Incidence rate of pathogen-specific clinical mastitis on conventional and organic Canadian dairy farms.

Mastitis is a common and costly production disease on dairy farms. In Canada, the incidence rate of clinical mastitis (IRCM) has been determined for conventionally managed dairy farms; however, no studies to date have assessed rates in organically managed systems. The objectives of this observational study were (1) to determine the producer-reported IRCM and predominant pathogen types on conventional and organic dairy farms in Southern Ontario, Canada, and (2) to evaluate the association of both mean overall IRCM and pathogen-specific IRCM with management system, housing type, and pasture access. Data from 59 dairy farms in Southern Ontario, Canada, distributed across conventional (n=41) and organic management (n=18) systems, were collected from April 2011 to May 2012. In addition to management system, farms were categorized by housing method (loose or tie-stall) and pasture access for lactating cows. Participating producers identified and collected samples from 936 cases of clinical mastitis. The most frequently isolated mastitis pathogens were coagulase-negative staphylococci, Bacillus spp., Streptococcus spp., Staphylococcus aureus, and Escherichia coli. The IRCM was higher on conventional farms than organic (23.7 vs. 13.2 cases per 100 cow-years) and was not associated with housing type (loose or tie-stall), pasture access, or herd-average milk yield. Bulk tank somatic cell count tended to be lower on conventional farms than organic (222,000 vs. 272,000 cells/mL). Pathogen-specific IRCM attributed to Staph. aureus, Bacillus spp., and E. coli was greater on conventional than organic farms, but was not associated with housing or any other factors. In conclusion, organic management was associated with reduced overall and pathogen-specific IRCM.

Health Heritage© a web-based tool for the collection and assessment of family health history: initial user experience and analytic validity.

A detailed family health history is currently the most potentially useful tool for diagnosis and risk assessment in clinical genetics. We developed and evaluated the usability and analytic validity of a patient-driven web-based family health history collection and analysis tool. Health Heritage(©) guides users through the collection of their family health history by relative, generates a pedigree, completes risk assessment, stratification, and recommendations for 89 conditions. We compared the performance of Health Heritage to that of Usual Care using a nonrandomized cohort trial of 109 volunteers. We contrasted the completeness and sensitivity of family health history collection and risk assessments derived from Health Heritage and Usual Care to those obtained by genetic counselors and genetic assessment teams. Nearly half (42%) of the Health Heritage participants reported discovery of health risks; 63% found the information easy to understand and 56% indicated it would change their health behavior. Health Heritage consistently outperformed Usual Care in the completeness and accuracy of family health history collection, identifying 60% of the elevated risk conditions specified by the genetic team versus 24% identified by Usual Care. Health Heritage also had greater sensitivity than Usual Care when comparing the identification of risks. These results suggest a strong role for automated family health history collection and risk assessment and underscore the potential of these data to serve as the foundation for comprehensive, cost-effective personalized genomic medicine.

Metabolic indicators of nutritional stress are not predictive of abnormal oral behavior in piglets.

Belly nosing is an abnormal oral-nasal behavior that can develop to high levels in newly weaned piglets and may signal nutritional need. The effects of feed restriction on both behavior and metabolic serum parameters were examined in 128 weaned piglets. All pigs were fed ad libitum during week 1, and during week 2, half of all pens (N=8) were restricted to 65% of ad libitum intake. Blood samples were collected on days 3 and 10 after weaning and behavior was observed from video recordings on days 5 and 12. Piglets were classified as early 'nosers' or early 'non-nosers' based on their behavior on day 5. Feed restriction resulted in elevated non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB) and both lower glucose and a NEFA/glucose ratio, but belly nosing was not affected. Piglets classified as 'nosers' did not have blood profiles indicating they were in greater nutritional need compared to 'non-nosers' in the first week of weaning, nor did they increase belly nosing or other piglet directed behaviors when restricted in week 2. Overall, no associations were found between blood parameters indicative of nutritional stress and belly nosing. This study identifies serum glucose, BHB and NEFA as well as the glucose/NEFA ratio as useful indicators of nutritional stress in newly weaned piglets.

The effect of dentition on feeding development in piglets and on their growth and behavior after weaning.

