Short communication: Investigation of antibiotic alternatives to improve health and growth of veal calves.

The inherent disease susceptibility of veal calves results in frequent antimicrobial use. Improvements in antimicrobial stewardship necessitate alternative therapies to improve calf health and growth, while reducing the need for antimicrobials important to human health. This study investigated the effect of 2 alternative therapies, lactoferrin (an iron-binding protein found in colostrum) and cinnamaldehyde (an essential oil of the cinnamon plant) on growth, disease incidence, and mortality risk in special-fed veal calves. On the day of arrival to the growing facility (3 to 7 d of age), calves (n = 80 per treatment) were randomized to 1 of 3 treatments: (1) control (no supplement), (2) lactoferrin (1 g/d in milk replacer for 7 d), or (3) cinnamaldehyde (1 g/d in milk replacer for 21 d). Body weight was measured on the day of arrival (d 0), 21, and 42 d postarrival. Health assessments were performed twice weekly through 21 d, and mortality records were obtained through 6 wk postarrival. A repeated-measures ANOVA was used to compare growth between treatment groups, and a Poisson regression model (PROC GENMOD, SAS v. 9.4, SAS Institute Inc., Cary, NC) was used to test differences between groups in the incidence of diarrhea (fecal score ≥2 with and without depression and temperature) and disease through 3 wk postarrival. Body weight and average daily gain were similar between treatments. Neither lactoferrin nor cinnamaldehyde had an effect on diarrhea incidence. However, the risk of navel inflammation was significantly lower for calves that received cinnamaldehyde compared with calves in the control group. Mortality through 6 wk postarrival was low, with 4, 1, and 0 deaths from the control, lactoferrin, and cinnamaldehyde treatment groups, respectively. Additional research is needed to investigate various doses of these alternative therapies on calf health and growth, in addition to different routes of administration.

Dairy calf management-A comparison of practices and producer attitudes among conventional and organic herds.

Dairy calves are at high risk for morbidity and mortality early in life. Understanding producer attitudes is important for implementation of best management practices to improve calf health. The objectives of this study were to evaluate usage frequency and producer attitudes on key calf management practices between conventional and organic dairy operations. A cross-sectional survey was mailed to conventional and organic dairy producers in Ohio and Michigan that included questions on cow-calf separation, colostrum management, and vaccination use. The overall survey response rate was 49% (727/1,488); 449 and 172 conventional and organic producer respondents, respectively, were included in the final analysis. Binary, cumulative, and multinomial logistic regression models were used to test differences within and between herd types for management practices and producer attitudes. The majority of conventional (64%, 279/439) producers reported separating the calf from the dam 30 min to 6 h after birth. More organic (34%, 56/166) than conventional (18%, 80/439) producers reported separation 6 to 12 h after birth, and organic producers were more likely to agree time before separation is beneficial. Few conventional (10%, 44/448) and organic (3%, 5/171) producers reported measuring colostrum quality. Most conventional producers (68%, 304/448) hand-fed the first feeding of colostrum, whereas the majority of organic producers (38%, 69/171) allowed calves to nurse colostrum. Last, 44% (188/430) of conventional producers reported vaccinating their calves for respiratory disease, compared with 14% (22/162) of organic producers; organic producers were more likely to perceive vaccines as ineffective and harmful to calf health. Thus, the usage frequency and perceived risks and benefits of calf management practices vary considerably between conventional and organic dairy producers. These findings provide helpful information to understand decision making at the herd level regarding key calf management and health practices, regardless of production systems.

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung.

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.

Biodegradability of dispersed crude oil at two different temperatures.

Laboratory experiments were initiated to study the biodegradability of oil after dispersants were applied. Two experiments were conducted, one at 20 degrees C and the other at 5 degrees C. In both experiments, only the dispersed oil fraction was investigated. Each experiment required treatment flasks containing 3.5% artificial seawater and crude oil previously dispersed by either Corexit 9500 or JD2000 at a dispersant-to-oil ratio of 1:25. Two different concentrations of dispersed oil were prepared, the dispersed oil then transferred to shake flasks, which were inoculated with a bacterial culture and shaken on a rotary shaker at 200 rpm for several weeks. Periodically, triplicate flasks were removed and sacrificed to determine the residual oil concentration remaining at that time. Oil compositional analysis was performed by gas chromatography/mass spectrometry (GC/MS) to quantify the biodegradability. Dispersed oil biodegraded rapidly at 20 degrees C and less rapidly at 5 degrees C, in line with the hypothesis that the ultimate fate of dispersed oil in the sea is rapid loss by biodegradation.

Intravenously administered oligonucleotides can be delivered to conducting airway epithelium via the bronchial circulation.

