Cassidy L. Klima

Effect of subtherapeutic vs. therapeutic administration of macrolides on antimicrobial resistance in Mannheimia haemolytica and enterococci isolated from beef cattle.

Macrolides are the first-line treatment against bovine respiratory disease (BRD), and are also used to treat infections in humans. The macrolide, tylosin phosphate, is often included in the diet of cattle as a preventative for liver abscesses in many regions of the world outside of Europe. This study investigated the effects of administering macrolides to beef cattle either systemically through a single subcutaneous injection (therapeutic) or continuously in-feed (subtherapeutic), on the prevalence and antimicrobial resistance of Mannheimia haemolytica and Enterococcus spp. isolated from the nasopharynx and faeces, respectively. Nasopharyngeal and faecal samples were collected weekly over 28 days from untreated beef steers and from steers injected once with tilmicosin or tulathromycin or continuously fed tylosin phosphate at dosages recommended by manufacturers. Tilmicosin and tulathromycin were effective in lowering (P < 0.05) the prevalence of M. haemolytica, whereas subtherapeutic tylosin had no effect. M. haemolytica isolated from control- and macrolide-treated animals were susceptible to macrolides as well as to other antibiotics. Major bacteria co-isolated with M. haemolytica from the nasopharynx included Pasteurella multocida, Staphylococcus spp., Acinetobacter spp., Escherichia coli and Bacillus spp. With the exception of M. haemolytica and P. multocida, erythromycin resistance was frequently found in other isolated species. Both methods of macrolide administration increased (P < 0.05) the proportion of erythromycin resistant enterococci within the population, which was comprised almost exclusively of Enterococcus hirae. Injectable macrolides impacted both respiratory and enteric microbes, whereas orally administered macrolides only influenced enteric bacteria.

Bringing plant-based veterinary vaccines to market: Managing regulatory and commercial hurdles.

The production of recombinant vaccines in plants may help to reduce the burden of veterinary diseases, which cause major economic losses and in some cases can affect human health. While there is abundant research in this area, a knowledge gap exists between the ability to create and evaluate plant-based products in the laboratory, and the ability to take these products on a path to commercialization. The current report, arising from a workshop sponsored by an Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme, addresses this gap by providing guidance in planning for the commercialization of plant-made vaccines for animal use. It includes relevant information on developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval with a focus on Canadian regulations.

Susceptibility to tulathromycin in Mannheimia haemolytica isolated from feedlot cattle over a 3-year period

Mannheimia haemolytica isolated from feedlot cattle were tested for tulathromycin resistance. Cattle were sampled over a 3-year period, starting 12 months after approval of tulathromycin for prevention and treatment of bovine respiratory disease. Nasopharyngeal samples from approximately 5,814 cattle were collected when cattle entered feedlots (N = 4) and again from the same cattle after ≥60 days on feed. The antimicrobial use history for each animal was recorded. Mannheimia haemolytica was isolated from 796 (13.7%) entry samples and 1,038 (20.6%) ≥ 60 days samples. Of the cattle positive for M. haemolytica, 18.5, 2.9, and 2.4% were administered therapeutic concentrations of tulathromycin, tilmicosin, or tylosin tartrate, respectively. In addition, 13.2% were administered subtherapeutic concentrations of tylosin phosphate in feed. In years one and two, no tulathromycin-resistant M. haemolytica were detected, whereas five isolates (0.4%) were resistant in year three. These resistant isolates were collected from three cattle originating from a single pen, were all serotype 1, and were genetically related (≥89% similarity) according to pulsed-field gel electrophoreses patterns. The five tulathromycin-resistant isolates were multi-drug resistant also exhibiting resistance to oxytetracycline, tilmicosin, ampicillin, or penicillin. The macrolide resistance genes erm(42), erm(A), erm(B), erm(F), erm(X) and msr(E)-mph(E), were not detected in the tulathromycin-resistant M. haemolytica. This study showed that tulathromycin resistance in M. haemolytica from a general population of feedlot cattle in western Canada was low and did not change over a 3-year period after tulathromycin was approved for use in cattle.

Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources

Bovine respiratory disease is a common health problem in beef production. The primary bacterial agent involved, Mannheimia haemolytica, is a target for antimicrobial therapy and at risk for associated antimicrobial resistance development. The role of M. haemolytica in pathogenesis is linked to serotype with serotypes 1 (S1) and 6 (S6) isolated from pneumonic lesions and serotype 2 (S2) found in the upper respiratory tract of healthy animals. Here, we sequenced the genomes of 11 strains of M. haemolytica, representing all three serotypes and performed comparative genomics analysis to identify genetic features that may contribute to pathogenesis. Possible virulence associated genes were identified within 14 distinct prophage, including a periplasmic chaperone, a lipoprotein, peptidoglycan glycosyltransferase and a stress response protein. Prophage content ranged from 2–8 per genome, but was higher in S1 and S6 strains. A type I-C CRISPR-Cas system was identified in each strain with spacer diversity and organization conserved among serotypes. The majority of spacers occur in S1 and S6 strains and originate from phage suggesting that serotypes 1 and 6 may be more resistant to phage predation. However, two spacers complementary to the host chromosome targeting a UDP-N-acetylglucosamine 2-epimerase and a glycosyl transferases group 1 gene are present in S1 and S6 strains only indicating these serotypes may employ CRISPR-Cas to regulate gene expression to avoid host immune responses or enhance adhesion during infection. Integrative conjugative elements are present in nine of the eleven genomes. Three of these harbor extensive multi-drug resistance cassettes encoding resistance against the majority of drugs used to combat infection in beef cattle, including macrolides and tetracyclines used in human medicine. The findings here identify key features that are likely contributing to serotype related pathogenesis and specific targets for vaccine design intended to reduce the dependency on antibiotics to treat respiratory infection in cattle.