The objectives of this study were to determine the effects of dentition on feed-oriented behavior and feed consumption before weaning at 28 d, and whether premolar eruption or occlusion at the time of weaning influenced postweaning growth or behavior. Over 3 trials, 24 litters of Yorkshire piglets (n = 233) were provided with creep feed marked with 1% chromic oxide on d 5. Dental exams were performed on d 2, 6, 9, 13, 16, 20, 23, and 27. Fecal samples were visually assessed for feed consumption (via fecal color) on the same day as dental exams, beginning on d 6. The duration of time spent at, and frequency of visits to, the creep feeder were determined from continuous video recordings on d 7, 10, 14, 17, 21, and 24 for 6 h/d (0700 to 1000 h, 1300 to 1600 h). After weaning, behavior was recorded every 5 min for three 2-h time periods (0600 to 0800 h, 1100 to 1300 h, and 1600 to 1800 h) on d 2, 4, 6, 8, 10, and 12. Piglets younger than 17 d with their premolars erupted and occluded spent less time at the creep feeder and visited it less often than piglets without their premolars erupted and occluded [duration: p(3) (premolar position 3 on maxilla), d 7 (P = 0.005); p(4) (premolar position 4 on mandible), d 7 (P < 0.0001), d 10 (P = 0.003); p(4 )(premolar position 4 on maxilla), d 17 (P = 0.012); occlusion, d 7 (P < 0.0001), d 10 (P = 0.0004); visits: p(3), d 7 (P < 0.0001); p(4), d 7 (P < 0.0001), d 10 (P = 0.001); p(3 )(premolar position 3 on mandible), d 14 (P = 0.037); p(4), d 17 (P = 0.024); occlusion, d 7 (P < 0.0001), d 10 (P = 0.003)]. By d 21 of age, this trend reversed such that piglets with premolars erupted and occluded spent more time at the feeder and visited it more frequently [duration: p(3), d 24 (P = 0.025); p(4), d 24 (P = 0.0005); occlusion, d 21 (P = 0.001), d 24 (P = 0.0001); visits: p(3), d 21 (P = 0.0002), d 24 (P < 0.0001); p(4), d 24 (P = 0.0002); occlusion, d 21 (P < 0.0001), d 24 (P < 0.0001)]. The percentages of piglets with positive fecal scores were 0, 1.4, 4.6, 8.0, 29.0, 44.9, and 60.6% on d 7, 10, 14, 17, 21, 24, and 27, respectively (P < 0.0001 between each day). No associations were found between the eruption or occlusion of premolars and feed consumption before weaning (P > 0.05), and no dental measures influenced growth rates (P > 0.10) or behavior (P > 0.10) after weaning. A more precise method may be necessary for detecting associations between dental eruption and feed consumption. However, the behavioral results indicate that, before weaning at 28 d, younger piglets are inhibited from feeding when their premolars first erupt, whereas older piglets with a more advanced dentition are more attracted to feed.

Normal profiles for deciduous dental eruption in domestic piglets: effect of sow, litter, and piglet characteristics.

The deciduous dentition of the domestic pig is comprised of 28 teeth (2 x incisors 3/3, canine 1/1, premolars 3/3, molars 0/0). The timing and sequence of deciduous dental eruption were determined from oral exams on 233 Yorkshire piglets from 0 to 5 wk of age. Eruption occurred sooner in gilts for all molariform premolars (p(3), p(4), and p(4), P < 0.01) and first incisor, i(1) (P = 0.004). Birth weight influenced eruption for all teeth except i(1) (i(1), p(3), p(3), p(4), and p(4); P < 0.01), with heavier piglets having earlier eruption. Average daily gain in wk 1 of life was associated with earlier eruption times of p(3) (P = 0.006), p(4) (P = 0.001), and i(1) (P = 0.001), whereas ADG during wk 2 was associated with earlier eruption for p(4) (P = 0.036). The parity (P = 0.025) and age (P = 0.013) of the sow were associated with earlier eruption of i(1). No litter characteristics were found to be significant. Sequence of eruption was determined to be i(1), p(3), p(4), i(1), p(3), p(4), although polymorphisms (reversals) were found to occur in over 40% of individuals of both sexes for mandibular i(1) and p(4) and maxillary p(3) and i(1). Size of the left i(3), which is already erupted at birth as part of the needle teeth dentition, was found to be larger in males (P = 0.026). Body weight gain was not associated with the size of i(3). Eruption times of p(3) and p(4) (the first premolars to erupt) occurred later than previously reported in the literature. Because these teeth are associated with initiation of feeding behavior for miniature breeds, implications of molar eruption on feeding behavior and feed intake should be considered.

