Reality Versus Grant Application Research "Plans".

This article describes the implementation of the American Indian mHealth Smoking Dependence Study focusing on the differences between what was written in the grant application compared to what happened in reality. The study was designed to evaluate a multicomponent intervention involving 256 participants randomly assigned to one of 15 groups. Participants received either a minimal or an intense level of four intervention components: (1) nicotine replacement therapy, (2) precessation counseling, (3) cessation counseling, and (4) mHealth text messaging. The project team met via biweekly webinars as well as one to two in-person meetings per year throughout the study. The project team openly shared progress and challenges and collaborated to find proactive solutions to address challenges as compared to what was planned in the original grant application. The project team used multiple strategies to overcome unanticipated intervention issues: (1) cell phone challenges, (2) making difficult staffing decisions, (3) survey lessons, (4) nicotine replacement therapy, (5) mHealth text messages, (6) motivational interviewing counseling sessions, and (7) use of e-cigarettes. Smoking cessation studies should be designed based on the grant plans. However, on the ground reality issues needed to be addressed to assure the scientific rigor and innovativeness of this study.

Increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3-10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.

Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5-6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.

Status of vaccines for porcine epidemic diarrhea virus in the United States and Canada.

In 2013, porcine epidemic diarrhea virus (PEDV) emerged in the United States as a rapidly spreading epidemic causing dramatic death losses in suckling piglets. Neonatal piglets are most vulnerable to clinical disease and their only protection is passive immunity from their dam. At the end of the third year of the PEDV outbreak, most US sow herds have been infected and many are entering into an endemic disease with much less, but still chronic losses. This endemic state and the occasional naïve herd that breaks with PEDV demonstrate a need to immunize sows to protect piglets. Stimulating PEDV immunity in the sow using safe and efficacious vaccines is the best course of action; however, conducting such studies to develop sow vaccines is very costly and logistically difficult. This manuscript reviews the status of PEDV vaccines available in the United States and Canada, and describes an experiment evaluating the potential use of young pigs as a surrogate model to evaluate potential sow vaccines.

Evaluation of two real-time polymerase chain reaction assays for Porcine epidemic diarrhea virus (PEDV) to assess PEDV transmission in growing pigs.

In April 2013, a Porcine epidemic diarrhea virus (PEDV) epidemic began in the United States. As part of the response, real-time reverse transcription polymerase chain reaction (RT-PCR) assays to detect PEDV were developed by several veterinary diagnostic laboratories. Our study evaluated RT-PCR PEDV assays that detect the N gene (gN) and S gene (gS) for their ability to detect PEDV infection and the transmission potential of pigs experimentally exposed to PEDV. Detection limits and quantification cycle (Cq) values of real-time RT-PCR were assayed for PEDV samples and positive controls for both gN and gS. The limit of detection for the gN assay was 10(-6) (mean Cq: 39.82 ± 0.30) and 10(-5) (mean Cq: 39.39 ± 0.72) for the gS assay with PEDV strain USA/Colorado/2013. Following recommended guidelines, rectal swabs (n = 1,064) were tested; 354 samples were positive by gN assay and 349 samples were positive by gS assay (Cq ≤ 34.99), 710 samples were negative by gN assay and 715 were negative by gS assay (Cq > 34.99) of which 355 and 344 were "undetermined" (i.e., undetected within a threshold of 40 RT-PCR cycles, by gN and gS assays, respectively). The coefficient of variation (intra-assay variation) ranged from 0.00% to 2.65% and interassay variation had an average of 2.75%. PEDV could be detected in rectal swabs from all pigs for ~2 weeks postinfection at which time the prevalence began to decrease until all pigs were RT-PCR negative by 5 weeks postinfection. Our study demonstrated that RT-PCR assays functioned well to detect PEDV and that the gN assay was slightly better.

Assessment of litter prevalence of Mycoplasma hyopneumoniae in preweaned piglets utilizing an antemortem tracheobronchial mucus collection technique and a real-time polymerase chain reaction assay.

