Robert W. Wills

Porcine Reproductive and Respiratory Syndrome Virus: Description of Persistence in Individual Pigs upon Experimental Infection

ABSTRACT We studied the persistence of porcine reproductive and respiratory syndrome virus (PRRSV) in individual experimentally infected pigs, during a period of up to 150 days postinfection (dpi). The results of this study suggest that the persistence of PRRSV involves continuous viral replication but that it is not a true steady-state persistent infection. The virus eventually clears the body and seems to do it in most of the animals by 150 dpi or shortly thereafter. High genetic stability was seen for several regions of the persistent PRRSVs genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.

The REFLECT Statement: Methods and Processes of Creating Reporting Guidelines for Randomized Controlled Trials for Livestock and Food Safety by Modifying the CONSORT Statement

The conduct of randomized controlled trials in livestock with production, health and food‐safety outcomes presents unique challenges that may not be adequately reported in trial reports. The objective of this project was to modify the CONSORT (Consolidated Standards of Reporting Trials) statement to reflect the unique aspects of reporting these livestock trials. A 2‐day consensus meeting was held on 18–19 November 2008 in Chicago, IL, USA, to achieve the objective. Prior to the meeting, a Web‐based survey was conducted to identify issues for discussion. The 24 attendees were biostatisticians, epidemiologists, food‐safety researchers, livestock‐production specialists, journal editors, assistant editors and associate editors. Prior to the meeting, the attendees completed a Web‐based survey indicating which CONSORT statement items may need to be modified to address unique issues for livestock trials. The consensus meeting resulted in the production of the REFLECT (Reporting Guidelines for Randomized Control Trials) statement for livestock and food safety and 22‐item checklist. Fourteen items were modified from the CONSORT checklist and an additional sub‐item was proposed to address challenge trials. The REFLECT statement proposes new terminology, more consistent with common usage in livestock production, to describe study subjects. Evidence was not always available to support modification to or inclusion of an item. The use of the REFLECT statement, which addresses issues unique to livestock trials, should improve the quality of reporting and design for trials reporting production, health and food‐safety outcomes.

Inter-relationships of Salmonella status of flock and grow-out environment at sequential segments in broiler production and processing.

In this study, we investigated how the likelihoods of Salmonella presence in various samples from broilers and their grow‐out environment throughout one production cycle were related. Sixty‐four broiler flocks from 10 complexes of two companies in the southern United States were included in the study. Samples from the gastrointestinal tracts of chicks, transport tray pads and litter and drag swabs from the house were collected on the day of placement of each flock. Approximately, 1 week before harvest, whole bird carcass rinses, caecum and crop samples were collected from birds from these same flocks. On the day of harvest, litter and drag swab samples were also taken from the house after the birds were removed. Upon arrival of the flocks at the processing plant, whole carcass rinses, caecum and crop samples were collected. As the flocks were processed, carcass rinses were collected just before the carcasses entered the immersion chill tank and as they exited the chill tank. Logistic regression was used to model the relationships between the likelihood of Salmonella in samples of each type collected at each sampling point and Salmonella frequencies in all the samples taken from the flock and grow‐out environment at preceding production stages. The analysis demonstrated that increased likelihood of Salmonella contaminated carcasses entering the immersion chill tank was associated with higher contamination of the exteriors and crops of birds at arrival for processing as well as house environmental samples at the time of harvest and prior to placement. The best predictors of post‐chill broiler carcass Salmonella status were the frequencies of Salmonella in the litter on the day of harvest and prior to placement. The immersion chilling appeared to disrupt some of the relationships between the processing plant and pre‐harvest samples.

Evaluating interventions against Salmonella in broiler chickens: applying synthesis research in support of quantitative exposure assessment

A scoping study and systematic review-meta-analyses (SR-MAs) were conducted to evaluate the effectiveness of various interventions for Salmonella in broiler chicken, from grow-out farm to secondary processing. The resulting information was used to inform a quantitative exposure assessment (QEA) comparing various control options within the context of broiler chicken production in Ontario, Canada. Multiple scenarios, including use of two separate on-farm interventions (CF3 competitive exclusion culture and a 2% lactose water additive), a package of processing interventions (a sodium hydroxide scald water disinfectant, a chlorinated post-evisceration spray, a trisodium phosphate pre-chill spray and chlorinated immersion chilling) a package consisting of these farm and processing interventions and a hypothetical scenario (reductions in between-flock prevalence and post-transport concentration), were simulated and compared to a baseline scenario. The package of on-farm and processing interventions was the most effective in achieving relative reductions (compared to baseline with no interventions) in the concentration and prevalence of Salmonella by the end of chilling ranging from 89·94% to 99·87% and 43·88% to 87·78%, respectively. Contaminated carcasses entering defeathering, reductions in concentration due to scalding and post-evisceration washing, and the potential for cross-contamination during chilling had the largest influence on the model outcomes under the current assumptions. Scoping study provided a transparent process for mapping out and selecting promising interventions, while SR-MA was useful for generating more precise and robust intervention effect estimates for QEA. Realization of the full potential of these methods was hampered by low methodological soundness and reporting of primary research in this area.

A systematic review-meta-analysis and meta-regression on the effect of selected competitive exclusion products on Salmonella spp. prevalence and concentration in broiler chickens.