Phospholemman does not participate in forskolin-induced swine carotid artery relaxation.

Phosphorylation of phospholemman (PLM) on ser68 has been proposed to at least partially mediate cyclic AMP (cAMP) mediated relaxation of arterial smooth muscle. We evaluated the time course of the phosphorylation of phospholemman (PLM) on ser68, myosin regulatory light chains (MRLC) on ser19, and heat shock protein 20 (HSP20) on ser16 during a transient forskolin-induced relaxation of histamine-stimulated swine carotid artery. We also evaluated the dose response for forskolin- and nitroglycerin-induced relaxation in phenylephrine-stimulated PLM-/- and PLM+/+ mice. The time course for changes in ser19 MRLC dephosphorylation and ser16 HSP20 phosphorylation was appropriate to explain the forskolin-induced relaxation and the recontraction observed upon washout of forskolin. However, the time course for changes in ser68 PLM phosphorylation was too slow to explain forskolin-induced changes in force. There was no difference in the phenylephrine contractile dose response or in forskolin-induced relaxation dose response observed in PLM-/- and PLM+/+ aortae. In aortae precontracted with phenylephrine, nitroglycerin induced a slightly, but significantly greater relaxation in PLM-/- compared to PLM+/+ aortae. These data are consistent with the hypothesis that ser19 MRLC dephosphorylation and ser16 HSP20 phosphorylation are involved in forskolin-induced relaxation. Our data suggest that PLM phosphorylation is not significantly involved in forskolin-induced arterial relaxation.

Gene structure and expression of phospholemman in mouse.

Phospholemman (PLM) is a small transmembrane cardiac protein that is the major sarcolemmal substrate for phosphorylation in response to adrenergic stimulation. PLM likely plays a role in muscle contractility and cell volume regulation through its function as a channel or a channel regulator. We are the first to describe the structure of the PLM gene and to demonstrate PLM cDNA splice variants. We cloned the murine PLM cDNA and used it as a probe to isolate the gene from a 129/SvJ genomic library. The gene contains seven introns and eight exons. The coding sequence is interrupted by five introns; the 5' untranslated region by two. Using rapid amplification of 5' cDNA ends we identified transcription start sites and four splice variants of the 5' untranslated domain. There was no TATA box or CAAT box in the putative promoter regions. The gene has several stretches of dinucleotide repeats. The 3' untranslated domains of mouse PLM cDNA clones show sequence differences not accounted for by alternative splicing. Mouse PLM shares 93, 83 and 80% amino acid identity with rat, dog, and human PLMs, respectively. Tissue expression of murine PLM parallels that in other species, being highest in heart, skeletal muscle, and liver.

Dominance of G(s) in doubly G(s)/G(i)-coupled chimaeric A(1)/A(2A) adenosine receptors in HEK-293 cells.

A(1) adenosine receptors inhibit adenylate cyclase by activating G(i)/G(o), whereas A(2A) receptors activate G(s). We examined how regions of A(1) and A(2A) receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to G(i)/G(o) and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A(1)/A(2A) 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A(1) 3ICL sequence (A(2A)/A(1)L or A(2A)/A(1)LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to G(i)/G(o). These chimaeras have basal cAMP levels lower than those of wild-type A(2A) receptors, similar to wild-type A(1) receptors. The A(1) C-terminus modulates the coupling of receptors with A(1) 3ICL such that A(2A)/A(1)LT is better coupled to G(i)/G(o) than A(2A)/A(1)L. The C-terminus has little impact on coupling to receptors containing A(2A) 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to G(i)/G(o) mediated by A(1) 3ICL and not by other intracellular domains that favour G(i) coupling. The C-terminus sequence has little or no effect on coupling to G(s). For doubly G(s)/G(i)-coupled adenosine receptors in HEK-293 cells, G(s)-mediated stimulation predominates over G(i)/G(o)-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus.

