SARS-CoV-2 monitoring at three sewersheds of different scales and complexity demonstrates distinctive relationships between wastewater measurements and COVID-19 case data.

Wastewater monitoring of SARS-CoV-2 presents a means of tracking COVID-19 community infection dynamics on a broader geographic scale. However, accounting for environmental and sample-processing losses may be necessary for wastewater measurements to readily inform our understanding of infection prevalence. Here, we present measurements of the SARS-CoV-2 N1 and N2 gene targets from weekly wastewater samples at three sites in Hamilton County, Ohio, during an increase and subsequent decline of COVID-19 infections. The concentration of N1 or N2 RNA in wastewater, measured over the course of six months, ranged from below the detection limit to over 104 gene copies/l, and correlated with case data at two wastewater treatment plants, but not at a sub-sewershed-level sampling site. We also evaluated the utility of a broader range of variables than has been reported consistently in previous work, in improving correlations of SARS-CoV-2 concentrations with case data. These include a spiked matrix recovery control (OC43), flow-normalization, and assessment of fecal loading using endogenous fecal markers (HF183, PMMoV, crAssphage). We found that adjusting for recovery, flow, and fecal indicators increased these correlations for samples from a larger sewershed (serving ~488,000 people) with greater industrial and stormwater inputs, but raw N1/N2 concentrations corresponded better with case data at a smaller, residential-oriented sewershed. Our results indicate that the optimal adjustment factors for correlating wastewater and clinical case data moving forward may not be generalizable to all sewersheds.

Isolation of transcripts from Diabrotica virgifera virgifera LeConte responsive to the Bacillus thuringiensis toxin Cry3Bb1.

Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (genus Diabrotica). The biological action of the Bt toxin in corn rootworms has not yet been clearly defined. Because development of resistance to Bt by corn rootworms will have huge economic and ecological costs, insight into larval response to Bt toxin is highly desirable. We identified 19 unique transcripts that are differentially expressed in D. virgifera virgifera larvae reared on corn transgenic for Cry3Bb1. Putative identities of these genes were consistent with impacts on metabolism and development. Analysis of highly modulated transcripts resulted in the characterization of genes coding for a member of a cysteine-rich secretory protein family and a glutamine-rich membrane protein. A third gene that was isolated encodes a nondescript 132 amino acid protein while a fourth highly modulated transcript could not be further characterized. Expression patterns of these four genes were strikingly different between susceptible and resistant western corn rootworm populations. These genes may provide useful targets for monitoring of Bt exposure patterns and resistance development in pest and non-target insect populations.

Chronic toxicity of 1,3,5-trinitrobenzene in Fischer 344 rats.

The chronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 (F344) rats was evaluated by feeding a diet containing 0, 5, 60, and 300 ppm of TNB for 2 years. The calculated average TNB intake over 2 years for males and females was 0.22, 2.64, 13.44 and 0.23, 2.68, 13.31 mg/kg body weight (BW)/day respectively. Terminal body weights were decreased and water intake was increased in both sexes (300 ppm), whereas food consumption was decreased in males (60 and 300 ppm groups) only. The relative spleen weights were significantly decreased in both sexes (300 ppm), whereas the relative brain weights were increased in females only (300 ppm). Hematological effects were not observed in animals killed at the 2-year time point, except significant decrease in the mean corpuscular hemoglobin (MCH) in males (300 ppm) and in females (60 and 300 ppm). Methemoglobin levels were increased in both sexes in the high dose group. Histopathological examination showed treatment-related changes in the kidney (hyaline droplets; 60 and 300 ppm) and the spleen (erythroid cell hyperplasia and pigment deposition; 300 ppm) of both sexes. Cytoplasmic hyaline droplets in the kidneys were characterized by immunohistochemistry as alpha-2mu-globulin. We propose a chronic, oral no-observable-adverse-effect level (NOAEL) of 2.68 mg/kg BW/day for TNB in the rat, based on the hematological and renal changes.

Fourteen-day toxicity study of 1,3,5-trinitrobenzene in Fischer 344 rats.

