High-resolution protein structure determination by serial femtosecond crystallography

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.     

Improvement in insulin resistance and reduction in plasma inflammatory adipokines after weight loss in obese dogs

Obesity is now a major disease of dogs, predisposing to numerous disorders including diabetes mellitus. Adipocytes are active endocrine cells, and human obesity is characterized by derangements in inflammatory adipokine production. However, it is unclear as to whether similar changes occur in dogs. The purpose of the current study was to assess insulin sensitivity and inflammatory adipokine profiles in dogs with naturally occurring obesity and to investigate the effect of subsequent weight loss. Twenty-six overweight dogs were studied, representing a range of breeds and both sexes. All dogs underwent a weight loss program involving diet and exercise. Body fat mass was measured by dual-energy x-ray absorptiometry; plasma concentrations of insulin, glucose, and a panel of inflammatory adipokines (including acute-phase proteins, cytokines, and chemokines) were also analyzed. Body fat mass before weight loss was positively correlated with both plasma insulin concentrations (Kendall τ = 0.30, P = 0.044) and insulin:glucose ratio (Kendall τ = 0.36, P = 0.022), and both decreased after weight loss (P = 0.0037 and 0.0063, respectively). Weight loss also led to notable decreases in plasma tumor necrosis factor-α (TNF-α), haptoglobin, and C-reactive protein concentrations (P < 0.05 for all), suggesting improvement of a subclinical inflammatory state associated with obesity. This study has demonstrated that in obese dogs, insulin resistance correlates with degree of adiposity, and weight loss improves insulin sensitivity. Concurrent decreases in TNF-α and adipose tissue mass suggest that in dogs, as in humans, this adipokine may be implicated in the insulin resistance of obesity.     

Outbreak of Lawsonia intracellularis on a Standardbred breeding farm in North Carolina

Lawsonia intracellularis is a cause of protein‐losing enteropathy in weanling foals commonly causing signs including diarrhoea, oedema and unthrifty appearance. It has previously been reported as sporadic cases throughout the USA, Canada and Europe. Only recently have outbreaks on a single farm location been reported. The cases reported here are an example of multiple foals on a single farm testing serologically positive for L. intracellularis. These foals ranged in severity from asymptomatic to severely hypoproteinaemic and septic; however, none of the foals ever displayed diarrhoea, which is the most common presenting sign of this disease process.     

Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella haemolytica and P trehalosi

OBJECTIVE: To determine whether Pasteurella haemolytica and P trehalosi isolates possess the structural gene for Pasteurella leukotoxin lktA and whether beta-hemolytic activity of these isolates correlated with detection of the lktA gene. SAMPLE POPULATION: 147 P haemolytica isolates from 21 biovariant groups and 101 P trehalosi isolates from 7 biovariant groups. In addition, P multocida and organisms from 7 other genera were tested to establish specificity of the procedure. PROCEDURE: Isolates were observed for beta-hemolysis. A polymerase chain reaction (PCR) procedure was used to amplify the RTX domain of the Pasteurella lktA gene. RESULTS: The lktA gene was detected in 108 (44%) isolates, including 15 associated with respiratory tract disease. All but 2 (98%) of the isolates that had the lktA gene were beta-hemolytic when grown on sheep blood agar. The remaining 140 isolates were negative for the lktA gene and hemolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Hemolytic activity of P haemolytica and P trehalosi isolates correlated with detection of the lktA gene for all but 2 isolates. However, 56% of isolates tested were negative for the lktA gene and beta-hemolytic activity. Leukotoxin production and secretion is a major virulence factor when other conditions are favorable for disease development. Therefore, identification of strains that possess the lktA gene may aid in the evaluation of the pathogenic potential of Pasteurella strains carried by wild and domestic animals.     

Position of inoculum in the canopy affects the risk of septoria tritici blotch epidemics in winter wheat

The spread of septoria leaf blotch in wheat caused by Mycosphaerella graminicola is widely reported to depend on the occurrence of splashy rainfall. Previous studies have also implicated an important effect of canopy architecture on the risk of disease spread. This is because architecture affects the proximity of the yield-forming leaves to inoculum present on older diseased leaves within the crop. This study demonstrated that infection of the final three leaves of winter wheat could occur in the absence of splashy rainfall. For cvs Riband and Longbow, the final two leaf layers emerged at a position ≈ 8 cm below older leaves containing sporulating lesions. Under these conditions, infection of new leaves occurred in treatments that simulated dew and nonsplashy rainfall. These treatments resulted in disease incidences of 10–40% above the untreated control on the final two leaf layers. Within a season, the distance between diseased and healthy leaves during the period of stem extension varied substantially across a range of 30 cultivars. While the magnitude of these differences was not the same across seasons, the relative differences between cultivars were generally consistent, suggesting a strong genotype influence on lesion proximity. This study shows how knowledge of the distribution of lesion proximity within a crop can be used to estimate the risk of inoculum transfer for a given maximum splash height. A rapid crop-monitoring method for estimating the distribution of lesion proximity was developed and tested. Lesion proximity was manipulated by plant growth regulator (PGR) treatments. Significant increases in disease, between 14 and 62%, were observed on the upper canopy leaves of plants treated with PGR. The largest differences were observed in treatments where lesion proximity was most affected.     

