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

The conduct of randomized controlled trials in livestock with production, health, and food-safety outcomes presents unique challenges that might 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 November 18–19, 2008 in Chicago, IL, to achieve the objective. Before 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. Before the meeting, the attendees completed a Web-based survey indicating which CONSORT statement items would 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 subitem 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.     

Evaluation of an arthroscopic approach for transection of the equine collateral sesamoidean ligament

Objective: To evaluate: (1) an arthroscopic technique for transection of the collateral sesamoidean ligament (CSL); and (2) the healing response using magnetic resonance (MR) and microscopic examination. Study Design: Experimental study. Animals: Adult horses (n=6). Methods: Six sound horses with normal front foot radiographic and MR examinations were used. Lameness examination was performed before surgery and monthly for 12 months. Front foot radiography was performed at 180 and 360 days after surgery. Front foot MR was performed before, and at 7, 90, 180, and 360 days after surgery. Arthroscopic CSL desmotomy was performed on 1 forelimb. Gross and microscopic examination was performed on the CSL from both forelimbs at 360 days after surgery. Lameness scores were compared over time using the nonparametric Friedman's test for paired groups. CSL measurements were compared using paired t‐tests with a 2‐tailed significance level of P<.05. Results: Radiographs remained normal throughout study period. Surgery resulted in lameness on the operated limb for up to 2 months, after which all horses returned to soundness. CSL transection was confirmed during arthroscopy and with MR examination 7 days after surgery. Gross and microscopic evaluation confirmed ligament healing. Conclusions: CSL desmotomy resulted in short‐term lameness after surgery followed by healing of the CSL confirmed by gross and microscopic analysis.     

Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses

The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-γ (IFN-γ) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1β (IL-1β) whilst confirming stable IFN-γ and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1β and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-γ responses.     

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.     

Mannan-binding lectin (MBL) in two chicken breeds and the correlation with experimental Pasteurella multocida infection

The present study is the first demonstration of an association of the genetic serum Mannan-binding lectin (MBL) concentration with bacterial infections in chickens. The genetic serum MBL concentration was determined in two chicken breeds, and the association with the specific Pasteurella multocida humoral immune response during an experimental infection was examined. Furthermore, we examined the association of the genetic serum MBL concentration with systemic infection. The chickens with systemic infection had a statistically significant lower mean serum MBL concentration than the rest of the chickens, suggesting that MBL plays an important role against P. multocida. A statistically significant negative correlation was found between the specific antibody response and the genetic serum MBL concentration for both breeds. This indicates that MBL in chickens is capable of acting as the first line of defence against P. multocida by diminishing the infection before the adaptive immune response takes over.     

Apocynin activity in spontaneously hypertensive rats (SHR): preliminary studies in vivo

An elevation in angiotensin II (Ang II) levels is a common occurrence in spontaneously hypertensive rats (SHRs). Infusions of Ang II and a high salt diet increase the activity of NADPH oxidase that stimulates superoxide anion (O⁻²) generation and increases the expression of certain subunits of NADPH oxidase. Apocynin, an NADPH oxidase inhibitor with antihypertensive effects, is able to inhibit the release of superoxide anion by inhibiting NADPH oxidase activity and blocking the migration of p47 phox to the mitochondrial membrane. The aim of our study was to evaluate the antihypertensive effects of apocynin in SHRs and Wistar rats (WKYs) using a micropuncture technique. After microperfusion of both the proximal and distal tubules, we found that SHRs treated with apocynin showed a decrease in the free-flow collection of the proximal tubule (PT), which was not affected in WKYs. Moreover, significant differences were not demonstrated in the distal tubule (DT), probably due a mechanism of compensation that occurs in the loop of Henle. In conclusion, it is possible that the mechanisms of reabsorption in the PT are controlled by the interactions of O⁻² and nitric oxide (NO). These data could suggest a higher activity of NADPH oxidase and increase in reactive oxygen species (ROS) production in the PT during hypertension.

     

The impact of the phyto‐oestrogen genistein on swine granulosa cell function

Soya and soybean products used in swine feeding contain genistein, a non‐steroidal phyto‐oestrogen which has been demonstrated to influence endocrine functions. This observation leads us to design this study to evaluate the effect of genistein on swine granulosa cell steroidogenesis and proliferation. In the attempt to unravel the genistein signal transduction mechanisms, we verified the effect of lavendustin, a Tyrosine Kinase (TK) inhibitor, and the potential involvement of NO/cGMP pathway. Finally, as angiogenesis is essential for follicle development, we tested the effect of the phyto‐oestrogen on vascular endothelial growth factor production and on granulosa cell redox status, because free‐radical species modulate neovascularization. Our data provide evidence that genistein interferes with granulosa cell steroidogenesis while it does not modulate cell growth: this effect could be at least partially produced by inhibiting TK‐dependent signalling systems. On the contrary, NO/cGMP pathway or vascular endothelial growth factor production can be excluded as signalling mechanism involved in phyto‐oestrogen effects. Remarkably, genistein stimulates hydrogen peroxide production thus potentially inhibiting follicular angiogenesis. Collectively, these results suggest that genistein consumption could potentially negatively impact swine reproductive function.     

Cysteamine Supplementation of In vitro Maturation Media: A Review

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti‐oxidant systems such as glutathione (GSH) can attenuate the deleterious effects of oxidative stress by scavenging ROS. It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur. Addition of low molecular weight compounds to culture media, such as cysteamine, can increase GSH levels by increasing cysteine uptake. Quite naturally, effects of supplementation of in vitro maturation (IVM) media with low molecular weight thiols have been studied in various species. This article reviews the use of cysteamine supplementation for IVM, its effects on maturation rates and further embryo development.     

Emesis in dogs: a review

Emesis is a common presenting sign in small animal practice. It requires a rational approach to management that is based upon a sound understanding of pathophysiology combined with logical decision making. This review, which assesses the weight of available evidence, outlines the physiology of the vomiting reflex, causes of emesis, the consequences of emesis and the approach to clinical management of the vomiting dog. The applicability of diagnostic testing modalities and the merit of traditional approaches to management, such as dietary changes, are discussed. The role and usefulness of both traditional and novel anti-emetic drugs is examined, including in specific circumstances such as following cytotoxic drug treatment. The review also examines areas in which common clinical practice is not necessarily supported by objective evidence and, as such, highlights questions worthy of further clinical research.     

A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs

A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55 degrees C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H(2)SO(4) catalysis for 1.5 h at 55 degrees C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.