Whirling disease: re-emergence among wild trout

Summary: Whirling disease of rainbow trout is caused by Myxobolus cerebralis, a myxozoan parasite possessing a life cycle well adapted to the natural environments where salmonid fish are found. Whirling disease was first described in Europe in 1898 among farmed rainbow trout but recent occurrences have been devastating to wild trout in North America. The disease is considered a major threat to survival of wild rainbow trout in the intermountain west of the United States. Difficulties in containing the spread and potentially eliminating the pathogen are tied to features of a complex life cycle involving two hosts, the salmonid fish and an aquatic oligochaete. Details of the morphologic development of the parasite have been described in each host but only now are we beginning to appreciate the breadth of interactions between these developmental forms and the sequential responses of the host. Fundamental mechanisms of the recognition and attachment of the parasite to the hosts, how host immunity is evaded and the unknown influences of environmental factors all contribute to a rather poor understanding of the biology of the parasite. Although the biology and ecology of the salmonid host are better known than for the oligochaete host, our knowledge is inadequate to interpret their complex interactions with the parasite. This uncertainty precludes the development of effective management activities designed to enhance the viability and productivity of wild trout populations in M. cerebralis- positive river systems. Improving our understanding of the hosts, the parasite and the environmental factors determining their interaction should provide for more focused and effective control methods for containing the spread and devastating effects whirling disease is causing to our wild trout populations.     

Production of pemphigus antibodyin vitro and analysis of T-cell subsets

T cells from nine patients in the active stage of pemphigus vulgaris and five in the inactive stage of the disease were studied with Leu-1, Leu-2, and Leu-3 monoclonal antibodies. No significant differences were observed in the proportions of total T cells or T cells expressing either helper or suppressor phenotype in peripheral blood leukocytes of patients compared to normal subjects. Immunoregulatory mechanisms were functionally studied using an assay measuring total IgG synthesizedin vitro. Peripheral blood leukocytes were separated into T- and B-cell fractions and cultured in various combinations. In nine experiments, the T cells were irradiated prior to culturing with B cells to remove their suppressor function. No statistically significant differences were observed in the total IgG synthesized by B cells obtained from patients and normal subjects when cultured with untreated T cells or irradiated T cells obtained from patients or normal controls. These results indicated that there was no loss of suppressor-cell function or increased helper-cell function when assessed by measuring the total IgG synthesized. The addition of serum from pemphigus patients to peripheral blood leukocyte cultures of pemphigus patients and normal controls had no statistically significant effect on the synthesis of total protein or on the amount of Ig synthesized and secreted. Peripheral blood leukocytes from six untreated patients with pemphigus vulgaris were stimulatedin vitro with pokeweed mitogen (PWM) to produce immunoglobulin. The IgG produced selectively bound to the intercellular cement substance of the epidermis of patients' perilesional skin, normal human skin, and monkey esophagus. The IgG was biosynthetically labeled by culturing the leukocytes in medium supplemented with [³H]leucine, and the binding of the radiolabeled IgG was visualized by autoradiography. The IgG nature of the protein was demonstrated by precipitation withStaphylococcus protein A and removal with rabbit anti-human IgG antisera. Peripheral blood leukocytes obtained from normal volunteers and control patients did not produce this antibody. Our studies indicate that there was no general functional or phenotypic alteration of suppressor or helper T cells in the peripheral blood. The peripheral blood leukocytes of pemphigus patients under PWM stimulation can produce an anti-intercellular cement substance antibodyin vitro. These results indicate that the abnormality of immunoregulation which resulted in the production of a pathogenetic autoantibody in pemphigus is highly specific.     

Determination of hepatitis C virus genotype by Pyrosequencing

A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Cryopreservation of human mononuclear cells for quality control in clinical immunology. I. Correlations in recovery of K- and NK-cell functions,surface markers,and morphology

Cryopreserved human peripheral blood mononuclear cells (PBMC) were tested for natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) and for high-affinity (29°C) and total (4°C) rosette formation with sheep erythrocytes. PBMC produced variable NK activity following freezing and thawing, but consistently reacted well in ADCC. A significant correlation was found between low NK activity and a decreased percentage of low-affinity rosette-forming cells. On the contrary, the number of large granular lymphocytes (LGL), among which NK cells are restricted, and the reactivity with the monoclonal antibody OKT10, which recognizes the majority of LGL in the peripheral blood, were not significantly altered by cryopreservation. Cryopreserved cells proved to be excellent controls for determining the day-to-day variability of the NK assay and for selecting optimum conditions for this test in the clinical immunology laboratory.     

January 1997 - 7 Year Old Girl with Seizures

A seven year old girl presented with six month history of seizures. An MRI scan showed a cortical lesion in the left temporal lobe which was resected. Neuropathology examination demonstrated meningioangiomatosis, an unusual hamartomatous condition sometimes associated with neurofibromatosis 2. Approximately 50 cases of meningioangiomatosis have been reported in the literature. These are reviewed and compared to the current case.     

Prevalence of<Emphasis Type="Italic"> Enterocytozoon bieneusi</Emphasis> in post-weaned dairy calves in the eastern United States

Fecal specimens were obtained from 3- to 8-month-old post-weaned dairy calves on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, 59 of 452 calves (13%) from 11 farms in six states were found positive for Enterocytozoon bieneusi by PCR and DNA sequence analysis. Based on gene sequence data this genotype of E. bieneusi found in post-weaned calves was 100% identical to that found in pre-weaned calves in North America and differed by only two positions in 1,069 base pairs from specimens analyzed from humans. However, compared with previous reports, the prevalence of E. bieneusi was significantly higher in post-weaned than in pre-weaned calves from many of the same farms.     

Oxygen and lactic acid transport in skeletal muscle

A mathematical model of oxygen and lactic acid transport in skeletal muscle is used to test the effects of reactive hyperemia on oxygen and lactic acid concentrations following a period of ischemia. The model is based on the Krogh cylinder as the geometrical representation of the functional unit of transport, i.e., a capillary and the tissue it supplies. Included in the mathematical development of the model are the convective and diffusive transport of the chemical species, the nonlinear aspects of oxygen and lactic acid kinetics, and the reversible reaction of oxygen with hemoglobin in capillary blood and myoglobin in the tissue. The steady-state solution to the model is obtained first as the baseline for the study. Ischemia is then simulated by the cessation of capillary blood flow. This is followed by a reactive hyperemic response that is a function of the occlusion duration. The general effect of reactive hyperemia is to shorten the time intervals for initial return of tissue oxygen levels and the washout of accumulated lactic acid and to maintain tissue oxygen levels above steady-state values.