging from 14- to 92-kilodaltons (kDa); 20 of which were adsorbed by strain 3865. The molecular masses of the three remaining proteins were 25-, 30- and 43-kDa. A genomic library of strain P1059 was constructed using the plasmid vector pUC-19 and screened with antisera against live strain P1059; 12 out of 4,100 clones were recognized. The inserts of the plasmids from these clones ranged from 0.48- to 6.S-kilobases (kb) in length. Five of the 12 clones expressed proteins with molecular masses of 34-, 37-, 42-, 46- and 55-kDa. Escherichia coli CSR603(pOP43- 2G) and CSR603(pOP33-SF) expressed proteins recognized by antisera directed against live strain P1059. E. coli CSR603(pOP43-2G) expressed an epitope(s) which was recognized by antisera directed against strains 3865 and 3866. Conditions for transformation were optimized and attempts were made to create a shuttle vector in order to establish a cloning system for gene expression in P. multocida. The highest efficiency of transformation (1.25 x 10⁷ CFU/μg DNA) was obtained when 7.6 x 10¹⁰ cells of P. multocida R473 were electroporated at 12.5 kV cm⁻¹ for 10 ms with 5 ng of the plasmid, p VM109. Of the six strains tested, representing serogroups A, B, D and E, all were transformed successfully. Vectors including pBR322, pUC19, pJFF224-NX and pSP329 were unable to transform P. multocida. To create a shuttle vector for gene expression in P. multocida, a Pasteurella plasmid (pLAR-1) was cloned in both orientations into the BamH I site of pBR322. These plasmids, pLRBR-21 and pLRBR-67, had a transformation efficiency of 4.5 to 8 x 10⁴ CFU/μg of DNA in strain R473. Chromosomal DNA containing the Brucella abortus copper-zinc superoxide dismutase gene was cloned into the Cla I site of pLRBR-21. The 1.8-kb fragment encoding a 42-kDa Pasteurella protein was cloned into an additional unique site (Nru 1) of pLRBR-21 to determine if this plasmid was a viable shuttle vector for gene expression in P. multocida. / Ph. D.

Abstract not available

The scope and application of high throughput techniques has expanded from studying a single genome, transcriptome or proteome to understanding complex environments at a greater resolution with the help of novel computational frameworks. Comprehensive structural annotation i.e. description of all functional elements in the genome, is required for measuring genome response accurately, using high throughput methods. Annotation of genome sequences using high throughput data from RNA-seq and proteomics experiments complement computational methods for identifying functional elements and can help validate existing in silico annotation, correct annotation errors, and could potentially identify novel functional elements. Re-annotation studies in recent times have revealed shortcomings of automated methods and the necessity to validate existing annotations using experimental data. This dissertation elucidates re-annotation of Mannheimia haemolytica, Pasteurella multocida and Histophilus somni, bacterial pathogens associated with bovine respiratory disease in cattle. Experimental re-annotation of these bacterial genomes using RNA-seq and proteomics enabled the validation of existing annotation and discovery of novel functional elements that can be utilized in future functional genomics studies. We also addressed the need for developing an automated bioinformatics workflow that is broadly applicable for bacterial genome re-annotation, by developing open source Perl pipeline that can use RNA-seq and proteomics data as input. Simultaneous analysis of host and pathogen gene expression profiling using metatranscriptomics approaches is necessary to improve our understanding of infectious diseases. Traditional methods for analysis of RNA-seq data do not address the impact of cross-mapping of reads to multiple genomes for data originating from a metatranscriptomic study. Analysis of sequence conservation between species can help determine a metric for cross mapping to correct for signal vs. noise. We generated artificial RNA-seq data and evaluated the impact of read length and sequence conservation on cross-mapping. Comparative genomics was used to identify a core and pan-genome for quantifying gene expression. Our results show that cross mapping between genomes can directly be related to evolutionary distance between these genomes and that an increase in RNA-seq read length tends to negate cross mapping.

Avian cholera, caused by infection with Pasteurella multocida, is an important infectious disease of wild birds in North America Since it was first confirmed in 2005, annual outbreaks of avian cholera have had a dramatic effect on common eiders on East Bay Island, Nunavut, one of the largest breeding colonies of northern common eiders (Somateria mollissima borealis) in the eastern Arctic. I investigated potential avian and environmental reservoirs of P. multocida on East Bay Island and other locations in the eastern Canadian Arctic by collecting cloacal and oral swabs from live or harvested, apparently healthy, common eiders, lesser snow geese, Ross’s geese, king eiders, herring gulls, and snow buntings. Water and sediment from ponds on East Bay Island were sampled before and during outbreaks. Avian and environmental samples were tested using a real-time polymerase chain reaction (PCR) assay to detect P. multocida. PCR positive birds were found in every species except for snow buntings, and PCR positive common eiders were found in most locations, supporting the hypothesis that apparently healthy wild birds can act as a reservoir for avian cholera. In all years, P. multocida DNA was detected in ponds both before and after the avian cholera outbreak began each year, suggesting that the environment also plays a role in outbreak dynamics. Contrary to our expectations, model results revealed that ponds were generally more likely to be positive earlier in the season, before the outbreaks began. Whereas average air temperature at the beginning of the breeding season was not an important predictor for detecting P. multocida in ponds, eiders were more likely to be PCR positive under cooler conditions, pointing to an important link between disease and weather. Potential origins of P. multocida causing avian cholera in Arctic eider colonies were investigated by comparing eastern Arctic isolates of P. multocida to isolates from wild birds across Canada, and the central flyway in the United States. Using repetitive extragenic palindromic-PCR (REP-PCR) and multi-locus sequence typing (MLST), we detected a low degree of genetic diversity among isolates, and P. multocida genotypes were correlated with somatic serotype. Isolates from East Bay Island were distinct from P. multocida from eider colonies in the St. Lawrence Estuary, Quebec, however, East Bay Island isolates were indistinguishable from isolates collected from a 2007 pelagic avian cholera outbreak on the east coast of Canada. Isolates from East Bay Island and Nunavik shared sequence types, indicating possible transmission of isolates among eider colonies in the eastern Arctic. Previously, feather corticosterone in eiders was found to be significantly associated with environmental temperature during the moulting period. In my study, path analysis revealed that environmental conditions experienced during the moulting period had direct impacts on arrival date and pre-breeding body condition of common eiders during the subsequent breeding period on East Bay Island, with indirect impacts on both reproductive success and survival. Higher temperatures experienced during the fall moulting period appear to impose significant costs to eiders, with subsequent carry-over effects on both survival and reproduction many months later during avian cholera outbreaks. This thesis describes several important features of the host, agent and environmental dynamics of avian cholera in North America with a particular focus on the disease in the eastern Canadian Arctic. Continued exploration of infectious wildlife disease dynamics is needed to better predict, detect, manage, and mitigate disease emergence that can threaten human and animal health and species conservation.