Bacterial Physiological State in Wastewater: Monitoring Maintenance and Production with Leu/TdR Ratio for Less Pollution

In domestic wastewater, bacterial physiology controls cell production (growth, replication) and cell maintenance, determining how energy is allocated between these two processes. The aim here was to develop a method to quantify these cellular processes so that the bacterial physiological state could be manipulated to lower this source of pollution. We simultaneously used the incorporation of radiolabelled thymidine into DNA (a measure of new cell synthesis) and leucine into protein in wastewater to quantitatively distinguish bacterial growth from maintenance processes. We found that DNA and protein syntheses were coupled in wastewater after substrate enrichment (with glucose or acetate)??balanced growth. Once the substrate was depleted, the two processes became uncoupled??unbalanced growth. In this physiological state, the bacteria were synthesising protein, but fewer bacteria were replicating. More energy was allocated to cell maintenance than replication. A mean Leu/TdR ratio of 7.4 was determined for wastewater and was similar to natural aquatic ecosystems. As the bacterial growth rate decreased, the Leu/TdR ratios increased. We show how the simultaneous measurement of [³H]Leu and [³H]TdR quantitatively distinguishes balanced from unbalanced growth. Low [³H]Leu/[³H]TdR ratios indicated bacteria were physiologically stressed, an ideal state for biological wastewater treatment processes (WWTP) as the bacteria divert more energy to maintenance activities instead of growth. Leu/TdR ratios of 70 have been recorded in natural aquatic ecosystems which suggests WWTP have potential to be manipulated to achieve much higher Leu/TdR ratios than we report here. Changes to plant operation to improve operation efficiency include finding the optimum rate of substrate (pollution supply) or alternating aerobic and anaerobic periods to maximise the Leu/TdR ratio to achieve less biomass production for land disposal and more cost-effective operation that generates less pollution in the effluent.     

Flow cytometric evaluation of canine bone marrow differential cell counts

Abstract: Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlo-rofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.     

Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens.

In May 1996 the Denver Zoological Gardens obtained two male and two female Pallas' cats (Otocolobus felis manul) that were wild-caught in the Ukraine. These animals were part of a group of 16 wild-caught adults (eight male and eight female) imported to the United States and Canada between 1995 and 1996. The Denver Zoological Gardens cats were quarantined at the zoo hospital for approximately I mo. During the quarantine period they were immobilized for physical examination, and sera were obtained from them to evaluate for exposure to Toxoplasma gondii. All cats were positive for T. gondii antibodies by latex agglutination (titers from 1:512 to 1:1,024). After being paired for breeding, one pair produced two litters, and another pair produced four litters, a total of 17 kittens between 1997 and 2001. Four kittens and two young adults died from a disseminated granulomatous and necrotizing inflammation consistent with toxoplasmosis. Toxoplasma gondii infection was confirmed in all six deceased cats by polymerase chain reaction performed on formalin-fixed tissues. An additional five kittens disappeared and were not available for necropsy. The fatality rate from toxoplasmosis was 35.3% (6/17) for cats that were available for necropsy and could have been as high as 64.7% (11/17) if it were assumed that the disappeared kittens were also affected. The Pallas' kitten survival rate at the Denver Zoological Gardens was 35.3%. This article describes the clinical and pathologic features of toxoplasmosis in a group of Pallas' cats at the Denver Zoological Gardens.     

Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation

The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.     

The Effect of Season on the Histologic and Histomorphometric Appearance of the Equine Pituitary Gland

The objective of this study was to determine the effect of season on the histologic and histomorphometric appearance of the normal equine pituitary gland. Pituitary glands were collected at necropsy from 121 horses throughout the year. Plasma was also collected from 59 of these horses before euthanasia. Hematoxylin and eosin stained median sagittal sections of each pituitary were evaluated and histologically graded by three pathologists. Histomorphometric analysis was performed on the same slides. Plasma α-melanocyte stimulating hormone was measured by radioimmunoassay in a subset of horses (n = 59). A total of 118 pituitary glands were included in the study after exclusions were made on the basis of the presence of pars intermedia (PI) adenomas (>5 mm). There was a positive correlation between PI hormone concentration (α-melanocyte stimulating hormone) and PI area. Pituitary gland measurements and grades from samples collected in the fall were compared with those collected in the nonfall months using t-test. The PI area, total pituitary area, and PI/total pituitary ratio were significantly greater in the fall compared with nonfall months (P < .0001, P < .01, P < .0001, respectively). Pituitary grades were also higher in the fall compared with nonfall months (P < .001). There was no seasonal difference in pars distalis or pars nervosa area. The results of this study show that the normal equine pituitary shows seasonal changes in appearance and size. These changes must be considered when using postmortem histologic evaluations in the diagnosis of pituitary pars intermedia dysfunction or for validation of antemortem diagnostic tests.     

