Rapid Identification of Scrapie-Infected Animals

Author Institution: Food Animal Health Research Program, The Ohio State Universit

Comparative Pathogenomics of Epidemiologically and Genetically Diverse Strains of Mycobacterium avium subspecies paratuberculosis

Author Institution: Food Animal Health Research Program, The Ohio State Universit

Cold Cas: reevaluating the occurrence of CRISPR/Cas systems in Mycobacteriaceae

Bacterial CRISPR/Cas systems target foreign genetic elements such as phages and regulate gene expression by some pathogens, even in the host. The system is a marker for evolutionary history and has been used for inferences in Mycobacterium tuberculosis for 30 years. However, knowledge about mycobacterial CRISPR/Cas systems remains limited. It is believed that Type III-A Cas systems are exclusive to Mycobacterium canettii and the M. tuberculosis complex (MTBC) of organisms and that very few of the >200 diverse species of non-tuberculous mycobacteria (NTM) possess any CRISPR/Cas system. This study sought unreported CRISPR/Cas loci across NTM to better understand mycobacterial evolution, particularly in species phylogenetically near the MTBC. An analysis of available mycobacterial genomes revealed that Cas systems are widespread across Mycobacteriaceae and that some species contain multiple types. The phylogeny of Cas loci shows scattered presence in many NTM, with variation even within species, suggesting gains/losses of these loci occur frequently. Cas Type III-A systems were identified in pathogenic Mycobacterium heckeshornense and the geological environmental isolate Mycobacterium SM1. In summary, mycobacterial CRISPR/Cas systems are numerous, Type III-A systems are unreliable as markers for MTBC evolution, and mycobacterial horizontal gene transfer appears to be a frequent source of genetic variation

Moraxella osloensis Gene Expression in the Slug Host Deroceras reticulatum

<p>Abstract</p> <p>Background</p> <p>The bacterium <it>Moraxella osloensis </it>is a mutualistic symbiont of the slug-parasitic nematode <it>Phasmarhabditis hermaphrodita</it>. In nature, <it>P. hermaphrodita </it>vectors <it>M. osloensis </it>into the shell cavity of the slug host <it>Deroceras reticulatum </it>in which the bacteria multiply and kill the slug. As <it>M. osloensis </it>is the main killing agent, genes expressed by <it>M. osloensis </it>in the slug are likely to play important roles in virulence. Studies on pathogenic interactions between bacteria and lower order hosts are few, but such studies have the potential to shed light on the evolution of bacterial virulence. Therefore, we investigated such an interaction by determining gene expression of <it>M. osloensis </it>in its slug host <it>D. reticulatum </it>by selectively capturing transcribed sequences.</p> <p>Results</p> <p>Thirteen <it>M. osloensis </it>genes were identified to be up-regulated post infection in <it>D. reticulatum</it>. Compared to the <it>in vitro </it>expressed genes in the stationary phase, we found that genes of ubiquinone synthetase (<it>ubiS</it>) and acyl-coA synthetase (<it>acs</it>) were up-regulated in both <it>D. reticulatum </it>and stationary phase <it>in vitro </it>cultures, but the remaining 11 genes were exclusively expressed in <it>D. reticulatum </it>and are hence infection specific. Mutational analysis on genes of protein-disulfide isomerase (<it>dsbC</it>) and <it>ubiS </it>showed that the virulence of both mutants to slugs was markedly reduced and could be complemented. Further, compared to the growth rate of wild-type <it>M. osloensis</it>, the <it>dsbC </it>and <it>ubiS </it>mutants showed normal and reduced growth rate <it>in vitro</it>, respectively.</p> <p>Conclusion</p> <p>We conclude that 11 out of the 13 up-regulated <it>M. osloensis </it>genes are infection specific. Distribution of these identified genes in various bacterial pathogens indicates that the virulence genes are conserved among different pathogen-host interactions. Mutagenesis, growth rate and virulence bioassays further confirmed that <it>ubiS </it>and <it>dsbC </it>genes play important roles in <it>M. osloensis </it>survival and virulence, respectively in <it>D. reticulatum</it>.</p

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system. RESULTS: Intracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFα at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFα compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565. CONCLUSION: Taken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease

Genomic characterization of Staphylococcus aureus at the swine-human interface

The epidemiology of S. aureus in swine held little interest until the ST398 lineage of MRSA was found to be prevalent in pigs and pig farmers in the Netherlands in 2004 (Voss et al. 2005). ST398 MRSA have since been detected in multiple livestock species and in many countries (EFSA, 2009; Smith and Pearson, 2011), while genetically distinct variants of ST398 S. aureus occur in some human populations independent of livestock reservoirs (Carrel et al., 2017). Furthermore, other genotypes of MRSA can occur in pigs, particularly ST9 MRSA in Asia, and ST5 MRSA in North America (Chuang and Huang, 2015; Frana et al. 2013). In the USA, methicillin susceptible variants of the ST398, ST9 and ST5 lineages are widespread in commercial swine, yet MRSA variants appear to occur at relatively low prevalence (Sun, et al., 2015). Despite common exposure to, and colonization of, swine workers by livestock associated S. aureus, significant clinical infections appear to be uncommon in occupationally exposed people. However, invasive and even fatal infections are reported at relatively low incidence in some countries, and medically compromised people appear to be at particular risk, even in the absence of animal contact (Larsen et al., 2017). There is evidence that ST398 MRSA of livestock origin are less transmissible among humans than MRSA of human origin. Also, genomic studies typically have indicated that livestock associated MRSA (both ST398 and ST5) lack most virulence factors that occur in human clinical isolates (Schijffelen et al. 2010; Price et al. 2012; Hau et al, 2015). However, to date there has been little genomic characterization of methicillin susceptible S. aureus (MSSA) that are prevalent in swine populations. The purpose of this study was to describe the occurrence of virulence factors and antibiotic resistance genes in S. aureus isolates from pigs and swine veterinarians in the USA

Elucidating the Regulon of a Fur-like Protein in \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e (MAP)

Intracellular iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis, and oxidative stress. A Mycobacterium avium subsp. paratuberculosis (MAP)-specific genomic island carries a putative metal transport operon that includes MAP3773c, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in MAP is unknown as this organism also carries IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism, and virulence that were likely regulated by MAP3773c. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of MAP3773c (Fur-like protein) in MAP. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron-replete and -deplete conditions, located mostly within open reading frames (ORFs). Three ChIP peaks were identified in genes that are directly related to iron regulation: MAP3638c (hemophore-like protein), MAP3736c (Fur box), and MAP3776c (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn2+ availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that MAP3773c is a Fur ortholog that recognizes a 19 bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of MAP Fur binding sites during iron-replete and -deplete conditions, highlighting unique properties of Fur regulon in MAP

Comparative genomic analysis of Mycobacterium avium subspecies obtained from multiple host species

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>(<it>M. avium</it>) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.</p> <p>Results</p> <p>A comparative genomic approach was used to identify large sequence polymorphisms among <it>M. avium </it>subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate <it>M. avium </it>subsp. <it>paratuberculosis </it>(MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. <it>M. avium </it>subsp. <it>silvaticum </it>isolates were observed to have a hybridization profile very similar to yet distinguishable from <it>M. avium </it>subsp. <it>avium</it>. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.</p> <p>Conclusion</p> <p>Genome diversity in <it>M. avium </it>subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of <it>M. avium </it>were distinguishable by the presence or absence of specific polymorphisms.</p