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity.

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

In silico identification and high throughput screening of antigenic proteins as candidates for a Mannheimia haemolytica vaccine

This study examined the use of comparative genomic analysis for vaccine design against Mannheimia haemolytica, a respiratory pathogen of ruminants. A total of 2,341genes were identified in at least half of the 23 genomes. Of these, a total of 240 were identified to code for N-terminal signal peptides with diverse sub-cellular localizations (78 periplasmic, 52 outer membrane, 15 extracellular, 13 cytoplasmic membrane and 82 unknown) and were examined in an ELISA assay using a coupled-cell free transcription/translation system for protein expressionwith antisera from cattle challenged with serovars 1, 2 or 6 of M. haemolytica. In total, 186 proteins were immunoreactive to at least one sera type and of these, 105 were immunoreactive to all sera screened. The top ten antigens based on immunoreactivity were serine protease Ssa-1 (AC570_10970), an ABC dipeptid transporter substrate-binding protein (AC570_04010), a ribonucleotide reductase (AC570_10780), competence protein ComE (AC570_11510), a filamentous hemagglutinin (AC570_01600), a molybdenum ABC transporter solute-binding protein (AC570_10275), a conserved hypothetical protein (AC570_07570), a porin protein (AC569_05045), an outer membrane assembly protein YeaT (AC570_03060), and an ABC transporter maltose binding protein MalE (AC570_00140). The framework generated from this research can be further applied towards rapid vaccine design against other pathogens involved in complex respiratory infections in cattle.

Antimicrobial Susceptibility of Bacteria That Cause Bovine Respiratory Disease Complex in Alberta, Canada

Bovine respiratory disease (BRD) is the most important illness of feedlot cattle. Disease management targets the associated bacterial pathogens, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida, Histophilus somni, and Trueperella pyogenes. We conducted a cross-sectional study to measure the frequencies of antimicrobial-resistant BRD pathogens using a collaborative network of veterinarians, industry, government, and a diagnostic laboratory. Seven private veterinary practices in southern Alberta collected samples from both living and dead BRD-affected animals at commercial feedlots. Susceptibility testing of 745 isolates showed that 100% of the M. haemolytica, M. bovis, P. multocida, and T. pyogenes isolates and 66.7% of the H. somni isolates were resistant to at least one antimicrobial class. Resistance to macrolide antimicrobials (90.2% of all isolates) was notable for their importance to beef production and human medicine. Multidrug resistance (MDR) was high in all target pathogens with 47.2% of the isolates resistant to four or five antimicrobial classes and 24.0% resistance to six to nine classes. We compared the MDR profiles of isolates from two feedlots serviced by different veterinary practices. Differences in the average number of resistant classes were found for M. haemolytica (p < 0.001) and P. multocida (p = 0.002). Compared to previous studies, this study suggests an increasing trend of resistance in BRD pathogens against the antimicrobials used to manage the disease in Alberta. For the veterinary clinician, the results emphasize the importance of ongoing susceptibility testing of BRD pathogens to inform treatment protocols. Surveillance studies that collect additional epidemiological information and manage sampling bias will be necessary to develop strategies to limit the spread of resistance.

A Novel aadA Aminoglycoside Resistance Gene in Bovine and Porcine Pathogens

Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species. ABSTRACT A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni. The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICEMh1, a mobile genetic element transmissible among members of the family Pasteurellaceae. The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.

A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6

Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.

Genomic approaches to characterizing and reducing antimicrobial resistance in beef cattle production systems

Abstract Antimicrobial resistance (AMR) is a global health threat, and a standstill in the discovery and design of new antibiotics has been linked to the growing number of human deaths attributed to AMR infections. Intensive beef production utilizes antimicrobials to promote health and growth efficiency. To understand the magnitude and risk of AMR in beef production, it is important to assess the prevalence and diversity of antimicrobial resistant genes (ARGs) within microbial populations. Antimicrobial resistant bacteria are traditionally identified by isolation and growth in the presence of selective antibiotics. Whole-genome, metagenomic, and RNA sequencing provide new avenues to detect and identify novel ARGs in both culturable and unculturable bacterial communities. Some of these approaches place ARGs within the context of mobile genetic elements, gauging their likelihood of transfer across genomes. Genomics can also mitigate AMR, contributing to rational drug design or the development of alternatives to antimicrobials such as vaccines and probiotics. RNA-seq-based transcriptomics and Tn-seq may provide new ways to examine mechanisms that promote or prevent AMR. Finally, clustered regularly interspaced short palindromic repeats (CRISPR) – Cas gene editing could directly reduce AMR by killing AMR-resistant bacteria without harming beneficial bacteria. Together, these technologies may provide new opportunities to identify, quantify, and mitigate AMR while developing alternatives to antimicrobials for beef production.