The swine industry currently lacks validated antemortem methods of detecting baseline herd prevalence of Mycoplasma hyopneumoniae. The focus of our study was to evaluate alternative antemortem detection techniques and to determine baseline litter prevalence in preweaned pig populations utilizing the selected technique and a real-time polymerase chain reaction (qPCR) assay. Preliminary data was analyzed on weaned piglets with evidence of respiratory disease (n = 32). Five sample types (antemortem nasal swab, tracheobronchial mucus, postmortem deep airway swab, bronchoalveolar lavage, and lung tissue) were collected from each pig. Individual samples were tested for M. hyopneumoniae using qPCR. Compared to nasal swabs, tracheobronchial mucus demonstrated higher test sensitivity (P < 0.0001). Tracheobronchial mucus was collected from apparently healthy preweaned piglets (n = 1,759; sow farms 1-3) and preweaned piglets exhibiting signs of respiratory disease (n = 32; sow farm 4), ranging in age from 12 to 25 days. Samples from sow farms 1-3 were pooled into 2 groups of 5 per litter (n = 360 pools from 180 litters), and qPCR was utilized to analyze each pool. A qPCR-positive result, threshold cycle <37, from either pool designated the litter positive for M. hyopneumoniae. Two out of 180 litters revealed a positive result (1.1%). Individual qPCR assays were run on the samples collected from sow farm 4. Five out of 30 samples revealed a positive result (16.7%). Tracheobronchial mucus collection in combination with qPCR is a sensitive antemortem sampling technique that can be used to estimate the prevalence of M. hyopneumoniae in preweaned pigs, thus providing insight into the infection dynamics across the entire farrow-to-finish process.

Evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs.

Clinical disease associated with porcine epidemic diarrhea virus (PEDV) infection in naïve pigs is well chronicled; however, information on endemic PEDV infection is limited. To characterize chronic PEDV infection, the duration of infectious virus shedding and development of protective immunity was determined. On Day 0 (D0), a growing pig was challenged with PEDV and 13 contacts were commingled. On D7, 9 contact pigs (principal virus group (PG)), were selected, moved to a separate room and commingled with one sentinel pig (S1). This process was repeated weekly with S2, S3 and S4. The PG was PEDV-positive by PCR from D3-11, with some pigs intermittently positive to D42. Pigs S1 and S2 were PEDV-positive within 24 hours of commingling. Antibodies were detected in all PG by D21 and by 7 days post-contact in S1 and S2. Pigs S3 and S4 were PCR and antibody negative following commingling. To evaluate protective immunity, 5 naïve pigs (N) and the PG were challenged (N/C, PG/C) with homologous virus on D49. All N/C pigs were PEDV PCR-positive by D52 with detection out to D62 in 3/5 N/C pigs. All PG/C pigs were PEDV PCR-negative post-challenge. By D63, all N/C seroconverted. Although PEDV RNA was demonstrated in pigs after primary infection until D42, infectious PEDV capable of horizontal transmission to naïve pigs was only shed 14-16 days after infection to age-matched pigs. Homologous re-challenge 49 days post initial PEDV exposure did not result in re-infection of the pigs. This demonstrates potential for an effective PEDV vaccine.

Preparing nurse educators in the country of Georgia.

This article describes a formal nurse educator train-the-trainer program initiated to educate qualified health professionals to teach contemporary nursing continuing education in the country of Georgia, formerly part of the Soviet Union. A 3-month intensive train-the-trainer program model was used to educate potential nurse educators to provide a foundation for introducing a higher level of continuing education to practicing nurses in Georgia. After the potential nurse educator candidates were interviewed and hired, they were required to attend at least 90% of the classes, achieve a score of 85% or higher on all train-the-trainer class posttests, and achieve a score of 90% or higher on the final examination. Sixteen of 17 nurse educators, who were physicians and nurses, successfully completed the program. These graduate nurse educators subsequently conducted formal continuing education for more than 2,900 practicing nurses, with a goal of implementing a baccalaureate nursing program as well. This program established a foundation for further nurse educator development and improvement in continuing education for currently practicing nurses in the country of Georgia.

Human cremation in Mexico 3,000 years ago.

Mixtec nobles are depicted in codices and other proto-historic documentation taking part in funerary rites involving cremation. The time depth for this practice was unknown, but excavations at the early village site of Tayata, in the southern state of Oaxaca, Mexico, recovered undisturbed cremation burials in contexts dating from the eleventh century B.C. These are the earliest examples of a burial practice that in later times was reserved for Mixtec kings and Aztec emperors. This article describes the burial contexts and human remains, linking Formative period archaeology with ethnohistorical descriptions of Mixtec mortuary practices. The use of cremation to mark elevated social status among the Mixtec was established by 3,000 years ago, when hereditary differences in rank were first emerging across Mesoamerica.

Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.