Diversity of aromatic hydroxylating dioxygenase genes in mangrove microbiome and their biogeographic patterns across global sites.

Aromatic hydrocarbons (AH), such as polycyclic aromatic hydrocarbons, are compounds largely found in nature. Aromatic-ring-hydroxylating dioxygenases (ARHD) are proteins involved in AH degradation pathways. We used ARHD functional genes from an oil-impacted mangrove area and compared their diversity with other sites around the world to understand the ARHD biogeographic distribution patterns. For this, a comprehensive database was established with 166 operational protein families (OPFs) from 1,758 gene sequences obtained from 15 different sites worldwide, of which twelve are already published studies and three are unpublished. Based on a deduced ARHD peptide sequences consensus phylogeny, we examined trends and divergences in the sequence phylogenetic clustering from the different sites. The taxonomic affiliation of the OPF revealed that Pseudomonas, Streptomyces, Variovorax, Bordetella and Rhodococcus were the five most abundant genera, considering all sites. The functional diversity analysis showed the enzymatic prevalence of benzene 1,2-dioxygenase, 3-phenylpropionate dioxygenase and naphthalene 1,2-dioxygenase, in addition to 10.98% of undefined category ARHDs. The ARHD gene correlation analysis among different sites was essentially important to gain insights on spatial distribution patterns, genetic congruence and ecological coherence of the bacterial groups found. This work revealed the genetic potential from the mangrove sediment for AH biodegradation and a considerable evolutionary proximity among the dioxygenase OPFs found in Antarctica and South America sites, in addition to high level of endemism in each continental region.

Nep1-like protein from Moniliophthora perniciosa induces a rapid proteome and metabolome reprogramming in cells of Nicotiana benthamiana.

NEP1 (necrosis- and ethylene-inducing peptide 1)-like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP-treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short-term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2-DE gels in each proteome, with one-third showing more than twofold differences in the expression values. Fifty-three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide-binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi-quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo-inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction-oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP-mediated cell death signaling in plants.

Osteopenic disease in growing pigs: diagnostic methods using serum and urine calcium and phosphorus values, parathoromone assay, and bone analysis.

This research was performed to evaluate the utility of several serum and urine parameters as well as bone ash and plasma parathormone assay to diagnose and monitor diet-related osteopenia in growing pigs. Five diets were tested as follows: calcium-deficient, phosphorus-replete; moderate-deficiency of calcium and phosphorus; marked deficiency of calcium and phosphorus; calcium replete, phosphorus deficient; and vitamin D deficient. Parameters monitored included serum calcium and phosphorus as well as ratios of urine calcium to creatinine, phosphorus to creatinine, calcium to phosphorus, and percent fractional excretions of calcium and phosphorus. Plasma parathormone (PTH) levels were monitored in 2 of 3 experiments. Osteopenic bone differences at necropsy were evaluated by bone density, percent ash, ash per milliliter bone, calcium per milliliter bone, and phosphorus per milliliter bone. Marked change in urine mineral parameters, especially the calcium-to-phosphorus ratio, typically occurred within 1 to 2 days of treatment and preceded significant change in serum mineral or plasma PTH by 2 to 3 weeks. When monitored, plasma PTH levels were elevated following treatment, which confirms the hyperparathyroid state induced by the test diets. Significant differences in bone mineralization between control and treatment diets at necropsy were generally observed. The results of this study indicate that the analysis of urine minerals offers an early, noninvasive technique to investigate diet-associated osteopenic disease in growing pigs, which can be supported further by bone mineral analysis at postmortem using techniques herein described. Several urine mineral reference intervals for application to field investigations are included. Research into application of similar techniques to evaluate calcium and phosphorus homeostasis in pigs of all ages, including gestating and lactating gilts and sows, appears warranted.

Application of multiplex polymerase chain reaction for rapid identification of Campylobacter jejuni and C coli associated with reproductive failure.

OBJECTIVE: To evaluate a multiplex polymerase chain reaction (PCR) to distinguish Campylobacter jejuni from C coli as causes of reproductive failure. PROCEDURE: Review of clinical cases of reproductive failure attributed to C jejuni or C coli. RESULTS: A case of swine abortion was attributable to infection with C coli. The porcine abortion isolates were verified as C coli by restriction fragment length polymorphism and multiplex PCR. Cases of endometritis in a fox and in mink caused by C jejuni were reviewed, and isolates were confirmed as C jejuni by results of the multiplex PCR. CONCLUSION: Multiplex PCR was useful in identifying C coli and C jejuni recovered from atypical cases of reproductive failure. Multiplex PCR in conjunction with conventional assays may be useful for verifying other unusual instances of campylobacteriosis.

An indirect microimmunofluorescence test for detection of chlamydial antibodies in ovine fetal fluids.