Clinical effects and plasma concentrations of fentanyl after transmucosal administration in three species of great ape.

Fentanyl is approved for transmucosal use in the United States as a preanesthetic agent in human pediatric patients and in adults for breakthrough cancer pain. Using this formulation in three species of great ape, including eight orangutans (Pongo pygmaeus), nine chimpanzees (Pan troglodytes), and two gorillas (Gorilla gorilla), fentanyl was offered transmucosally at an intended dose of 10-15 microg/kg based on estimated body weight. The animals were trained to accept and suck slowly on a piece of placebo candy, given as a treat, after an overnight fast. On the day of the study, the animals were given the lollipop formulation of fentanyl. The resulting plasma concentrations of fentanyl supported transmucosal absorption, similar to that reported in humans. This study provides an alternative sedative regimen and yielded half-life data of transmucosal fentanyl in great apes. Although transmucosal fentanyl was a useful adjunct for sedating orangutans and gorillas, its acceptance by chimpanzees before chemical immobilization was suboptimal and unpredictable.     

Timing of Early Foetal Loss for Single and Twin Pregnancies in Dairy Cattle

The incidence of early foetal loss is increasing under intensive management systems for dairy cattle. The aims of the present study were to determine whether there is any peak period of pregnancy loss during the early foetal period and to evaluate possible differences between single and twin pregnancies. The study population consisted of 1442 pregnant cattle from a single herd. Pregnancy was diagnosed by transrectal ultrasonography between 36 and 42 days after insemination, and then weekly until day 90 of gestation or until pregnancy loss. A total of 1310 cows (90.8%) bore single embryos and 132 (9.2%) carried twins. Pregnancy loss was registered in 139 (9.6%) cows before day 90 of pregnancy: 101 (7.7%) in single and 38 (28.8%) in twin pregnancies. The average time of pregnancy loss for all animals was 58.4 ± 12.6 days and ranged from 45 to 90 days. Seventy‐five per cent of the pregnancy losses were registered between 45 and 60 days of gestation. The average time of pregnancy loss for cows with singletons was 52.1 ± 4.1 days and ranged from 45 to 61 days and that for those with twins was 75.1 ± 12.4 days and ranged from 46 to 90 days. Seventy‐five per cent of the twin pregnancy losses were registered between 68 and 90 days of gestation. Our data show that the foetal loss in singleton pregnancies occurs earlier than in twin pregnancies. Assessment of normal development of gestation on days 60 and 90 after insemination is suggested.     

Supplemental betaine and peroxide-treated feather meal for finishing cattle

These studies evaluated the effects of betaine, provided either as feed-grade betaine or as concentrated separator by-product (CSB; desugared beet molasses), on performance and carcass characteristics of finishing cattle. In Exp. 1, 175 steers (410 kg initial BW) were fed a finishing diet based on steam-flaked and dry-rolled corn, and treatments included 10.5 and 21 g/d feed-grade betaine and 250 and 500 g/d CSB (supplying 15.5 and 31 g/d of betaine, respectively). Steers fed feed-grade betaine had greater (linear and quadratic effects, P < 0.1) DMI than control steers, but ADG and gain efficiencies were not affected by treatment. Dressing percent and backfat thickness was greater (P < 0.1) for steers that received feed-grade betaine than for controls. Longissimus muscle area was lower (P < 0.1) for steers supplemented with either feed-grade betaine or CSB than for control steers. Yield grades were higher for cattle receiving feed-grade betaine (quadratic effect, P < 0.1) than for control steers. Marbling scores were not affected by supplemental betaine, but the percentage of carcasses grading USDA Select was lower (linear and quadratic effects, P < 0.1) for steers fed feed-grade betaine than for control steers, predominantly due to a greater percentage grading USDA Choice. In Exp. 2, 312 heifers (343 kg initial BW) were used in a finishing study to evaluate the effects of graded levels of feed-grade betaine and peroxide-treated feather meal on performance and carcass characteristics. Treatments included two finishing diets (containing peroxide-treated or untreated feather meal) and four levels (0, 4, 8, and 12 g/d) of feed-grade betaine arranged in a 2 x 4 factorial. No significant interactions occurred between treatment of feather meal and betaine. Treatment of feather meal with hydrogen peroxide (5% wt/wt) increased in situ protein degradability but did not alter DMI, ADG, gain efficiencies, or carcass characteristics of heifers when it replaced untreated feather meal in the diet. Top-dressing feed-grade betaine to the diets had no effect on DMI, ADG, and gain efficiencies. Marbling scores were greater (cubic effect, P < 0.05) for heifers fed diets top-dressed with 4 and 12 g/d of feed-grade betaine, but other carcass characteristics were not altered significantly. Overall, feed-grade betaine and CSB did not alter growth performance, but did have minor effects on carcass characteristics.     

O.I.E. Manual of Standards for Diagnostic Tests and Vaccines,List A and B diseases of mammals,birds and bees, 3rd edn

Tropical Animal Health and Production -