Staphylococccus pseudintermedius surface proteins SpsD and SpsO mediate adherence to ex vivo canine corneocytes

The Gram-positive bacterium Staphylococcus pseudintermedius is regarded as the major cause of canine bacterial pyoderma. Despite its clinical importance, there is only very limited knowledge about the pathogenesis of S. pseudintermedius infection and the specific bacterial virulence factors involved in causing disease. Using a whole-genome approach, we have previously identified 18 predicted cell-wall-anchored surface proteins representing possible virulence factors in a clinical isolate of S. pseudintermedius (strain ED99). They were designated S. pseudintermedius surface proteins A-R (SpsA-SpsR). The present study tested three of the putative Sps proteins (SpsD, SpsL and SpsO) for their ability to mediate adherence of bacteria to canine corneocytes. The three proteins were expressed on the surface of the nonpathogenic surrogate host Lactococcus lactis, a Gram-positive bacterium that does not adhere to canine corneocytes. Adherence assays were performed using corneocytes from different healthy canine donors (n = 5), and bacterial cells were quantified using computerized image analysis. Two of the proteins, SpsD and SpsO, mediated adherence of L. lactis to canine corneocytes, suggesting that they contribute to S. pseudintermedius pathogenesis and may represent novel therapeutic targets to combat infection.     

Protein structures of common bean (Phaseolus vulgaris) alpha-amylase inhibitors

Two nucleotide sequences for genes that encode alpha-amylase inhibitor 4 (alphaAI-4) from white kidney bean (WKB) cv. 858, designated gene alphaAI-4 (Accession No. ), and alpha-amylase inhibitor 5 (alphaAI-5) from black bean (BB), designated gene alphaAI-5 (Accession No. ), were determined. Genes alphaAI-4 and alphaAI-5 encode 244 amino acid prepro-alphaAI-4 and prepro-alphaAI-5 polypeptides that are 93 and 95% identical with alpha-amylase inhibitor l (alphaAI-l; Hoffman, L. M.; Ma, Y.; Barker, R. F.Nucleic Acids Res. 1982, 10, 7819-7828), 40 and 43% identical with red kidney bean lectin, and 52 and 55% identical with arcelin l of wild-type bean. The high degree of sequence similarity indicates the evolutionary relationship among these genes. PCR analysis of genomic DNA purified from six genotypes of Phaseolus vulgaris showed very similar band patterns in 2% agarose gel, another indication of the conserved size homology among these genes. Proteolytic processing sites were located between Asn77 and Ser78 for pro-alphaAI-4 and pro-alphaAI-5. A bend next to Asn77 in three-dimensional model structures of alphaAI-4 and alphaAI-5 proinhibitors indicates that the proteolytic cleavage is necessary to remove the conformational constraint for activation to the mature protein. Mature WKB alphaAI-4 was composed of four subunits (2alpha2beta) and had a molecular weight of 50000 determined by multiangle laser light scattering and 56714 determined by laser-assisted time-of-flight mass spectrometry.     

Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Amphibian therapeutics.

The treatment of diseases in amphibians presents unique challenges for the clinician. This article describes the basic principles in selecting and administering therapeutics, and provides drug dose rates used successfully in amphibians. The importance of proper husbandary and hygiene, as it relates to amphibian health and medical management of disease, is also emphasized.     

Paramyxovirus infection in caiman lizards (Draecena guianensis).

Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were imported into the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizards demonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus that was consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolate of ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity within pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards were placed in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later, syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similar syncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracytoplasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundant filamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed in culture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples were collected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determine presence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of < or =1:20 and 10 had titers of >1:20 and < or =1:80. This report is only the second of a paramyxovirus identified in a lizard and is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.     

Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1). A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS) detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.     

Outbreak of wildebeest-associated malignant catarrhal fever in Ankole cattle

During an outbreak of malignant catarrhal fever in a herd of Ankole cattle in a zoological collection, two adult cows and one adult bull from a herd of 15 died or were euthanased between July and November 2004. The clinical, gross postmortem and histological findings were typical of the disease in uk native domestic cattle. The diagnosis was confirmed by serology in two animals, and by pcr in all three; the pcr provided evidence of alcelaphine herpesvirus type 1 infection in all three animals and also of ovine herpesvirus type 2 infection in one.     

Multiple predator effects and native prey responses to two non‐native Everglades cichlids

Abstract – Non‐native predators may have negative impacts on native communities, and these effects may be dependent on interactions among multiple non‐native predators. Sequential invasions by predators can enhance risk for native prey. Prey have a limited ability to respond to multiple threats since appropriate responses may conflict, and interactions with recent invaders may be novel. We examined predator–prey interactions among two non‐native predators, a recent invader, the African jewelfish, and the longer‐established Mayan cichlid, and a native Florida Everglades prey assemblage. Using field enclosures and laboratory aquaria, we compared predatory effects and antipredator responses across five prey taxa. Total predation rates were higher for Mayan cichlids, which also targeted more prey types. The cichlid invaders had similar microhabitat use, but varied in foraging styles, with African jewelfish being more active. The three prey species that experienced predation were those that overlapped in habitat use with predators. Flagfish were consumed by both predators, while riverine grass shrimp and bluefin killifish were eaten only by Mayan cichlids. In mixed predator treatments, we saw no evidence of emergent effects, since interactions between the two cichlid predators were low. Prey responded to predator threats by altering activity but not vertical distribution. Results suggest that prey vulnerability is affected by activity and habitat domain overlap with predators and may be lower to newly invading predators, perhaps due to novelty in the interaction.     

Soil enzyme activities and abundance of microbial functional genes involved in nitrogen transformations in an organic farming system

Organic farming systems receive organic amendments to maintain soil fertility and supply nutrients for plant growth. This study investigated the effect of organic fertilizers (no amendment as control, compost, and manure), and their interaction with cover crops (millet, buckwheat, and black turtle bean) on soil enzyme activities, N transformation rates, and functional gene abundances under an organic production system. Organic N fertilizers had a stronger effect than cover crop type on soil function and functional gene abundances. Soil enzyme activities were increased by both compost and manure, but there were few differences between these treatments. Nitrification potential, nitrite oxidation potential, and denitrification potential were significantly higher in manure-treated than in control and compost-treated soils, indicating application of manure had a higher N loss potential than compost application in this organic farming system. Organic N fertilizers significantly increased the abundance of some genes involved in N mineralization, ammonification, and nitrification (sub, ureC, bacterial amoA and nxrB). The activity of ammonia-oxidizing bacteria and archaea were both increased by organic N fertilizers, and their activities were higher in manure-treated than in compost-treated soils. Overall, the abundance of functional genes was significantly correlated with their corresponding enzyme activity. However, functional gene abundance was less important than soil chemical and microbiological properties in explaining the variation in the corresponding enzyme activity.