The objective of this study was to evaluate an indirect microimmunofluorescence test (IMIF) for detection of chlamydial antibodies in serum and/or thoracic fluids of aborted ovine fetuses. One hundred eighty-two ovine fetuses, including 64 fetuses from 40 ewes that were experimentally infected with an ovine abortion strain of Chlamydia psittaci at gestation days 90-100, 10 fetuses from 6 normal ewes, and 108 fetuses selected from those received at the Iowa Veterinary Diagnostic Laboratory, were evaluated in this study. Fetuses from experimentally infected ewes were examined 4-60 days after inoculation. The IMIF findings were compared with the results of complement fixation serology for chlamydiae and concentrations of immunoglobulin (IgG). Chlamydiae-specific antibodies were detected by IMIF in 28 of 38 fetuses infected with C. psittaci. Elevated levels of IgG and IMIF titers > or = 1:8 were consistent findings in ovine fetuses infected with chlamydiae for more than 24 days. IgG levels and titers of chlamydial antibodies increased with maturity of the fetus and duration of chlamydial infection. Chlamydial antibodies were not detected with the complement fixation test. Fluids from ovine fetuses aborted as a result of other causes also were examined, and IMIF results were negative. The results of this study indicate that the IMIF is a useful and relatively rapid test for identification of chlamydial antibodies in ovine fetuses.

Excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars.

Four boars intranasally inoculated with porcine reproductive and respiratory syndrome (PRRS) virus were monitored for 56 days after exposure for changes in semen characteristics and for the presence of virus in the semen. Clinically, 2 of 4 boars had mild respiratory signs of 1 day's duration after infection. Changes in appetite, behavior, or libido were not detected. All boars seroconverted on the indirect fluorescent antibody and serum virus neutralization tests by day 14 after inoculation. Virus was isolated from serum between days 7 and 14 after inoculation. During the monitoring period, semen volume decreased and pH correspondingly increased; however, this change began 7 to 10 days prior to infection. Differences in sperm morphologic features, concentration, or motility between the preinfection and postinfection samples were not observed. The PRRS virus was detected in semen at the first collection in each of the 4 boars (ie, 3 or 5 days after challenge exposure). Virus was detected in nearly all semen samples collected from the 4 infected boars through days 13, 25, 27, and 43, respectively. Neither gross nor microscopic lesions attributable to PRRS virus were observed in tissues collected at the termination of the experiment (day 56), and virus isolation results from reproductive tissues were negative.

Experimental transmission of an apparent viral pneumonia in conventional and gnotobiotic pigs.

Endemic pneumonia in five- to eight-week-old pigs induced microscopic lesions of proliferative interstitial pneumonia which were compatible with a viral aetiology. The disease was transmitted experimentally to conventional and gnotobiotic pigs by means of a lung homogenate filtered through a 0.22 micron filter. No common viral respiratory pathogens of pigs were isolated. Two types of virus particles were observed in cell culture by electron microscopy; one was about 70 nm in diameter and had an envelope and short surface spicules, the other also had an envelope, was elongated, pleomorphic, measured 80 x 320 nm and was coated by antibodies.

Metabolic changes in blood and liver during development and early treatment of experimental fatty liver and ketosis in cows.

Eighteen cows were assigned in equal numbers to three groups: control, ketosis induction by using feed restriction plus dietary 1,3-butanediol to provide ketone bodies, and glucose treatment with 484 g/d of glucose infused intraduodenally starting 7 d after beginning ketosis induction. Ketosis induction, begun at d 15 postpartum, caused ketonemia and gradual development of clinical ketosis by d 40 to 45. None of the cows in the control or glucose-treated groups became ketotic. Concentrations of NEFA in plasma of cows that became ketotic increased 3.0-, 2.6-, and 1.9-fold at 3 wk before, 2 wk before, and at ketosis, respectively, but increased nonsignificantly for glucose-treated cows. Concurrently, beta-hydroxybutyrate increased 3.5-, 5.8- and 8.4-fold for cows that became ketotic but 1.6-fold or less for glucose-treated cows. Plasma acetate increased dramatically 2 wk before ketosis. Liver glycogen content decreased to nearly 0 by 2 wk before ketosis occurred, but it increased to prepartal values in glucose-treated cows. Liver triglycerides averaged 2.0% of wet weight at d 5 for all cows but increased to 8 to 10% for about 2 wk before ketosis occurred. Microscopy of liver samples demonstrated progressive accumulation of lipid globules, which began in hepatocytes near the central vein and progressed toward the portal triad. Visible lipid content reached a peak 2 wk before ketosis. Hepatic in vitro gluconeogenic capacity decreased significantly for ketosis induction protocol cows when clinical ketosis was detected. Results indicate that experimental ketosis was preceded by metabolic abnormalities up to 2 wk before clinical ketosis occurred. The key events for onset of clinical ketosis, however, were not elucidated.

Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes.

A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.