Topical gene transfer to the airways of cystic fibrosis (CF) patients has been inefficient, partly due to extracellular barriers such as sputum. In an attempt to circumvent these, we assessed whether airway epithelial cells can be transfected by intravenous (i.v.) administration of liposome-complexed or "naked" oligonucleotides (ODNs). The conducting airways are the likely target for CF therapy and are supplied by the bronchial circulation. Consequently, we assessed ODN transfer in the mouse trachea and main bronchi as these are supplied by the bronchial circulation. Liposome-protamine-DNA (LPD) complexes were detected in the bronchial circulation but did not transfect conducting airway epithelial cells, even in the presence of microvascular leakage. In contrast, 'naked' ODNs were delivered to 17% (inter-quartile range (IQR) 10-34%) and 35% (IQR 24-59%) of epithelial cells when injected at 500 microg/animal, without and with microvascular leakage, respectively. Two types of nuclear signal were observed; punctate in cells throughout the airways (3%, IQR 2-6%, and 6%, IQR 4-7%, of cells when delivered without and with microvascular leakage, respectively) and diffuse in a small number of epithelial cells in the proximal trachea. ODNs may be relevant to CF in a variety of ways and these data suggest one way towards implementing their use.

Protocol for laboratory testing of crude-oil bioremediation products in freshwater conditions.

In 1993, the Environmental Protection Agency, National Risk Management Research Laboratory (EPA, NRMRL), with the National Environmental Technology Application Center (NETAC), developed a protocol for evaluation of bioremediation products in marine environments [18]. The marine protocol was adapted for application in freshwater environments by using a chemically defined medium and an oil-degrading consortium as a positive control. Four products were tested using the modified protocol: two with nutrients and an oleophilic component; one with nutrients, sorbent, and organisms; and one microbial stimulant. A separate experiment evaluated the use of HEPES and MOPSO buffers as replacements for phosphate buffer. The oleophilic nutrient products yielded oil degradation similar to the positive control, with an average alkane removal of 97.1+/-2.3% and an aromatic hydrocarbon removal of 64.8+/-1.2%. The positive control, which received inoculum plus nutrients, demonstrated alkane degradation of 98.9+/-0.1% and aromatic degradation of 52.9+/-0.1%. The sorbent-based product with inoculum failed to demonstrate oil degradation, while the microbial stimulant showed less oil degradation than the positive control. Replacement of phosphate buffer with other buffers had no significant effect on one product's performance. Differences in product performance were easily distinguishable using the protocol, and performance targets for alkane and aromatic hydrocarbon degradation are suggested.

Lengthening osteotomy at the intertrochanteric level with simultaneous correction of angular deformities.

Fixed multiplane deformities around the hip representing a combination of rotational, angular, and leg-length discrepancies are disabling for the patient and pose a challenge for the orthopaedic surgeon. We describe a joint-preserving, one-stage procedure to address these complex problems using an intertrochanteric osteotomy with a step-cut to allow for corrective lengthening, angulation, and rotation. We present our results in nine patients. The aim of this paper is to provide the orthopaedic surgeon with a detailed approach to treating these deformities with reproducible results.

Microtubules modulate cardiomyocyte beta-adrenergic response in cardiac hypertrophy.

The role of microtubules in modulating cardiomyocyte beta-adrenergic response was investigated in rats with cardiac hypertrophy. Male Sprague-Dawley rats underwent stenosis of the abdominal aorta (hypertensive, HT) or sham operation (normotensive, NT). Echocardiography and isolated left ventricular cardiomyocyte dimensions demonstrated cardiac hypertrophy in the HT rats after 30 wk. Cardiomyocyte microtubule fraction was assayed by high-speed centrifugation and Western blot. In contrast to previous reports of increased microtubules after acute pressure overload, microtubule fraction for HT was significantly lower than that for NT. Cardiomyocytes were exposed to either 1 microM colchicine, 10 microM taxol, or equivalent volume of vehicle. Colchicine decreased microtubules, and taxol increased microtubules in both groups. Cardiomyocyte cytosolic calcium ([Ca2+]c) and shortening/relaxation dynamics were assessed during exposure to increasing isoproterenol concentrations. The beta-adrenergic response for these variables in the HT group was blunted compared with NT. However, increased microtubule assembly by taxol partially recovered the normal beta-adrenergic response for time to peak [Ca2+]c, time to peak shortening, and mechanical relaxation variables. Microtubule assembly may play a significant role in determining cardiomyocyte beta-adrenergic response in chronic cardiac hypertrophy.

Two-dimensional visualization of liquid layers on transparent walls.