The effectiveness of various competitive exclusion (CE) products for reducing Salmonella colonization in broiler chickens was evaluated using systematic review-meta-analysis-meta-regression (SR-MA-MR). Relevance screening identified 201 relevant studies that were subjected to methodological assessment. Of these studies, 159 were suitable for data extraction, 66 were presented in a number of MAs and 130 were examined in a meta-regression (MR). Fourteen different CE products were identified, 9 of them commercial products, and the most common route of administration was oral gavage (63.7% of trials). Meta-analyses indicated that a number of CE products reduce Salmonella colonization in broilers, the most effective one being Preempt™ which was formerly known as CF-3. Five study characteristics (publication year, CE type, CE route, sample origin, and Salmonella serovar administered/recovered) and three methodological soundness characteristics (treatment assignment, intervention and laboratory methods description) were retained as statistically significant (p<0.05) in the final MR model. The MR analysis indicated that, undefined CE products outperformed all commercial products, except for: Preempt™ and Broilact(®). Both were considered comparable to the undefined chicken source CE culture products in effectiveness. The administration of CE through spraying the chicks at the hatchery was determined to be just as effective as the oral gavage treatment, and more practical for farmers to administer. The results of this study could be useful in decision-making concerning the on-farm use of CE products in broiler chickens, and as inputs for risk assessments as the industry pushes for more antibiotic-free alternatives. Out of the various interventions to reduce Salmonella colonization in broilers on-farm, CE was the most studied; its inability to be licenced in certain countries and proof of consistent efficacy remains a barrier.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs

OBJECTIVE To determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. STUDY DESIGN Randomized, blocked, crossover design with a 14-day washout period. ANIMALS Healthy intact female Walker Hounds aged 1-6 years and weighing 20.5-24.2 kg. METHODS Dogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg(-1), PO, every 12 hours), carprofen (4.4 mg kg(-1), PO, every 24 hours), meloxicam (0.2 mg kg(-1), PO, on the 1st day then 0.1 mg kg(-1), PO, every 24 hours), and deracoxib (2 mg kg(-1), PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B(2) was also measured before and during administration of each drug. RESULTS All NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg(-1) every 12 hours), carprofen (4.4 mg kg(-1) every 24 hours), meloxicam and deracoxib, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Oral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding.

Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon.

BackgroundSalmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogens growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum.ResultsWe found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies.ConclusionsThis study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

Development of bioluminescent Salmonella strains for use in food safety

BackgroundSalmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, we developed bioluminescent Salmonella strains that can be used for real-time monitoring of the pathogens growth on food products. To accomplish this, twelve Salmonella strains from the broiler production continuum were transformed with the broad host range plasmid pAKlux 1, and a chicken skin attachment model was developed.ResultsSalmonella strains carrying pAKlux 1 constitutively expressed the luxCDABE operon and were therefore detectable using bioluminescence. Strains were characterized in terms of bioluminescence properties and plasmid stability. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, we developed an attachment model using chicken skin. The effect of washing on attachment of Salmonella strains to chicken skin was tested using bioluminescent strains, which revealed the attachment properties of each strain.ConclusionThis study demonstrated that bioluminescence is a sensitive and effective tool to detect Salmonella on food products in real-time. Bioluminescence imaging is a promising technology that can be utilized to evaluate new food safety measures for reducing Salmonella contamination on food products.

Use of the Cortisol‐to‐ACTH Ratio for Diagnosis of Primary Hypoadrenocorticism in Dogs

Background The ACTH stimulation test is currently required for definitive diagnosis of hypoadrenocorticism. Increased cost of synthetic ACTH (cosyntropin) has prompted a search for alternative diagnostic methods. Objective The purpose of this study was to determine whether a cortisol‐to‐ACTH ratio (CAR) can be used to differentiate dogs with hypoadrenocorticism from normal dogs and those with nonadrenal illness. Animals Eight healthy dogs (H), 19 dogs with nonadrenal illness (NAI), and 15 dogs with hypoadrenocorticism (HAD). Methods Dogs in the HAD group were retrospectively identified from PUVTH medical records. The NAI group consisted of hospitalized dogs with clinical signs, clinicopathologic findings, or both, consistent with a diagnosis of hypoadrenocorticism, but in which hypoadrenocorticism was ruled out based on ACTH stimulation test results. Healthy dogs were recruited from hospital staff and students. Endogenous ACTH concentrations and cortisol concentrations before and after ACTH stimulation were measured in all dogs. Results Baseline cortisol concentration was significantly lower, and ACTH concentration was significantly higher, in the HAD group versus the H and NAI group (P < .001). However, there was overlap among groups. Cortisol‐to‐ACTH ratio was significantly lower in the HAD group versus the H and NAI groups (P < .001), and there was no overlap between the HAD group and the other 2 groups. Conclusions and Clinical Importance CAR can be used for definitive diagnosis of primary hypoadrenocorticism.

Platelet Cyclooxygenase Expression in Normal Dogs

BACKGROUND Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. HYPOTHESIS/OBJECTIVES The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. ANIMALS Eight female, intact hounds. METHODS A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. RESULTS Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. CONCLUSIONS AND CLINICAL IMPORTANCE Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness.