Michael J. Toscano

Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth

Catecholamines may stimulate enteric bacteria including the foodborne pathogen Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by two mechanisms in vivo: as a quorum sensing signal and a supplier of iron. To identify genes of Salmonella Typhimurium that respond to norepinephrine, transposon mutagenesis and DNA microarray analysis were performed. Insertional mutations in the following genes decreased norepinephrine-enhanced growth: degS, entE, entF, fes, gpmA, hfq, STM3846. DNA microarray and real-time RT-PCR analyses revealed a decrease in the expression of several genes involved in iron acquisition and utilization during norepinephrine exposure, signifying the iron-limiting conditions of serum-SAPI minimal medium and the siderophore-like activity of norepinephrine. Unlike the wild-type parent strain, growth of neither a fepA iroN cirA mutant nor a fepC mutant, harboring deletional mutations in the outer and inner membrane transporters of enterochelin, respectively, was enhanced by norepinephrine. However, growth of the fepC and the fepA iroN cirA mutants could be rescued by an alternative siderophore, ferrioxamine E, further validating the role of norepinephrine in supplying the organism with iron via the catecholate-specific iron transport system. Contrary to previous reports using small animal models, the fepA iroN cirA mutant of Salmonella Typhimurium colonized the swine gastrointestinal tract, as did the fepC mutant.

PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities: A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.

Continuous monitoring of pop hole usage by commercially housed free-range hens throughout the production cycle

Free-range laying hens are able to move between the indoor house and range through exits termed pop holes. The aim of this study was to examine the proportion of the flock that used the pop holes and to identify patterns of movement throughout the flock cycle. Four flocks of free-range hens each of 1500 birds were studied. Ten per cent of each flock were tagged with RFID (radio-frequency identification) transponders and their pop hole activity studied throughout the production cycle. Within two weeks of tagging at 25, 35, 45, 55 and 65 weeks of age, approximately 80 per cent of the tagged birds were seen in the pop holes and 50 per cent of the tagged birds were seen on 80 per cent of the days available to them after tagging. Within the flock, subpopulations of birds could be identified: those that never ventured to the pop holes (approximately 8 per cent), those that used the pop holes very infrequently (approximately 8 per cent), those that sat in the pop holes (approximately 4 per cent), and those that used the pop holes frequently (approximately 80 per cent). There was an effect of age of the birds, time of day and daily mean temperature on pop hole usage. Additional factors affecting activity on particular days were wind speed, rainfall and hours of sunshine. The findings show that a significant proportion of the flock accesses the pop holes on a regular basis with only a very small proportion preferring to stay in the house.

Causes of keel bone damage and their solutions in laying hens

Keel bone damage (KBD) is a critical issue facing the contemporary laying hen industry due to the likely pain leading to compromised welfare and reduced productivity. Recent reports suggest that KBD, while highly variable and likely dependent on a host of factors, extends to all housing systems (including traditional battery cages, furnished cages and non-cage systems), genetic lines, and management styles. Despite the extent of the problem, the research community remains uncertain as to the causes and influencing factors of KBD. To combat these issues, the current review was produced following discussions from the 1st International Keel Bone Damage Workshop held in Switzerland in April 2014. This exercise sought to assess current knowledge, foster novel collaborations, propose unique methodologies and highlight the key areas where innovative research is needed. The following paper is based on the content of those discussions and presents nine recommendations for future research efforts.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets

INTRODUCTION The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Soft Perches in an Aviary System Reduce Incidence of Keel Bone Damage in Laying Hens

Keel bone fractures and deviations are one of the major welfare and health issues in commercial laying hens. In non-cage housing systems like aviaries, falls and collisions with perches and other parts of the housing system are assumed to be one of the main causes for the high incidence of keel bone damage. The objectives of this study were to investigate the effectiveness of a soft perch material to reduce keel bone fractures and deviations in white (Dekalb White) and brown laying hens (ISA Brown) kept in an aviary system under commercial conditions. In half of 20 pens, all hard, metal perches were covered with a soft polyurethane material. Palpation of 20 hens per pen was conducted at 18, 21, 23, 30, 38, 44 and 64 weeks of age. Production data including egg laying rate, floor eggs, mortality and feed consumption were collected over the whole laying period. Feather condition and body mass was assessed twice per laying period. The results revealed that pens with soft perches had a reduced number of keel bone fractures and deviations. Also, an interaction between hybrid and age indicated that the ISA hybrid had more fractured keel bones and fewer non-damaged keel bones compared with the DW hybrid at 18 weeks of age, a response that was reversed at the end of the experiment. This is the first study providing evidence for the effectiveness of a soft perch material within a commercial setting. Due to its compressible material soft perches are likely to absorb kinetic energy occurring during collisions and increase the spread of pressure on the keel bone during perching, providing a mechanism to reduce keel bone fractures and deviations, respectively. In combination with genetic selection for more resilient bones and new housing design, perch material is a promising tool to reduce keel bone damage in commercial systems.