A new two-dimensional visualization technique based on laser-induced fluorescence for investigation of liquid-fuel films on transparent walls was applied in an SI engine. For optical access the upper part of the engine cylinder is replaced with a quartz ring. An UV laser beam is coupled into the ring, forcing total reflection at quartz-air interfaces. Because of the similar indices of refraction of quartz and fuel, laser light penetrates into the liquid. The corresponding fluorescence signal visualizes areas where wall film is present. It is shown that the technique is an excellent tool for investigation of wall-film development in combustion engines.

Dilated cardiomyopathy associated with deficiency of the cytoskeletal protein metavinculin.

BACKGROUND: The cytoskeleton plays an important role in maintaining cell structure and integrity. Defects in cytoskeletal proteins can cripple cell strength and may cause cardiomyopathy. We analyzed heart tissues from subjects with dilated cardiomyopathy for abnormalities in the cardiac cytoskeleton. Metavinculin, a cardiac isoform of the cytoskeletal protein vinculin, connects actin microfilaments to the intercalated disk and membrane costameres of the heart. METHODS AND RESULTS: Metavinculin and vinculin transcripts and protein were analyzed by polymerase chain reaction (PCR) and Western blotting. Thirty-three human heart specimens were studied, including 5 normal controls, 4 subjects with ischemic cardiomyopathy, 1 with X-linked cardiomyopathy, and 23 with idiopathic dilated cardiomyopathy (IDC). PCR of cardiac cDNA detected absence of the metavinculin transcript in cardiac tissue from a subject with IDC. PCR of genomic DNA showed that the metavinculin exon was present but not utilized in the cardiac transcript. Western blot analysis demonstrated absence of metavinculin protein in the heart from this subject. Immunostaining of cardiac vinculin in this heart showed disorganized intercalated disk structures. Metavinculin deficiency was associated with normal cardiac expression of the cytoskeletal proteins vinculin, alpha-actinin, and dystrophin. Normal metavinculin expression in the other heart specimens suggests that the defect is specific in the IDC subject identified. CONCLUSIONS: These results demonstrate an association between metavinculin deficiency and dilated cardiomyopathy due to a defect in alternative mRNA splicing.

Nonsteroidal anti-inflammatory drugs and necrotising fasciitis. An update.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen and others are the treatment of choice for mild to moderate pain. Because of the relative safety and efficacy of NSAIDs, many of the agents are now available in the US and in other parts of the world without a physician prescription. While these drugs are relatively well tolerated, adverse effects resulting from their use can occur. One such adverse effect recently linked to NSAID use is necrotising fasciitis. Reports of necrotising fasciitis possibly associated with NSAID use have been published in both the medical and lay literature. Several hypotheses regarding a possible association between NSAIDs and the development of necrotising fasciitis have appeared in the literature. One hypothesis is a simple masking of the signs and symptoms of an existing infection, leading to a delay in diagnosis. Some authors have speculated that in certain skin and soft-tissue infections, particularly those caused by group A beta-haemolytic streptococci, this delay in diagnosis may have allowed a simple infection to progress to necrotising fasciitis. Other postulated mechanisms of NSAID involvement in the development of necrotising fasciitis include an impairment of natural host defense mechanisms. A review of the medical literature for reports of possible NSAID-associated necrotising fasciitis revealed that the events were rare, but clinically significant. From the available evidence, a causal relationship between NSAIDs and necrotising fasciitis cannot be established.

A 5' dystrophin duplication mutation causes membrane deficiency of alpha-dystroglycan in a family with X-linked cardiomyopathy.

5'-mutations in the dystrophin gene can result in cardiomyopathy without clinically-apparent skeletal myopathy. The effect of dystrophin mutations on the assembly and stability of the dystrophin associated protein (DAP) complex in human heart are not fully understood. The molecular defect in the dystrophin complex was explored in a family with an X-linked pedigree and severe dilated cardiomyopathy. Dystrophin gene analysis demonstrated a 5' duplication involving exons 2-7, which encodes the N-terminal actin binding domain of dystrophin. Ribonuclease protection and PCR assays demonstrated a reduction in muscle promoter transcribed dystrophin mRNA in the heart compared to skeletal muscle. A deficiency of cardiac dystrophin protein was observed by Western blot and lack of membrane localization by immunocytochemistry. The cardiac expression of the dystrophin related protein utrophin was increased, and the 43 kDa (beta-dystroglycan), 50 kDa (alpha-sarcoglycan) and 59 kDa (syntrophin) dystrophin associated proteins (DAPs) were co-isolated and present in nearly normal amounts in the membrane. However, cardiac dystrophin deficiency and increased utrophin expression were associated with loss of extracellular 156 kDa dystrophin associated glycoprotein (alpha-dystroglycan) binding to the cardiomyocyte membrane. alpha-Dystroglycan is responsible for linkage of the dystrophin complex to the extracellular matrix protein laminin. Therefore, 5' dystrophin mutations can reduce cardiac dystrophin mRNA, protein expression, and dystrophin function in X-linked cardiomyopathy (XLCM). The presence of membrane-associated beta-dystroglycan, alpha-sarcoglycan, syntrophin, and utrophin are insufficient to maintain cardiac function. This XLCM family has a 5' dystrophin gene mutation resulting in cardiac dystrophin deficiency and a loss of alpha-dystroglycan membrane binding.