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively influences the binding of the ribose-substituted adenosine analog, 5'-N-ethylcarboxamidoadenosine to a variable extent, depending on the nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Cloned receptors and cardiovascular responses to adenosine.

Four subtypes of adenosine receptors have recently been cloned from thyroid, brain and testis. In this review we have summarised properties of these purinergic receptors. The cloned A1 and A2 subtypes are probably similar or identical to receptors that exist on cardiac and vascular tissues, respectively. A comparison of the amino acid sequences of A1, A2a, and A2b receptors reveals several stretches of conserved amino acids that are unique to adenosine receptors, primarily in the membrane spanning regions. Species differences in A1 receptors indicate that minor changes in receptor structure can produce marked changes in ligand binding properties and may facilitate the identification of amino acids involved in ligand recognition. A confusing A1 receptor subclassification system of putative A1a, A1b, and A3 subtypes has emerged based on subtle rank order potency differences for various ligands among tissues. cDNAs corresponding to these A1 subtypes have not yet been isolated. Atrial A1 receptors activate K+ channels and inhibit adenylyl cyclase. These two pathways appear to be independently up and down regulated, suggesting the existence either of atrial A1 receptor subtypes or of differential regulation of the coupling of a single receptor to distinct GTP binding proteins. An adenosine receptor distinct from A1 and A2 receptors has been cloned from testis and designated TGPCR, or A3, although it differs from the pharmacologically defined A3 receptor. We suggest that the current A1/A3 receptor subtype nomenclature be abandoned and superseded by a nomenclature based solely on receptor cDNAs. In addition to the cloned adenosine receptors, a novel A4 subtype has been proposed based on pharmacological and electrophysiological criteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a bovine adenosine A1 receptor cDNA.

A bovine brain adenosine A1 receptor cDNA encoding a 326 amino acid protein has been identified. This cDNA, which encodes a protein greater than 90% identical to analogous rat and dog receptors, was transiently expressed in COS-1 cells. Recombinant receptors exhibited the features of bovine A1 receptors that distinguish it from rat and canine receptors, including subnanomolar Ki for 1,3-dipropyl-8-cyclopentylxanthine, R-phenylisopropyl- adenosine (R-PIA) and xanthine amino conjugate, and the distinct potency order: R-PIA greater than S-PIA much greater than 5'-N-ethylcarboxamidoadenosine greater than 2'-chloroadenosine. The results indicate that the pharmacological differences between A1 adenosine receptors among species result from only minor differences in receptor structures.

Molecular characterization of a rat alpha 2B-adrenergic receptor.

Alpha 2-adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. We have isolated a cDNA clone (pRNG alpha 2) encoding a rat alpha 2-adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNG alpha 2 DNA display high affinity and saturable binding to [3H]rauwolscine (Kd = 2 nM). Competition curve data analysis shows that RNG alpha 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine greater than or equal to chlorpromazine greater than or equal to prazosin greater than or equal to clonidine greater than norepinephrine greater than or equal to oxymetazoline. RNG alpha 2 RNA accumulates in both rat kidney and neonatal rat lung (predominant species is 4000 nucleotides). When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNG alpha 2 protein fails to bind [3H]rauwolscine after expression in COS cells. We conclude that pRNG alpha 2 likely represents a cDNA for a rat alpha 2B-adrenergic receptor.

Heat stroke in infancy.

We observed a 16-month-old infant with residual brain damage following a heat stroke from being left in a parked automobile. In contrast with adults, in whom heat stroke usually follows strenuous exercise, the condition in infants usually results from excessive environmental temperature and/or dehydration. Early recognition of the illness is imperative. Three cardinal freatures are hot, dry skin, central nervous system disturbance, and hyperpyrexia. Immediate treatment should be aimed at improving circulation with volume expanders and rapid cooling. Other supportive measures may be necessary to control seizures, renal failure, hematologic abnormalities, or hepatic involvement.