Conservative Versus Arthroscopic Management for Medial Coronoid Process Disease in Dogs: A Prospective Gait Evaluation

OBJECTIVE To investigate, using objective gait analysis, the long-term outcome of dogs with medial coronoid process disease (MCPD) treated with conservative management (CM) versus arthroscopic treatment (AT). STUDY DESIGN Prospective clinical trial. ANIMALS Dogs (n = 20) with unilaterally confirmed MCPD. METHODS Eleven dogs were treated arthroscopically with removal of coronoid fragments and burring of any associated chondromalacic cartridge and 9 dogs were managed conservatively. All dogs were administered a 6-week course of oral tepoxalin on enrollment. Inverse dynamics gait analysis was performed at initial presentation and at 4, 8, 26, and 52 weeks. The gait variables analyzed were elbow moment (EM), elbow power (EP), total support moment (TSM), and total support moment ratio (TSMR) as a measure of forelimb asymmetry. RESULTS Affected peak EM increased from 0.58 to 0.76 Nm/kg in the AT dogs, and from 0.66 to 0.81 Nm/kg in the CM dogs and there was no significant difference between groups. Affected peak EP increased marginally in the AT dogs, but was unchanged in the CM dogs and there was no significant difference between groups. TSM increased from 1.49 to 1.92 Nm/kg in the AT dogs and from 1.52 to 2.06 Nm/kg in the CM dogs and there was no significant difference between groups. TSMR was statistically different between treatment groups at 1 (P = .003) and 2 months (P = .048) with the AT group more asymmetric and hence more lame. TSMR at 12 months was 0.83 (AT) and 0.86 (CM) implying a failure of return to soundness by either group. CONCLUSIONS AT dogs had increased mechanical asymmetry at 4 and 8 weeks compared to the CM group revealing surgery worsened limb function. There was no significant difference in mechanical symmetry between groups at 26 and 52 weeks.

Methods for assessment of keel bone damage in poultry

Keel bone damage (KBD) is a critical issue facing the laying hen industry today as a result of the likely pain leading to compromised welfare and the potential for reduced productivity. Recent reports suggest that damage, while highly variable and likely dependent on a host of factors, extends to all systems (including battery cages, furnished cages, and non-cage systems), genetic lines, and management styles. Despite the extent of the problem, the research community remains uncertain as to the causes and influencing factors of KBD. Although progress has been made investigating these factors, the overall effort is hindered by several issues related to the assessment of KBD, including quality and variation in the methods used between research groups. These issues prevent effective comparison of studies, as well as difficulties in identifying the presence of damage leading to poor accuracy and reliability. The current manuscript seeks to resolve these issues by offering precise definitions for types of KBD, reviewing methods for assessment, and providing recommendations that can improve the accuracy and reliability of those assessments.

Development of an ex vivo protocol to model bone fracture in laying hens resulting from collisions.

Fractures of the keel bone, a bone extending ventrally from the sternum, are a serious health and welfare problem in free range laying hens. Recent findings suggest that a major cause of keel damage within extensive systems is collisions with internal housing structures, though investigative efforts have been hindered by difficulties in examining mechanisms and likely influencing factors at the moment of fracture. The objectives of this study were to develop an ex vivo impact protocol to model bone fracture in hens caused by collision, to assess impact and bird-related factors influencing fracture occurrence and severity, and to identify correlations of mechanical and structural properties between different skeletal sites. We induced keel bone fractures in euthanized hens using a drop-weight impact tester able to generate a range of impact energies, producing fractures that replicate those commonly found in commercial settings. The results demonstrated that impact energies of a similar order to those expected in normal housing were able to produce fractures, and that greater collision energies resulted in an increased likelihood of fractures and of greater severity. Relationships were also seen with keel’s lateral surface bone mineral density, and the peak reactive force (strength) at the base of the manubrial spine. Correlations were also identified between the keel and long bones with respect to both strength and bone mineral density. This is the first study able to relate impact and bone characteristics with keel bone fracture at the moment of collision. Greater understanding of these relationships will provide means to reduce levels of breakage and severity in commercial systems.

Effects of imprint training procedure at birth on the reactions of foals at age six months

REASONS FOR PERFORMING STUDY While imprint training procedures have been promoted in popular magazines, they have received limited scientific investigation. OBJECTIVES To determine the effects of a neonatal imprint training procedure on 6-month-old foals and to determine if any one session had a greater effect than others. METHODS Foals (n = 131) were divided into the following treatments: no imprint training, imprint training at birth, 12, 24 and 48 h after birth or imprint training only at birth, 12, 24, 48, or 72 h after birth. Foals then received minimal human handling until they were tested at 6 months. RESULTS During training, time to complete exposure to the stimulus was significant for only 2 of 6 stimuli. Percentage change in baseline heart rate was significant for only 2 of 10 stimuli. These 4 effects were randomly spread across treatments. CONCLUSIONS Neither the number of imprint training sessions (0, 1, or 4) nor the timing of imprint training sessions (none, birth, 12, 24, 48, or 72 h after birth) influenced the foals behaviour at 6 months of age. POTENTIAL CLINICAL RELEVANCE In this study, imprint training did not result in better behaved, less reactive foals.