High deoxynivalenol and ergot alkaloid levels in wheat grain: effects on growth performance, carcass traits, rumen fermentation, and blood parameters of feedlot cattle.

This study was designed to assess the impacts of a mixture of deoxynivalenol (DON) and ergot alkaloids (EAs) on growth performance, rumen function, blood parameters, and carcass traits of feedlot cattle. Forty steers (450 ± 6.0 kg) were stratified by weight and randomly allocated to 1 of 4 treatments; control-low (CON-L), control-high (CON-H) which contained low or high wheat screenings that lacked mycotoxins at the same level as the mycotoxin-low (MYC-L; 5.0 mg/kg DON, 2.1 mg/kg EA), and mycotoxin-high (MYC-H: 10 mg/kg DON, 4.2 mg/kg EA) diets that included wheat screening with mycotoxins. Steers were housed in individual pens for a 112-day finishing trial. Intake was 24.8% lower (P < 0.001) for MYC steers compared to CON steers. As a result, average daily gains of MYC steers were 42.1% lower (P < 0.001) than CON steers. Gain to feed ratio was also lower (P < 0.001) for MYC steers compared to CON steers. Platelets, alanine aminotransferase, globulins, and blood urea nitrogen were lower (P ≤ 0.008), and lymphocytes, glutathione peroxidase activity (GPx), and interleukin-10 (IL-10) were elevated (P ≤ 0.002) in MYC steers compared to CON steers. Hot carcass weights and backfat thickness were reduced (P < 0.001) in MYC steers, resulting in leaner (P < 0.001) carcasses and higher (P < 0.007) meat yield compared to CON steers. Results suggest that a mixture of DON and EAs negatively impacted health, performance, and carcass traits of feedlot steers, with the majority of this response likely attributable to EAs. However, more research is needed to distinguish the relative contribution of each mycotoxin to the specific responses observed.

Invited review: Application of meta-omics to understand the dynamic nature of the rumen microbiome and how it responds to diet in ruminants.

Ruminants are unique among livestock due to their ability to efficiently convert plant cell wall carbohydrates into meat and milk. This ability is a result of the evolution of an essential symbiotic association with a complex microbial community in the rumen that includes vast numbers of bacteria, methanogenic archaea, anaerobic fungi and protozoa. These microbes produce a diverse array of enzymes that convert ingested feedstuffs into volatile fatty acids and microbial protein which are used by the animal for growth. Recent advances in high-throughput sequencing and bioinformatic analyses have helped to reveal how the composition of the rumen microbiome varies significantly during the development of the ruminant host, and with changes in diet. These sequencing efforts are also beginning to explain how shifts in the microbiome affect feed efficiency. In this review, we provide an overview of how meta-omics technologies have been applied to understanding the rumen microbiome, and the impact that diet has on the rumen microbial community.

Characterization of the rumen and fecal microbiome in bloated and non-bloated cattle grazing alfalfa pastures and subjected to bloat prevention strategies.

Frothy bloat is an often fatal digestive disorder of cattle grazing alfalfa pastures. The aim of this study was to investigate ruminal and fecal microbiota dynamics associated with development of alfalfa-induced frothy bloat and to further explore how bloat prevention strategies influence the composition of these microbial communities. In a 3 × 3 crossover experiment, twelve rumen-cannulated steers were sequentially subjected to: (1) pure alfalfa pasture, (2) pure alfalfa pasture supplemented with the pluronic detergent ALFASURE, and (3) alfalfa - sainfoin mixed pasture. Eleven out of 12 steers in pure alfalfa pasture developed clinical bloat, whereas ALFASURE treatment prevented the development of bloat in all 12 steers and alfalfa - sainfoin prevented bloat in 5 out of 11 steers. Development of bloat was associated with considerable shifts in the microbiota profile of rumen contents. In particular, the microbiota of solid rumen contents from bloated steers contained higher species richness and diversity. Streptococcus, Succinivibrio and unclassified Myxococcales were enriched in the rumen microbiota of bloated steers, whereas Fibrobacter and Ruminococcus were overrepresented in the rumen contents of non-bloated steers. Our results provide novel insights into bloat-associated shifts in the composition and predicted functional properties of the rumen microbiota of cattle grazing alfalfa pasture.