Expression and regulation of the dystrophin Purkinje promoter in human skeletal muscle, heart, and brain.

Dystrophin mRNA transcripts from the P (Purkinje) promoter were shown to be differentially expressed in human skeletal muscle, heart, and brain. The expression pattern was characteristic of tissue type and developmental stage. Polymerase chain reaction (PCR) analysis of the P promoter transcripts in adult skeletal muscle and adult brain identified two alternatively spliced sequences, one that encodes a full-length dystrophin mRNA and a second that transcribes a termination codon 27 nucleotides (8 amino acids) after the ATG initiation site. Alternative splicing of this truncated coding transcript was developmentally regulated, and it was expressed as the major form in adult cortical brain and adult heart. The biological significance of this peptide remains unclear. The full-length transcript was the major form in fetal cortical brain and adult skeletal muscle. Ribonuclease protection assay demonstrated that as much as 20% of dystrophin transcription in normal adult skeletal muscle was derived from the full-length transcript from the P promoter. In contrast, adult heart did not express significant levels of P promoter derived transcripts. Thus, transcripts from the P promoter were found to be developmentally regulated in the brain, and its activity was differentially expressed in skeletal versus cardiac muscle tissues. These data show that the P promoter transcript displays a broader scope of expression, regulation, and complexity than previously appreciated.

Nonsteroidal anti-inflammatory drugs and necrotizing fasciitis.

The clinical features, etiology, and treatment of necrotizing fasciitis are reviewed, and the possible involvement of nonsteroidal anti-inflammatory drugs (NSAIDs) in the pathogenesis of this disease is discussed. Symptoms of necrotizing fasciitis include edema, erythema, warmth, and tenderness in the affected area, spreading rapidly to involve entire limbs or portions of the abdominal wall. Gangrene may develop, followed by multi-organ failure and death. The most common causative organisms are group A beta-hemolytic streptococci. Treatment includes tissue debridement, intravenous antibiotics, and supportive treatment with fluids and electrolytes. A number of case reports and articles in the lay press have suggested an association between the use of NSAIDs and the development of necrotizing fasciitis, although this association has not been substantiated in the scientific literature. Inhibition of the immune response has been proposed as an explanation for the reported association between necrotizing fasciitis and NSAIDs. NSAIDs should be administered with caution, if at all, to patients with inflammatory soft-tissue lesions, especially if concurrent infection is likely.

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure.

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbon-degrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

Molecular remodelling in hypertrophied hearts from polyomavirus large T-antigen transgenic mice.

Polyomavirus large T-antigen transgenic mice develop cardiac hypertrophy characterized by an increase in atrial natriuretic factor and beta-myosin heavy chain isoform expression. The aim of this study was to examine changes in proto-oncogene expression in hypertrophied hearts from the transgenic mice. Expression of early growth response-1 (Egr-1) mRNA was detected in hearts from all 15 transgenic mice, but was not detectable in 13 control mice. Reverse transcriptase-polymerase chain reaction experiments using Egr-1-specific primers confirmed the increase in Egr-1 mRNA in enlarged hearts from the transgenic mice. Expression of c-jun, junD and Ha-ras mRNAs was increased in the transgenic hearts 3, 17 and 2.8-fold respectively. Western blots showed an increase in c-myc, c-jun and ras protein in hypertrophied transgenic hearts. Immunofluorescence analyses confirmed an increase in Egr-1 and c-jun protein in transgenic cardiomyocytes. Proliferating cell nuclear antigen, Ki-ras and HSP 90 mRNAs were decreased 22, 2.7 and 3-fold, respectively in the transgenic hearts. Not altered in most hypertrophied hearts was expression of c-fos, junB, p53, c-neu, c-myc, HSP70, HSP27, TGF-beta or IGF 1 mRNAs. Proto-oncogene and growth factor gene expression in hypertrophy induced by PVLT expression is modulated with some proto-oncogenes increased and others decreased in expression.

Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein.

The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.