Toxic effects of 1,3,5-trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg-1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high-dose groups (800 and 1200 mg TNB kg-1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg-1 diet) was 92 mg kg-1 BW day-1 for male rats and 80 mg kg-1 BW day-1 for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high-dose groups was noted. In addition, histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg-1 BW day-1 was established based on the findings of this study.

Body temperature response to cocaine and diazepam in morphine-treated rats.

Since cocaine or diazepam are used clinically and/or abused concomitantly with narcotics, this study was designed to determine if morphine-dependent, morphine-withdrawn, or acute morphine rats exhibit an altered core body temperature at 24 or 4 degrees C to either cocaine (10, 20, or 30 mg/kg i.p.) or diazepam (2 or 4 mg/kg i.p.). At 24 degrees C, each dose of cocaine manifested hyperthermia in all groups of rats except morphine-dependent whereas at 4 degrees C cocaine produced hypothermia in all groups except morphine-dependent. At both 24 and 4 degrees C, diazepam, 2 or 4 mg/kg, caused a hypothermic response in control, dependent, and withdrawn animals. At 24 degrees C, administration of acute morphine alone induced a hyperthermia which was antagonized by both doses of diazepam. At 4 degrees C, acute morphine alone induced an initial hypothermia followed by a hyperthermia; both doses of diazepam potentiated the hypothermic response to acute morphine. Thus, significant alterations in core body temperature may be induced following the administration of cocaine or diazepam to morphine-treated rats.

Locomotor activity in morphine-treated rats: effects of and comparisons between cocaine, procaine, and lidocaine.

The effects of cocaine, procaine, and lidocaine on open field and spontaneous (actophotometer) locomotor activities were assessed and compared in rats (1) treated acutely with morphine (single injection), (2) made dependent on morphine (SC pellets), (3) implanted with morphine and withdrawn at the time of peak dependence, and (4) implanted SC with lactose-containing pellets (sham). Cocaine-induced (10 or 30 mg/kg) open field and spontaneous locomotor activities were significantly greater in each of the four groups than those of the corresponding groups administered saline. Procaine (50 or 100 mg/kg) significantly reduced open field locomotor activity in all morphine-treated rats and spontaneous locomotor activity in acute rats. Lidocaine (30 mg/kg) significantly depressed spontaneous locomotor activity in acute rats. Upon comparison of the activities induced by the three local anesthetics, open field locomotor activity of sham-implanted rats was greater following cocaine (10 or 30 mg/kg) than following procaine (50 or 100 mg/kg). Only morphine withdrawn rats manifested greater activity following cocaine (10 mg/kg) than following either procaine (50 mg/kg) or lidocaine (10 mg/kg); activities were equivalent in dependent and acute rats. In contrast, cocaine-induced (30 mg/kg) open field locomotor activity of all morphine-treated rats was greater than either procaine- (100 mg/kg) or lidocaine- (30 mg/kg) induced activities. Spontaneous locomotor activity of all groups except acute morphine was greater following both doses of cocaine than following both doses of either procaine or lidocaine. In acute rats, only cocaine (10 mg/kg, induced greater activity than the other local anesthetics. Thus, stimulation of locomotor activity following cocaine treatment is a pharmacological property unique to cocaine and not shared by either procaine or lidocaine. Further, the data indicate that the methods selected for assessing locomotor activity may not give comparable results.

Effect of steroid hormones and diethylstilbestrol on adrenomedullary catecholamine secretion.

Acetylcholine-induced catecholamine secretion from isolated, perfused bovine adrenal medulla was strongly inhibited by hydrocortisone (30 micro M), estradiol (30 micro M), and estriol (30 micro M). Diethylistilbestrol (DES) (10 and 30 micro M) inhibited acetylcholine-induced secretion, and the effect of the higher dose was prolonged. Inhibition of high potassium-induced secretion by DES (30 micro M) indicated that hormonal inhibition of evoked secretion was not by competition for acetylcholine-sensitive receptor sites. Dehydrotestosterone (30 micro M) was ineffective in inhibiting acetylcholine-induced secrection. Hydrocortisone, estradiol, and estriol treatments enhanced nonstimulated release. It is suggested that incorporation of certain steroid into plasma and granule membranes, thereby modifying membrane function, enhances nonstimulated release and may also block evoked release of secretoy material.