Isolation of High Quality RNA for Metatranscriptomic Analysis of Lignocellulose Digestion in the Rumen.

Metatranscriptomics can be used to examine both the composition of a microbial community as well as its metabolic activity under a particular set of conditions and complement metagenomic studies. The availability of low-cost, high-throughput next-generation sequencing has led to a rapid increase in the number of metatranscriptomic studies being undertaken. One of the primary difficulties when conducting transcriptomics is the ability to isolate high-quality RNA from samples of interest. The application of metatranscriptomics in rumen microbiology is still relatively novel but there is a significant push toward applying this technology in this field. In this protocol, we outline the method that is used routinely in our laboratory to purify high quality RNA from rumen contents that are suitable for metatranscriptomic sequencing using RNA-seq.

Biochemical and kinetic characterization of the multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase, Bgxa1.

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, ß-glucosidase/ß-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of ß-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-ß-D-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-α-D-arabinofuranoside (pNPAf), suggesting it is a multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of ß-glucosidase activity was 100× greater than ß-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0-10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.

Invasive group A streptococcal infections in North Carolina: epidemiology, clinical features, and genetic and serotype analysis of causative organisms.

During 1994 and 1995, an increase in the number and severity of group A streptococcal (GAS) infections was noted in North Carolina. Ninety-six patients had GAS recovered from blood and other sterile body fluids, abscesses, and soft tissue. The overall case fatality rate was 11% but was much higher in patients with toxic shock syndrome (55%) and necrotizing fasciitis (58%). Recent invasive GAS isolates were compared with pre-1994 invasive isolates and temporally related pharyngeal isolates by M protein serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction amplification of the streptococcal pyrogenic exotoxin A gene. Serotypes M1 and M3 accounted for 50% of recent invasive isolates (1994-1995) and 58% of pharyngeal isolates (1994). The latter isolates demonstrated PFGE patterns that were identical to invasive M1 and M3 strains, suggesting that pharyngeal infections may have served as a reservoir for virulent GAS clones.

Mutagenesis studies of the human erythropoietin receptor. Establishment of structure-function relationships.

Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding. We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR. Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats. In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I-EPO binding in receptor binding assays. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand.

Use of viral cultures and serologic tests for cytomegalovirus infection. Rational or random?

To assess their applied clinical utility, viral cultures and serological tests for cytomegalovirus (CMV) were reviewed at Duke University Medical Center (DUMC), a 1,125-bed tertiary-care hospital. Less than 1% (3 of 1,216) of CMV cultures were positive, and 8% of serum samples (45 of 587) were positive by single sera IgM ELISA. Sixteen percent (32 of 199) of IgG acute to convalescent sera pairs were positive. Four hundred five of 588 (69%) serum samples were positive for the IgM/IgG passive latex agglutination test, consistent with the results for random blood donors. Review of hospital records showed that fewer than 1% of the positive-test patients (excluding the latex test) received treatment for CMV. Comparisons of tests ordered on individual patients did not disclose a coherent diagnostic strategy. The authors conclude that the majority of testing for CMV in their medical center does not yield useful clinical information, but carries a substantial financial burden. A new diagnostic strategy to attempt to diagnose CMV disease is needed.

Identification of a critical ligand binding determinant of the human erythropoietin receptor. Evidence for common ligand binding motifs in the cytokine receptor family.

The erythropoietin receptor (EPOR) is a member of a family of cytokine and growth factor receptors that share conserved features in their extracellular and cytoplasmic domains. We have used site-specific mutagenesis within the extracellular domain of the EPOR to search for amino acid residues involved in erythropoietin (EPO) binding. Mutant proteins were expressed in bacteria as soluble EPO binding proteins (EBP) and characterized for EPO binding activity in a number of different assays. Substitution of phenylalanine at position 93 (Phe93) with alanine (F93A mutation) resulted in a drastic reduction in EPO binding in the EBP. More conservative tyrosine or tryptophan substitutions at Phe93 resulted in much less dramatic effects on EPO binding. Biophysical studies indicated that the F93A mutation does not result in gross structural alterations in the EBP. Furthermore, the F93A mutation in full-length EPOR expressed in COS cells abolished detectable EPO binding. This was not a result of processing or transport defects, since mutant receptor was present on the surface of the cells. Mutations in the region immediately around Phe93 and in residues homologous to other reported ligand binding determinants of the cytokine receptor family had small to moderate effects on EPO binding. These data indicate that Phe93 is a critical EPO binding determinant of the EPOR. Furthermore, since Phe93 aligns with critical ligand binding determinants in other receptors of the cytokine receptor family, these data suggest that receptors of this family may use common structural motifs to bind their cognate ligands.

