Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples.

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's Disease (JD), which is endemic to dairy cattle and also incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative polymerase chain reaction (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD disease. MAP IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry and TaqMan technology was used in 107 reports. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

Effects of probiotics Pediococcus acidilactici strain MA18/5M and Saccharomyces cerevisiae subsp. boulardii strain SB-CNCM I-1079 on fecal and intestinal microbiota of nursing and weanling piglets.

In this study, the influence of (PA) and subsp. (SCB) on fecal and intestinal microbiota of piglets during lactation and after weaning was monitored. Forty sows and their litters were used and allocated to the following dietary treatments: 1) PA, 2) SCB, 3) a mixture of the 2 probiotics (PA+SCB), 4) antibiotics (ATB), and 5) control (CTRL). Four weeks before parturition, probiotic-treated sows started receiving a daily probiotic dose of at least 2.5 × 10 cfu mixed in 500 g of feed until the end of lactation. The other groups were fed a diet without probiotics and ATB. Two days after birth, piglets received, daily, 1 × 10 cfu of the same probiotics as their mother. At weaning (d 21), these piglets were fed a basal diet enriched with the same probiotics whereas piglets from untreated litters were fed the basal diet with or without ATB. Two piglets per litter were randomly chosen to evaluate the influence of treatments on fecal microbial composition (d 10 and 28) and on ileum and colon microbiota at d 37. The microbiota was characterized by culture on selective media and by 16S rRNA gene diversity assessment using the terminal RFLP technique and clone library analysis to evaluate diversity index and phylum affiliation. Terminal RFLP profiles were also analyzed to determine differences in microbial composition between animals receiving different treatments and to identify diet-specific terminal restriction fragments (TRF) using pairwise multiresponse permutation procedures (MRPP) and indicator species analysis. Before weaning, administration of probiotics to sows and piglets had minor effect on fecal microbiota of piglets. Most modulatory effects of probiotics on ileum and colon microbiota were observed on d 37. Results revealed that PA or ATB treatments reduced ileal microbiota diversity compared with the CTRL ( < 0.05) and promoted the establishment of Firmicutes whereas SCB consumption positively influenced the establishment of the Porphyromonadaceae and Ruminococcaceae bacterial families in the colon. Moreover, pairwise MRPP analysis indicated that ileum bacterial communities of pigs treated with PA or ATB differed from those of CTRL pigs ( < 0.05). In conclusion, PA and SCB supplements, respectively, influenced, in a strain-dependent manner, the ileum and colon microbiota of weaned piglets. Results also suggest that PA and SCB have the potential as feed additives to modulate bacterial populations associated with gut health.

XIAP-mediated neuroprotection in retinal ischemia.

Retinal ischemia results in the loss of vision in a number of ocular diseases including acute glaucoma, diabetic retinopathy, hypertensive retinopathy and retinal vascular occlusion. Recent studies have shown that most of the neuronal death that leads to loss of vision results from apoptosis. XIAP-mediated gene therapy has been shown to protect a number of neuronal types from apoptosis but has never been assessed in retinal neurons following ischemic-induced cell death. We injected an adeno-associated viral vector expressing XIAP or GFP into rat eyes and 6 weeks later, rendered them ischemic by raising intraocular pressure. Functional analysis revealed that XIAP-treated eyes retained larger b-wave amplitudes than GFP-treated eyes up to 4 weeks post-ischemia. The number of cells in the inner nuclear layer (INL) and the thickness of the inner retina were significantly preserved in XIAP-treated eyes compared to GFP-treated eyes. Similarly, there was no significant reduction in optic nerve axon numbers in XIAP-treated eyes. There were also significantly fewer TUNEL (TdT-dUTP terminal nick end labeling) positive cells in the INL of XIAP-treated retinas at 24 h post-ischemia. Thus, XIAP-mediated gene therapy imparts both functional and structural protection to the retina after a transient ischemic episode.

Evaluation of margin angles of collarless metal ceramic restorations.

This study was designed to measure the porcelain margin angles of completed collarless metal ceramic restorations and determine if these angles correspond to the most frequently recommended porcelain margin angle of 90 degrees. A sample of 99 metal ceramic restorations with porcelain labial margins were evaluated. A 1.0 mm slice taken from the midfacial impression of each restoration was evaluated by tooth location with a stereomaster microscope and the angle of the labial porcelain margin was calculated. The comparison of margin angles by tooth location showed no statistical difference between the groups. Comparison of the average margin angle with the most suggested 90-degree angle was analyzed with a One-Sample t-test and found to be statistically significant (p = 0.000). Although the majority of dental schools teach the shoulder preparation for collarless metal ceramic restorations, only 43% of the restorations measured fell within 10 degrees of this standard.

Evaluation of resistance form of dislodged crowns and retainers.

STATEMENT OF PROBLEM: The concept of limiting taper has been described as a boundary between tapers that do and those that do not provide resistance form for a preparation. There is controversy as to whether this boundary that divides preparations with from those without resistance form translates clinically into a boundary for success. PURPOSE: This investigation evaluated the resistance form of abutments of crowns or retainers that have been dislodged to determine the clinical correlation between restoration dislodgment and lack of resistance form in the preparation. MATERIAL AND METHODS: Dies were fabricated from single crowns and retainers of fixed partial dentures that came loose and evaluated for resistance form. A total of 44 abutments were evaluated and included 1 incisor, 15 premolars, and 28 molars. Data from a previous study on the percentage of abutments lacking resistance form for restorations leaving a large dental laboratory was used for comparative statistical tests. RESULTS: Forty-two of the 44 abutments (95%) lacked resistance form. All molar abutments and 93% of premolar abutments lacked resistance form in one or more directions. The one incisor abutment did not lack resistance form. Chi-square test revealed a statistically significant difference in the percentage lacking resistance form between the group composed of clinical failure (uncemented crowns) and the group leaving a dental laboratory with P = .0005 for the molars, and P = .0005 for the premolars. CONCLUSION: The clinical dislodgment of cast restorations is associated with the lack of resistance form in the preparations. In this study, there was a relationship between clinical success or failure and the all-or-none nature of resistance form; dislodged crowns come almost exclusively from preparations with tapers that did not provide resistance form.

Radiographic verification of implant abutment seating.

STATEMENT OF PROBLEM: It is possible to incorrectly seat an abutment on an external hex implant. PURPOSE: This study investigated the theoretical and practical limits of radiographic verification of the complete seating of implant abutments on external hex implants. MATERIAL AND METHODS: A positioning device that could accurately place the film and/or tube head in any vertical relation desired relative to the implant and abutment was fabricated. For all records, a standard abutment was secured to the implant in both a fully seated position (closed) and with the abutment turned approximately 30 degrees and seated atop the implant external hex (open). Digital radiographs were made with the tube head fixed perpendicular to the implant and the film angled at 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 degrees. The film was then positioned parallel to the implant and the tube head was angled at 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 degrees. An image of each position was shown to 36 dentists and they were asked if the image was diagnostic for determining the status of the implant/abutment interface. RESULTS: The data were analyzed with a computer statistics program. The group with the tube head changed was analyzed with the Cochran's Q test and revealed a statistically significant (p < 0.0001) change in the diagnostic value of radiographs beginning at 20 degrees. The group with the film angle changed was analyzed with a Pearson chi-square and showed no statistical difference (p = 0.394) for the diagnostic value of any of the film angles. CONCLUSION: Maintaining the tube head to less than 20 degrees from perpendicular to the long axis of the implant resulted in a diagnostic radiograph, regardless of the angle of the film.