ULTRASONOGRAPHIC AND CLINICOPATHOLOGIC FINDINGS IN 21 DOGS WITH INTESTINAL ADENOCARCINOMA

In a retrospective study of 21 dogs with intestinal adenocarcinoma, the signalment, clinical presentation, laboratory findings, ultrasonographic features, treatment, and outcome were reviewed. Anorexia (n = 16), vomiting (n = 15), diarrhea (n = 10), and weight loss (n = 9) were the most common clinical signs reported. Ultrasonographic features that were evaluated included location, length, wall thickness, echogenicity, regional motility, layering, regional lymphadenopathy, and fluid accumulation proximal to the lesion site. All lesions were transmural and associated with complete loss of wall layering. Maximum wall thickening at the lesion site ranged from 7 to 17 mm (median 12 mm, mean 11.9 mm). Most of the dogs had a lesion measuring from 23 to 63 mm in length, (median 40 mm, mean 42 mm). Most intestinal lesions were poorly echogenic and had an irregular lumen. Fluid accumulation proximal to the lesion site was identified in 17 of 21 dogs, and in 13 of 17 dogs the fluid accumulation was considered moderate to severe. Regional lymphadenopathy and/or nodular mesentery/omentum were noted in 12 of 21 dogs. The tumor was located in small intestine for 15 dogs and in the colon for the remaining 6 dogs. Fifteen dogs were treated by surgical resection of the intestinal mass. Their median survival time was 233 days. Only gender appeared to influence survival. Female dogs lived a median of 28 days, whereas male dogs lived a median of 272 days.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Evaluation of cardiac lesions and risk factors associated with myocarditis and dilated cardiomyopathy in southern sea otters (Enhydra lutris nereis)

OBJECTIVE: To describe cardiac lesions and identify risk factors associated with myocarditis and dilated cardiomyopathy (DCM) in beach-cast southern sea otters. ANIMALS: Free-ranging southern sea otters. PROCEDURE: Sea otters were necropsied at the Marine Wildlife Veterinary Care and Research Center from 1998 through 2001. Microscopic and gross necropsy findings were used to classify sea otters as myocarditis or DCM case otters or control otters. Univariate, multivariate, and spatial analytical techniques were used to evaluate associations among myocarditis; DCM; common sea otter pathogens; and potential infectious, toxic, and nutritional causes. RESULTS: Clusters of sea otters with myocarditis and DCM were identified in the southern aspect of the sea otter range from May to November 2000. Risk factors for myocarditis included age, good body condition, and exposure to domoic acid and Sarcocystis neurona. Myocarditis associated with domoic acid occurred predominantly in the southern part of the range, whereas myocarditis associated with S. neurona occurred in the northern part of the range. Age and suspected previous exposure to domoic acid were identified as major risk factors for DCM. A sample of otters with DCM had significantly lower concentrations of myocardial L-carnitine than control and myocarditis case otters. CONCLUSIONS AND CLINICAL RELEVANCE: Cardiac disease is an important cause of death in southern sea otters. Domoic acid toxicosis and infection with S. neurona are likely to be 2 important causes of myocarditis in sea otters. Domoic acid-induced myocarditis appears to progress to DCM, and depletion of myocardial L-carnitine may play a key role in this pathogenesis.     

Genome sequence diversity and clues to the evolution of variola (smallpox) virus

Comparative genomics of 45 epidemiologically varied variola virus isolates from the past 30 years of the smallpox era indicate low sequence diversity, suggesting that there is probably little difference in the isolates' functional gene content. Phylogenetic clustering inferred three clades coincident with their geographical origin and case-fatality rate; the latter implicated putative proteins that mediate viral virulence differences. Analysis of the viral linear DNA genome suggests that its evolution involved direct descent and DNA end-region recombination events. Knowing the sequences will help understand the viral proteome and improve diagnostic test precision, therapeutics, and systems for their assessment.