Erythropoietin receptor: application in drug development.

Erythropoietin (EPO) is the primary hormone responsible for the growth and maturation of red blood cells in mammals. In contrast to many other growth factors, the specificity of EPO for mature erythroid cells has lead to its development as a safe and efficacious therapeutic, EPREX. The medical benefits of EPREX have been well established in the treatment of anaemic chronic renal failure patients, anaemic HIV patients treated with AZT, cancer chemotherapy patients, and patients wishing to donate their own blood prior to elective surgery (autologous predonation). Due to the chronic nature of EPO therapy, it would be desirable to have an orally administered 'second generation' molecule. An understanding of the structural basis of the interaction of EPO with its receptor will aid in the design of an oral anaemia drug. In this study, a series of mutations have been generated in a truncated form of the receptor comprising the extracellular region, termed EPO binding protein (EBP). One mutant, in which alanine replaces phenylalanine at position 93 (F93A) has a 500-fold reduction in binding compared to wild-type EBP. A neutralizing anti-EBP antibody binds poorly to the F93A mutant, while a non-neutralizing anti-EBP antibody binds wild-type and F93A equally well. Information from this mutational analysis can be applied to a receptor 3-D model and ultimately used in drug development.

Erythropoietin structure-function relationships: high degree of sequence homology among mammals.

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

A randomized study of ciprofloxacin versus ceftriaxone in the treatment of nursing home-acquired lower respiratory tract infections.

OBJECTIVE: To compare the efficacy and safety of ciprofloxacin and ceftriaxone in patients with nursing home-acquired lower respiratory tract infections requiring initial hospitalization. DESIGN: Prospective, randomized trial. SETTING: Extended care nursing homes affiliated with a teaching hospital. PATIENTS: Fifty patients aged 60 years or older with normal or mildly impaired renal function admitted to the hospital for treatment of lower respiratory tract infections. INTERVENTIONS: Twenty-four patients received initial therapy with intravenous ciprofloxacin, 200 mg every 12 hours (19 patients) or 400 mg every 12 hours (5 patients) during the acute phase followed by 750 mg orally every 12 hours during the convalescence phase. Twenty-six patients received initial therapy with intravenous ceftriaxone, 2 g every 24 hours during the acute phase followed by 1 g administered intramuscularly every 24 hours during the convalescent phase. The total duration of therapy was 14 days. MAIN OUTCOME MEASUREMENTS: Successful outcome was defined as resolution or marked improvement in clinical signs and symptoms of lower respiratory tract infection upon completion of the treatment course. RESULTS: Twelve (50%) of the ciprofloxacin-treated and 14 (54%) of ceftriaxone-treated patients had successful outcomes. Recurrent oropharyngeal aspiration was the reason for treatment failure in most patients refractory to either antibiotic. Mortality during therapy was 8% in each group. From 21 satisfactory sputum specimens collected, S. pneumoniae was the most common isolate, followed by H. influenzae and other Gram-negative bacteria. Ciprofloxacin therapy was well tolerated; ceftriaxone therapy was discontinued in two patients (8%) due to adverse reactions (intramuscular pain and drug fever). CONCLUSIONS: Sequential intravenous/oral ciprofloxacin appears to be as safe and effective as sequential intravenous/intramuscular ceftriaxone. The optimal dosage of intravenous ciprofloxacin in this patient population appears to be 400 mg every 12 hours; however, additional clinical and pharmacokinetic studies with this regimen are warranted.

The effect of anaerobiosis on antistaphylococcal antibiotics.

