Impacts of the 2014 severe drought on the Microcystis bloom in San Francisco Estuary.

The increased frequency and intensity of drought with climate change may cause an increase in the magnitude and toxicity of freshwater cyanobacteria harmful algal blooms (CHABs), including Microcystis blooms, in San Francisco Estuary, California. As the fourth driest year on record in San Francisco Estuary, the 2014 drought provided an opportunity to directly test the impact of severe drought on cyanobacteria blooms in SFE. A field sampling program was conducted between July and December 2014 to sample a suite of physical, chemical, and biological variables at 10 stations in the freshwater and brackish reaches of the estuary. The 2014 Microcystis bloom had the highest biomass and toxin concentration, earliest initiation, and the longest duration, since the blooms began in 1999. Median chlorophyll a concentration increased by 9 and 12 times over previous dry and wet years, respectively. Total microcystin concentration also exceeded that in previous dry and wet years by a factor of 11 and 65, respectively. Cell abundance determined by quantitative PCR indicated the bloom contained multiple potentially toxic cyanobacteria species, toxic Microcystis and relatively high total cyanobacteria abundance. The bloom was associated with extreme nutrient concentrations, including a 20-year high in soluble reactive phosphorus concentration and low to below detection levels of ammonium. Stable isotope analysis suggested the bloom varied with both inorganic and organic nutrient concentration, and used ammonium as the primary nitrogen source. Water temperature was a primary controlling factor for the bloom and was positively correlated with the increase in both total and toxic Microcystis abundance. In addition, the early initiation and persistence of warm water temperature coincided with the increased intensity and duration of the Microcystis bloom from the usual 3 to 4 months to 8 months. Long residence time was also a primary factor controlling the magnitude and persistence of the bloom, and was created by a 66% to 85% reduction in both the water inflow and diversion of water for agriculture during the summer. We concluded that severe drought conditions can lead to a significant increase in the abundance of Microcystis and other cyanobacteria, as well as their associated toxins.

Susceptibility to Myxobolus cerebralis among Tubifex tubifex populations from ten major drainage basins in Colorado where Cutthroat Trout are endemic.

Establishment of Myxobolus cerebralis (Mc) resulted in declines of wild Rainbow Trout Oncorhynchus mykiss populations in streams across Colorado during the 1990s. However, the risk for establishment and spread of this parasite into high-elevation habitats occupied by native Cutthroat Trout O. clarkii was unknown. Beginning in 2003, tubificid worms were collected from all major drainages where Cutthroat Trout were endemic and were assayed by quantitative PCR to determine the occurrence and distribution of the various lineages of Tubifex tubifex (Tt) oligochaetes. Over a 5-year period, 40 groups of Tt oligochaetes collected from 27 streams, 3 natural lakes, 2 private ponds, and a reservoir were evaluated for their relative susceptibility to Mc. Exposure groups were drawn from populations of pure lineage III Tt, mixed-lineage populations where one or more of the highly resistant (lineage I) or nonsusceptible lineages (V or VI) were the dominant oligochaete and susceptible lineage III worms were the subdominant worm, or pure lineage VI Tt. Experimental replicates of 250 oligochaetes were exposed to 50 Mc myxospores per worm. The parasite amplification ratio (total triactinomyxons [TAMs] produced / total myxospore exposure) was very high among all pure lineage III Colorado exposure groups, averaging 363 compared with 8.24 among the mixed-lineage exposure groups. Lineage III oligochaetes from Mt. Whitney Hatchery in California, which served as the laboratory standard for comparative purposes, had an average parasite amplification ratio of 933 among 10 exposed replicates over a 5-year period. Lineage I oligochaetes were highly resistant to infection and did not produce any TAMs. Lineages V and VI Tt did not become infected and did not produce any TAMs. These results suggest that the risk of establishment of Mc is high for aquatic habitats in Colorado where Cutthroat Trout and lineage III Tt are sympatric.

Differential in vitro activity of anidulafungin, caspofungin and micafungin against Candida parapsilosis isolates recovered from a burn unit.

