A novel Henneguya species from channel catfish described by morphological, histological, and molecular characterization.

Channel catfish Ictalurus punctatus from a commercial farming operation in the Mississippi Delta were submitted for examination for the presence of infection by the trematode Bolbophorus damnificus. The fish were instead found to possess skin nodules suggestive of Henneguya pellis, a species previously described in the blue catfish I. furcatus. Despite the dermal location and distribution of lesions, morphological characteristics of the myxospores were inconsistent with H. pellis. Spores possessed a lanceolate spore body 15.4 +/- 1.5 microm (mean +/- SD; range = 12.2-19.3 microm) in length and 5.5 +/- 0.6 microm (range = 4.5-6.8 microm) in width in valvular view, and 4.7 +/- 0.2 microm (range = 4.2-5.0 microm) in width in sutural view. Polar capsules were pyriform and unequal in both length and width and contained polar filaments with six coils. Polar capsules measured 6.1 +/- 0.8 microm (range = 4.0-7.9 microm) long and 1.7 +/- 0.3 microm (range = 1.0-2.2 microm) wide. The caudal appendages were 50.5 +/- 8.3 microm (range = 34.8-71.4 micorm) long and the total length of the spore was 65.9 +/- 8.6 microm (range = 48.2-90.0 microm). The "blister like" plasmodia were round or ovoid, up to 2 mm in diameter, and randomly distributed throughout the epidermis of the fish. Histologically, plasmodia were confined to the dermis and elicited no inflammatory reaction from the fish. A blast search of the 18S small subunit rDNA sequence obtained by polymerase chain reaction amplification resulted in no identical sequence matches but indicated a close relationship to H. gurlei, H. ictaluri, and H. exilis. The unique host record, spore morphology, and novel genetic sequence derived from this isolate lead us to propose this isolate as a novel species, H. sutherlandi.

Seasonal values of selected blood parameters of farm-raised channel catfish (Ictalurus punctatus) in the Mississippi Delta.

Hematocrit, sodium, chloride, potassium, calcium, glucose, and pH were measured in whole blood of 1,522 channel catfish collected from 3 commercial food-fish ponds in the Mississippi Delta. Samples were collected from March 1995 to March 1996 to monitor seasonal fluctuations. A total of 10-20 fish were arbitrarily collected with snag lines from each pond on each sample day. The mean monthly hematocrits fluctuated seasonally from a low of 14.5% in midwinter to a high of 25.7% in midsummer (annual x = 21%, SE = 0.15). Sodium levels were consistent throughout the year with a mean (SE) of 134 (0.13) mM/liter. Mean chloride values for the year were 120 (0.14) mM/liter but increased to 132 mM/liter in midwinter. By March 1996, the chloride levels had returned to levels observed during spring 1995. Potassium and glucose levels varied throughout the year with means of 4.43 (0.06) mM/liter and 26.9 (0.46) mg/dl, respectively, and coefficients of variation of 51.8% and 63.3%, respectively. Calcium and pH values were fairly stable with means of 1.31 (0.004) mM/liter and 7.13 (0.004), respectively. All parameters except glucose and potassium may be adequately evaluated with a sample size of 25 or less. These data were collected to provide baseline information for ongoing pond health studies.

Toxin production by Pasteurella granulomatis.

Pasteurella granulomatis (Pg) is a recently identified bacterium associated with proliferative fibrogranulomatous panniculitis (also called "lechiguana") in Brazilian cattle. Recent attempts to experimentally reproduce this disease have only been partially successful. We hypothesized that Pg may produce hemolysin(s) and/or cytotoxin(s) which could contribute to its pathogenicity in susceptible cattle. The objective of this study was to determine the presence and degree of hemolytic and leukotoxic activity of selected isolates of Pg. Either ovine or bovine blood agar plates were streaked with 1 of 7 Pg isolates, incubated at 37 degrees C +/- 1 C for 48 hours, and examined for hemolysis. Two of seven isolates showed hemolytic activity on bovine plates, while all seven showed hemolytic activity on ovine plates. By use of the CAMP reaction, involving simultaneous intersecting cultures of Staphylococcus aureus and Pg, all seven Pg isolates showed enhanced (positive CAMP) hemolysis within 24 hours on bovine blood agar plates. Preliminary results using tetrazolium (MTT) dye reductions with bovine neutrophils showed leukotoxicity in 13 of 16 Pg cultures. Alamar blue tests indicate leukotoxic activity for all 7 Pg isolates. We conclude that some Pg isolates have variable hemolytic and/or leukotoxic properties and that this variability (presence and/or degree) of these 2 properties may affect the relative pathogenicity of Pg in susceptible cattle.

Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase.

Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD+ oxidoreductase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific activity of 222 units/mg protein. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic parameters for all four substrates in the forward and reverse reactions indicated a sequential mechanism for this enzymic process. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH the second product released. Protection against thermodenaturation of the enzyme by NAD and not by malate was supportive of this mechanism. A pronounced product inhibition by NADH (K(i) = 9.0 microM) was observed. Although NADP did not serve as a coenzyme, a number of analogs of NAD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the purified malate dehydrogenase. Coenzyme-competitive inhibition of the malate dehydrogenase was observed with five adenosine derivatives and six structural analogs of NAD. Of the NAD analogs studied as inhibitors, 3-pyridylcarbinol adenine dinucleotide was the most effective (K(i) = 18 microM). Although inhibition of growth of H. parasuis by this analog was observed, it was less effective (K(i) = 136 microM) than the inhibition of the purified dehydrogenase.

Azotobacter vinelandii glucose 6-phosphate dehydrogenase properties of NAD- and NADP-linked reactions.

Glucose 6-phosphate oxidation, catalyzed by purified Azotobacter vinelandii glucose 6-phosphate dehydrogenase, was studied with respect to the selective utilization of NAD, NADP, thionicotinamide adenine dinucleotide or thionicotinamide adenine dinucleotide phosphate as coenzyme. A sigmoidal relationship was observed for the effect of substrate concentration on initial velocities when either NAD, NADP or thionicotinamide adenine dinucleotide was used as coenzyme, with N values from the Hill equation equalling 2.0, 1.7, and 1.7, respectively. The thionicotinamide analogs of NAD and NADP both functioned as coenzyme-competitive inhibitors of the enzyme-catalyzed NAD- and NADP-linked reactions. A dual wavelength assay, using a combination of NADP and thio-NAD, was established and was used to demonstrate that increasing glucose 6-phosphate concentration did not change the enzyme preference for the coenzyme form used. Sigmoidal relationships were observed for reduction of both dinucleotides, and N values were the same as those observed when each dinucleotide was studied as the only coenzyme form present in reaction mixtures. Using the dual wavelength assay, inhibition by isocitrate, 6-phosphogluconate, ATP, and palmitoyl-CoA was shown to be equally effective in both NAD- and NADP-linked reactions. An enzyme activator, glucosamine 6-phosphate, altered the glucose 6-phosphate sigmoidicity through activation at low substrate concentrations.

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth.

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.

Detection of the Lyme disease bacterium, Borrelia burgdorferi, by using the polymerase chain reaction and a nonradioisotopic gene probe.

A 419-bp region of the flagellin gene sequence of Borrelia burgdorferi was used as a target for the polymerase chain reaction. With a nonradioactively labeled gene-specific probe, sensitivity to as few as 1 to 10 spirochetes was observed. The targeted gene fragment was conserved in the American and European strains of B. burgdorferi tested and among several other pathogenic borreliae.

Cystic lesions of the adrenals: diagnosis and management.

Until recently, adrenal cysts were diagnosed at surgery or autopsy. Current imaging methods allow accurate preoperative diagnosis in symptomatic patients and may obviate surgery in the asymptomatic. Eighteen cystic lesions of the adrenal glands are described. Computed tomography was the most useful diagnostic test. Many cysts had typical appearances of a thin-walled, fluid-filled lesion with or without thin septa and mural calcification. Atypical features included central densities, which may be characteristic of intracystic hemorrhage, and wall thickening which should initiate a search for endocrinopathy and tumor.

Localized colonic perforation following renal transplantation.

Well-contained abdominal abscesses developed in two renal transplant patients ten years and six months, respectively, after transplantation. Investigations including gastrointestinal series and CT scan demonstrated cystic collections within the abdominal cavity. Laparotomy in each case revealed an infected cystic mass arising from the sigmoid colon. In both patients deroofing the abscess and, in one patient, defunctioning colostomy were performed. Both patients did well in the postoperative period.

Computed tomography in congenital anomalies of the aortic arch and great vessels.

Computed tomography can help in the evaluation of congenitally anomalous aortic arches and subclavian arteries. The authors discuss the role of computed tomography in the evaluation of such anomalies in seven patients.

Computed tomography of absent left pericardium.

Pericardial defects rarely cause clinical symptoms and thus are not often recognized. A case is reported in which the CT appearance of complete absence of the left pericardium reflects the abnormal relationship of the heart, pericardium, lung, and great vessels.

Lung function in diaphragm pacing.

Electric stimulation of the diaphragm via the phrenic nerve to induce ventilation has recently been used for the long-term management of chronic ventilatory insufficiency. Since 1973 three patients with inadequate alveolar ventilation have been treated with diaphragm pacing at the Toronto Western Hospital. Two, who had quadriplegia due to lesions of the spinal cord in the upper cervical region and a severe restrictive ventilatory defect, were treated with continuous diaphragm pacing. The third patient required assisted nocturnal ventilation because of primary alveolar hypoventilation. All three patients tolerated the diaphragm pacing well, and pulmonary function tests showed satisfactory gas exchange with the patients breathing room air. This form of therapy seems to be a practical clinical method of managing chronic ventilatory failure in patients with lesions of the upper cervical cord or primary alveolar hypoventilation.