application-crispr-interference-crispri-for-gene-silencing-pathogenic

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of <em>Leptospira</em>

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

United States Department of Agriculture

Bovine digital dermatitis (BDD) is a multifactorial polymicrobial infectious disease associated with multiple species and phylotypes of treponemes. However, despite the abundance of molecular signatures for treponemes that are identified in bovine lesions, relatively few isolates are cultured, and even fewer have been characterized at the level of protein expression. Here we report the successful isolation and characterization of novel strains of T. brennaborense and T. phagedenis from cases of BDD in Iowa dairy cows, and compare them to a well characterized strain of T. phagedenis, and the type strain of the more recently recognized T. pedis. Propagation of T. brennaborense was only possible at room temperature in Cooked Meat Medium, and not in oral treponeme enrichment medium at 37 °C as used for T. phagedenis and T. pedis. A prominent and rapid motility is observed by T. brennaborense under dark-field microscopy. The highly motile T. brennaborense strain 11-3 has an identical enzymatic profile to that of the only other isolate of T. brennaborense to be cultured from a lesion of BDD. Outer membrane protein profiles of each strain were compared by 2-D gel electrophoresis, and the five most abundant proteins in each strain were identified by mass spectrometry. All identified proteins are predicted to have signal peptides. Results identified outer membrane proteins specific to each strain including predicted membrane lipoproteins, ABC transporters and, as yet, uncharacterized proteins. Collectively, our results provide for the identification and characterization of outer membrane components of multiple phylotypes of treponemes associated with BDD which can facilitate development of vaccines and diagnostics in our efforts to eradicate the disease.

Phenotypic and proteomic characterization of treponemes associated with bovine digital dermatitis.

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

J. E. Nally

Infectious Bacterial Diseases Research Unit,
National Animal Disease Center,
Agricultural Research Service,
Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service

United States Department of Agriculture

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

J. E. Nally

Infectious Bacterial Diseases Research Unit,National Animal Disease Center,Agricultural Research Service,Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service

United States Department of Agriculture

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Infectious Bacterial Diseases Research Unit,
National Animal Disease Center,
Agricultural Research Service,
Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service