Application of a CRISPR Sequence-Based Method for a Large-Scale Assessment of Salmonella Serovars in Ontario Poultry Production Environments.

Accurate detection of all Salmonella serovars present in a sample is important in surveillance programs. Current detection protocols are limited to detection of a predominant serovar, missing identification of less abundant serovars in a sample. An alternative method, called CRISPR-SeroSeq, serotyping by sequencing of amplified CRISPR spacers, was employed to detect multiple serovars in a sample without the need of culture isolation. The CRISPR-SeroSeq method successfully detected 34 most frequently reported Salmonella serovars in pure cultures and target serovars at 104 CFU/mL in 27 Salmonella-negative environmental enrichment samples post-spiked with one of 15 different serovars, plus 2 additional serovars at 1 log CFU/mL higher abundance. When the method was applied to 442 naturally contaminated environmental samples collected from 192 poultry farms, 25 different serovars were detected from 430 of the samples. In 73.1% of the samples, 2 to 7 serovars were detected, with Salmonella Kiambu (55.7%), Salmonella Infantis (48.4%), Salmonella Kentucky (27.1%), Salmonella Livingstone (26.6%), and Salmonella Mbandaka/Montevideo (23.4%) being the most prevalent on the farms. Single isolates from 384 samples were also analyzed using a traditional serotyping method, and the same serovar identified by culture was detected by CRISPR-SeroSeq in 96.1% (369/384) of samples, with the former missing detection of additional and sometimes critical serovars. The surveillance data obtained via CRISPR-SeroSeq revealed a significant emergence of Salmonella Kiambu and Salmonella Rissen on poultry farms in Ontario. The results highlight the effectiveness of the CRISPR-SeroSeq approach in detecting multiple Salmonella serovars in poultry environmental samples under applied conditions, providing updated surveillance information on Salmonella serovars on poultry farms in Ontario. IMPORTANCE The CRISPR-SeroSeq method represents an alternative molecular tool to the traditional culture-based serotyping method that can detect multiple Salmonella serovars in a sample and provide rapid serovar results without the need of selective enrichment and culture isolation. The evaluation results can facilitate implementation of the method in routine Salmonella surveillance on poultry farms and in outbreak investigations. The application of the method can increase the accuracy of current serovar prevalence information. The results highlight the effectiveness of the validated method and the need for monitoring Salmonella serovars in poultry environments to improve current surveillance programs. The updated surveillance data provide timely information on emergence of different Salmonella serovars on poultry farms in Ontario and support on-farm risk assessment and risk management of Salmonella.

A comparative study of hearing aids and round window application of the vibrant sound bridge (VSB) for patients with mixed or conductive hearing loss.

OBJECTIVE: This study was undertaken to determine the efficacy of the round window (RW) application of the vibrant soundbridge (VSB) in patients with mixed or conductive hearing loss. DESIGN: Speech in quiet and in noise were compared to preoperative data attained with conventional hearing aids so that each subject served as his or her own control in a single test protocol. STUDY SAMPLE: Eighteen adults implanted monaurally with the VSB in the poorer hearing ear. Experience with the VSB ranged from nine to 25 months. RESULTS: Sixteen of the 18 subjects were successful VSB users, wearing their device all waking hours. There was no significant deterioration in the averaged bone conduction results preoperatively versus post-operatively (p>0.05). Speech recognition in quiet results were not significantly different to performance attained whilst wearing hearing aids (p>0.05). Speech recognition in noise performance was substantially improved with use of the VSB in most test conditions. CONCLUSIONS: For the majority of the subjects, the VSB was an effective method of hearing restoration for their mixed and conductive hearing loss.

Expression of chemokine decoy receptors and their ligands at the porcine maternal-fetal interface.

Successful pregnancy requires coordinated maternal-fetal cross-talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30-40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub-category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non-signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal-fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest.

Angiogenic DC-SIGN(+) cells are present at the attachment sites of epitheliochorial placentae.