The activity of eight antimicrobial agents which might be used in the treatment of staphylococcal osteomyelitis was tested under anaerobic conditions similar to those found in chronically infected bone. An agar-dilution method was employed to determine the minimum inhibitory concentrations of tobramycin, vancomycin, teicoplanin, ciprofloxacin, clindamycin, ceftriaxone, ticarcillin-clavulanic acid, and amoxicillin-clavulanic acid against 25 coagulase-positive and 25 coagulase-negative staphylococcal strains. The activity of tobramycin against coagulase-positive staphylococci, and of amoxicillin-clavulanic acid and ticarcillin-clavulanic acid against coagulase-negative staphylococci was markedly decreased with anaerobiosis. Vancomycin, teicoplanin, and ciprofloxacin were active against coagulase-positive and coagulase-negative staphylococci under both aerobic and anaerobic conditions. It was also found that antibiotic concentrations comparable to the high levels which might be achieved with local antibiotic therapy of osteomyelitis were not sufficient to overcome the level of resistance (100 micrograms/ml) of staphylococci which were not susceptible to tobramycin, clindamycin, ceftriaxone, and ticarcillin-clavulanic acid.

Prospective study of lower respiratory tract infections in an extended-care nursing home program: potential role of oral ciprofloxacin.

PURPOSE: Infections of the lower respiratory tract pose an important problem in nursing homes. Despite the magnitude of this problem, few, if any, antibiotic studies have been targeted specifically at nursing home-acquired bronchopulmonary infections. Following the establishment of a teaching Extended-Care Nursing Home Program, which facilitated the early diagnosis and therapy of bronchopulmonary infections, a comparative trial of oral ciprofloxacin and intramuscular cefamandole was initiated in elderly patients with lower respiratory tract infections. In addition to assessing the relative efficacy and safety of ciprofloxacin and cefamandole, our goals were to identify problems and pitfalls associated with conducting clinical research in this nursing home setting, evaluate selected clinical and laboratory features of lower respiratory tract infection in this patient population, and measure outcomes in all study groups. PATIENTS AND METHODS: During a 20-month period, 40 patients with pneumonia and 20 patients with acute bronchitis were enrolled in this randomized study. Sixty-three patients with pneumonia who were ineligible for the randomized study were also followed prospectively. The mean age of the 111 participants (123 cases) was 80.8 years; all patients had at least one chronic medical condition. RESULTS: Although Streptococcus pneumoniae was the single most common isolate, gram-negative bacteria were cultured from 81 percent of the cases that yielded pathogens from a satisfactory sputum specimen. The in-hospital mortality rate was strikingly low (6.5 percent), and a large majority of patients in all study groups were discharged safely back to their nursing homes well within the Diagnosis-Related Group length of stay. CONCLUSION: Ciprofloxacin appeared to be as safe and effective as cefamandole in this nursing home program; however, additional studies are needed to determine its role in the treatment of elderly patients with bronchopulmonary infections.

Multicenter comparative evaluation of two rapid microscopic methods and culture for detection of Chlamydia trachomatis in patient specimens.

Four hundred and seventy-three men and women at high risk for sexually transmitted disease were tested for the presence of Chlamydia trachomatis in the urethra or the endocervix. Four groups were involved in this multicenter study of two direct fluorescent-antibody microscopy tests, Kallestad Pathfinder and Syva Microtrak, compared with culture techniques. Results from the test sites indicated that there was no significant difference overall in the sensitivity and specificity of the two test kits. However, there was some interlaboratory variation seen in the sensitivity of the microscopy, but little difference in the specificity. Either kit could be an effective screening method for C. trachomatis in high-risk populations.

Classification of type III (severe) open fractures relative to treatment and results.

Severe type III open fractures were subtyped according to the differences in prognosis for sepsis, amputation, and treatment: IIIA (adequate soft-tissue coverage of bone with extensive soft-tissue laceration or flaps), IIIB (extensive soft-tissue loss with periosteal stripping and bone exposure), and IIIC (arterial injury requiring repair). Analysis of 303 open fractures revealed a sepsis rate of 0% in type I, 2.5% in type II, and 13.7% in type III. The rate of amputation was 18.7%, and the rate of nonunion was 18.5% in type III open fractures. Type IIIA, IIIB, and IIIC open fractures had sepsis rates of 5%, 28%, and 8%, and amputation rates of 2.5%, 5.6%, and 25%, respectively. The overall wound sepsis rate in the 303 open fractures was 4.4%, and the nonunion rate was 8.6%.