Recent studies suggest that differences in antifungal activity among echinocandins may exist. In this study, the activities of three echinocandins (anidulafungin, caspofungin, and micafungin) against Candida parapsilosis isolates from burn unit patients, healthcare workers and the hospital environment were determined. Additionally, the effect of these echinocandins on the cell morphology of caspofungin-susceptible and caspofungin-non-susceptible isolates was assessed using scanning electron microscopy (SEM). The C. parapsilosis isolates obtained from patients were susceptible to anidulafungin, but were less so to caspofungin and micafungin. Isolates obtained from healthcare workers or environmental sources were susceptible to all antifungals. SEM data demonstrated that although anidulafungin and caspofungin were equally active against a caspofungin-susceptible C. parapsilosis strain, they differed in their ability to damage a caspofungin-non-susceptible strain, for which lower concentrations of anidulafungin (1 mg/L) than of caspofungin (16 mg/L) were needed to induce cellular damage and distortion of the cellular morphology. To determine whether the difference in the antifungal susceptibility of C. parapsilosis isolates to anidulafungin as compared to the other two echinocandins could be due to different mutations in the FKS1 gene, the sequences of the 493-bp region of this gene associated with echinocandin resistance were compared. No differences in the corresponding amino acid sequences were observed, indicating that differences in activity between anidulafungin and the other echinocandins are not related to mutations in this region. The results of this study provide evidence that differences exist between the activities of anidulafungin and the other echinocandins.

Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex.

Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations.

Detection of the intranuclear microsporidium Nucleospora salmonis in naturally and experimentally exposed Chinook salmon Oncorhynchus tshawytscha by in situ hybridization.

Nucleospora salmonis, an intranuclear microsporidian parasite of salmonid fish, is often difficult to observe in histological sections or wet mount preparations from lightly infected tissues because of its small size and location within the nuclei of lymphoblast-type cells. Diagnosis of infections by conventional light microscopy is directly dependent upon distinguishing different stages of the parasite from host cell nuclear material or vacuoles. To assist detection of stages of the parasite in tissues of its primary host, the Chinook salmon (Oncorhynchus tshawytscha), we developed a nonradioactive in situ hybridization (ISH) method. The new method was then used to detect N. salmonis among Chinook salmon after both natural and experimental exposures to the parasite. Probes derived from the small subunit ribosomal DNA (ssu-rDNA) sequence of the microsporidium were labeled with digoxigenin deoxyuridine triphosphate (DIG-dUTP) and hybridized to parasite DNA present in infected tissues. The ISH procedure effectively identified merogonic and spore stages of N. salmonis in paraffin-embedded tissues of clinically and subclinically infected fish. A Nucleospora-like microsporidium was also detected by ISH in tissues of a nonsalmonid fish, the English sole (Pleuronectes vetulus), using probes designed to a region of the ssu-rDNA of N. salmonis.

Evaluation of malacosporean life cycles through transmission studies.

Myxozoans, belonging to the recently described Class Malacosporea, parasitise freshwater bryozoans during at least part of their life cycle, but no complete malacosporean life cycle is known to date. One of the 2 described malacosporeans is Tetracapsuloides bryosalmonae, the causative agent of salmonid proliferative kidney disease. The other is Buddenbrockia plumatellae, so far only found in freshwater bryozoans. Our investigations evaluated malacosporean life cycles, focusing on transmission from fish to bryozoan and from bryozoan to bryozoan. We exposed bryozoans to possible infection from: stages of T. bryosalmonae in fish kidney and released in fish urine; spores of T. bryosalmonae that had developed in bryozoan hosts; and spores and sac stages of B. plumatellae that had developed in bryozoans. Infections were never observed by microscopic examination of post-exposure, cultured bryozoans and none were detected by PCR after culture. Our consistent negative results are compelling: trials incorporated a broad range of parasite stages and potential hosts, and failure of transmission across trials cannot be ascribed to low spore concentrations or immature infective stages. The absence of evidence for bryozoan to bryozoan transmissions for both malacosporeans strongly indicates that such transmission is precluded in malacosporean life cycles. Overall, our results imply that there may be another malacosporean host which remains unidentified, although transmission from fish to bryozoans requires further investigation. However, the highly clonal life history of freshwater bryozoans is likely to allow both long-term persistence and spread of infection within bryozoan populations, precluding the requirement for regular transmission from an alternate host.

Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae.

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 micro m and height of 14 microm, and possessed two soft valves surrounding two spherical polar capsules (2 microm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 microm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.

Changes in virus load markers during AIDS-associated opportunistic diseases in human immunodeficiency virus-infected persons.

Human immunodeficiency virus (HIV) load markers are being used increasingly to monitor disease progression and evaluate antiretroviral therapy. This study examined plasma HIV RNA and p24 antigen levels before, during, and after 15 AIDS-associated opportunistic disease events in patients with AIDS (median CD4 cell count = 65/microL). Plasma HIV RNA was detected during 13 of the 15 events (median level before an event = 21,000 copies/mL). There was an increase in the level of plasma HIV RNA with the onset of an AIDS-associated opportunistic disease during 11 of 13 events for which HIV RNA was detectable (median level during an event = 145,000 copies/mL). There was a decline in the level of HIV RNA with the recovery from disease (median level after an event = 29,700 copies/mL). In contrast, there was no consistent or significant change in p24 antigen levels or CD4 cell counts with either the onset of or recovery from an event. Clinical interpretation of plasma HIV RNA changes must take into account this reversible elevation during AIDS-associated opportunistic disease.

Gender is not a factor in serum human immunodeficiency virus type 1 RNA levels in patients with viremia.

We investigated gender as a factor in viral load measurements for human immunodeficiency virus-infected patients. Forty antiretroviral-therapy-naive, age- and CD4-matched women and men were tested for serum RNA and p24 antigen levels prior to antiretroviral therapy and at approximately 12 weeks after therapy. No gender differences were observed for these two markers of viral load.

Comparison of HIV type 1 RNA plasma viremia, p24 antigenemia, and unintegrated DNA as viral load markers in pediatric patients.

Better surrogate markers need to be developed to evaluate therapy in HIV-infected children. This study evaluated plasma RNA, immune complex-dissociated p24 antigenemia, and unintegrated DNA (uDNA) in HIV-infected pediatric patients. Ten children were followed from initiation of nucleoside antiretroviral therapy at intervals up to 24 months. Prior to initiation of therapy, HIV RNA was detected in 10 of 10 patients (median, 76,000 Eq/ml), p24 antigen was detected in 8 of 10 patients (median, 193 pg/ml), and uDNA was detected in 6 of 7 patients (median, 10% uDNA). After 12 months the RNA decreased in all patients and became undetectable in six. In contrast, p24 antigenemia decreased in 6 of 10 patients, remained undetectable in 1, and increased in 3. HIV uDNA decreased in six of six patients and became undetectable in three. There was no overall change in CD4 cell count. Plasma RNA and uDNA levels are both sensitive markers of nucleoside therapy in children; however, they do not covary strongly.

Rapid decrease in unintegrated human immunodeficiency virus DNA after the initiation of nucleoside therapy.

Better markers for determining therapeutic efficacy of antiretroviral drugs are needed for human immunodeficiency virus (HIV) infection. The amounts of unintegrated HIV DNA (uDNA) were sequentially determined in peripheral blood mononuclear cells (PBMC) from 20 HIV-infected patients starting nucleoside therapy. HIV copy number was determined using a quantitative polymerase chain reaction assay. Before therapy, 19 of 20 patients had detectable HIV uDNA. The average percentage of uDNA was 42%. After 1, 4, and 8 weeks of nucleoside therapy the average decreased to 23% (P < .001), 7%, and 3%, respectively. The amount of HIV uDNA decreased in all 19 patients during the first week and was undetectable in 14 by 8 weeks. Thus, measurement of HIV uDNA has many characteristics needed for a good marker of therapeutic efficacy of antiretroviral drugs, including detectability in a high proportion of patients, large and rapid response to initiation of therapy, and a biologically plausible mechanism.