Spontaneous early and mid-gestation fetal losses occur in swine. At both stages, endometrial lymphocytes associated with smaller, paler conceptuses have fewer pro-angiogenic and more pro-inflammatory cytokine transcripts compared with robust conceptuses. We hypothesized that similar differences occur in conceptus-associated dendritic cells (DCs). Using laser capture-microdissection, dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN)(+) cells were isolated from attachment sites of healthy and arresting conceptuses at gestation day (gd)20 and 50. DC-SIGN(+) cells were screened using real-time PCR for vascular endothelial cell growth factor (Vegf), its receptors, semaphorins (Sema) and plexins (Plxn), and for toll-like receptor (Tlr) transcripts to address potential activation pathways. Homogenized endometrial and trophoblast biopsies were quantified for type 1/type 2 cytokine transcripts/proteins. DC-SIGN(+) cells from healthy and arresting conceptuses had more Vegf transcripts at early than mid gestation whereas transcripts for Vegfr1 and Vegfr2 were stable. In gd20 arresting site DC-SIGN(+) cells, Neuropilin-2 transcripts were elevated, whereas at gd50 arresting sites, Plxn-A2 increased and Sema3A transcripts were lost. Tlr-1, Tlr-4 and Tlr-6 transcript abundance was independent of conceptus health. At gd20, type 1 cytokines were prevalent, whereas at gd50 type 2 cytokines predominated in endometrium and trophoblast. Thus, gestational features, characteristic of haemochorial placentation, are present in species with distinctly different placentation.

Immunological mechanisms affecting angiogenesis and their relation to porcine pregnancy success.

Prenatal mortality due to loss of lymphocyte-promoted endometrial angiogenesis is being investigated as a major cause of litter reductions during pregnancy in pigs. This review discusses immune mechanisms influencing porcine endometrial angiogenesis as well as additional signalling molecules that may play important roles in the compromise of peri-implantation and mid-gestation fetal pig survival. These include dendritic cells, signalling molecules such as toll-like receptors, chemokines and ficolins. Together these cells and molecules regulate immune responses and, ideally, protect the mother and prevent immune-based conceptus losses. Dendritic cells were recently shown to be angiogenic. Their tolerogenic role at the maternal-fetal interface coupled with the ability to secrete and respond to angiogenic factors suggests that dendritic cells are the key coordinators of angiogenesis at the porcine maternal-fetal interface. Chemokines coordinate the localization of immune effector and endothelial cells. The balance between pro-angiogenic and anti-angiogenic chemokines is addressed in relation to conceptus viability. Ficolins, components of the lectin-mediated complement activation pathway, are used for self/non-self recognition. Together, these components of the immune system could regulate lymphocyte- and non-lymphocyte-promoted endometrial angiogenesis to determine conceptus survival.

A review of molecular contrasts between arresting and viable porcine attachment sites.

Significant spontaneous fetal loss of unknown cause occurs in North American commercial swine. About 30% of conceptuses, thought to be genetically normal, are lost during the peri-attachment period. An additional 20% are lost at mid-pregnancy. Littermate endometrial and trophoblast biopsies were studied by quantitative real-time PCR for gene expression, and immunohistochemistry for protein expression at gestation day (gd)15-23 and 50. RNA analyses were also conducted on endometrial lymphocytes and arterial endothelial cells removed from biopsies by laser capture microdissection. Genes were selected for study from human literature and cloned as required. As in humans, angiogenic, cytokine, chemokine and chemokine decoy receptor gene expression occurs at the porcine maternal-fetal interface. In each tissue studied, distinct patterns of expression are found between early and mid-pregnancy, as well as between viable and arresting conceptus attachment sites. These changes involve both endometrial lymphocytes and dendritic cells. Restriction in endometrial angiogenesis, reduction in expression of the chemokine decoy receptor D6, and reduction in dendritic cell numbers contribute to fetal arrest. In peri-attachment loss, interferon-gamma is more abundantly transcribed than tumor necrosis factor-alpha, but this ratio is reversed during midgestation failure. Further characterization of spontaneous fetal loss in pigs will identify targets for modification by hog producers and may provide a model for identification of antecedents to fetal loss in humans.