Norfloxacin in the treatment of uncomplicated gonococcal infections.

Norfloxacin, an oral fluoroquinolone antibacterial, is active in vitro against a variety of gram-positive and gram-negative pathogens, including both penicillinase-producing and non-penicillinase-producing strains of Neisseria gonorrhoeae. An earlier study demonstrated that a two-dose regimen of norfloxacin was as effective as standard therapy with spectinomycin for treating gonococcal urethritis, including infections caused by penicillinase-producing organisms. In this randomized study of treatment for uncomplicated gonococcal infection in men and women, three oral treatment regimens were compared: patients received either two doses of norfloxacin (600 mg twice daily), a single dose of norfloxacin (800 mg), or a single-dose ampicillin (3.5 g)/probenecid (1.0 g) regimen (as recommended by the Centers for Disease Control). All three treatment regimens achieved similar cure rates. Although the number of patients treated was too small to yield statistically significant conclusions, it appears that norfloxacin may be slightly better treatment for rectal and pharyngeal gonococcal infections than ampicillin and probenecid. Additionally, norfloxacin was well tolerated in this study. Thus, based on a review of these data, norfloxacin appears to be an alternative, single-dose, oral treatment regimen for uncomplicated gonococcal infection.

Elevated serum angiotensin-converting enzyme (SACE) activity in acute pulmonary histoplasmosis.

Serum angiotensin-converting enzyme (SACE) levels were measured in 44 subjects six weeks after acute pulmonary histoplasmosis. All patients were infected in a common-source outbreak of histoplasmosis which occurred on one day. All patients had both strictly defined clinical and serologic evidence of infection. The SACE activity was elevated at six weeks compared to normal controls, and seven of the 44 had levels more than 2 SD above the normal mean. SACE levels were also measured at three and 24 weeks after acute infection in a smaller number of the same subjects. Serial observations demonstrated that all subjects (including those with normal and elevated SACE at six weeks) had a rise and fall in SACE activity following symptomatic acute pulmonary histoplasmosis. Our findings suggest that elevated SACE does not reliably separate sarcoidosis from histoplasmosis, although elevations in histoplasmosis are much less common and may occur only briefly following acute pulmonary histoplasmosis. More important, it seems that SACE activity rises acutely in all patients with symptomatic acute histoplasmosis and then falls gradually toward baseline over several months, coinciding temporally with the granulomatous response.

Comparative in vitro activity of Sch 29,482, a new oral penem, against Neisseria gonorrhoeae.

The in vitro activity of Sch 29,482, a new oral beta-lactam antimicrobial agent, was compared with those of norfloxacin, rosoxacin, ampicillin, erythromycin, and tetracycline against 142 Neisseria gonorrhoeae strains. Sch 29,482 was as active as norfloxacin and rosoxacin. Its activity was greater than the other three antimicrobial agents. It inhibited 90% of the isolates, regardless of beta-lactamase activity, at a concentration of less than or equal to 0.06 micrograms/ml.

Comparative efficacy of cefmenoxime versus penicillin in the treatment of gonorrhea.

A total of 121 men with complicated infections caused by beta-lactamase-negative Neisseria gonorrhoeae were included in this study. They were randomly assigned to regimens of either cefmenoxime (1.0 g) or procaine penicillin G (4.8 X 10(6) U) intramuscularly. Only the penicillin group also took 1.0 g of probenecid orally. A total of 99 patients completed the study, providing data from 108 infected sites. In the cefmenoxime group, there were 49 urethral, 1 rectal, and 2 pharyngeal infections; in the penicillin group, there were 49 urethral, 4 rectal, and 3 pharyngeal infections. In the cefmenoxime group, all except one urethral infection were eradicated. This patient admitted having had sexual intercourse during the follow-up period and was considered to be reinfected. In the penicillin group, all except one pharyngeal infection were cured. No adverse reactions were noted in either group. In this study, cefmenoxime was as effective as penicillin in the treatment of gonococcal urethritis in men.