R. D. Wyatt

Mitochondrial dysfunction is an early event in ochratoxin a but not oosporein toxicity to rat renal proximal tubules

Ochratoxin A (OA) and oosporein (OSN) are two mycotoxins that may cause nephrotoxicity through either mitochondrial dysfunction or lipid peroxidation. Using isolated rat renal proximal tubules in suspension, the cellular events preceding OA- or OSN-induced cytotoxicity were investigated. OA and OSN decreased tubule viability in a concentration (0-1 mM)- and time (0-4 hr)-dependent manner, with initial decreases occurring 1 hr after exposure. Tubule basal and nystatin-stimulated oxygen consumption decreased before cell death after OA (0.5 and 1 mM) and 0.25 mM t-butyl hydroperoxide (TBHP) exposure, but did not decrease after OSN exposure (0.25-1 mM). The oxidant TBHP was used as a positive control in these studies. Direct probing of mitochondrial function within proximal tubules confirmed the toxicity of OA to mitochondria. Respiration was reduced in the absence and presence of a phosphate acceptor using site I (glutamate/malate) and site II (succinate) respiratory substrates 15 and 30 min after exposure to 1 mM OA. Lipid peroxidation preceded cell death after exposure to 1 mM OA and 0.25 mM TBHP, but did not occur after exposure to 1 mM OSN. Deferoxamine (1 mM) pretreatment before the addition of 1 mM OA or OSN prevented OA-induced lipid peroxidation, but did not prevent OA- or OSN-induced cytotoxicity. In contrast, deferoxamine pretreatment prevented lipid peroxidation, mitochondrial dysfunction, and the loss of tubule viability after exposure to 0.25 mM TBHP. This study shows that mitochondrial dysfunction is an early event during the development of OA toxicity, but not in OSN-induced toxicity. Furthermore, iron-mediated lipid peroxidation does not contribute to OA- or OSN-induced proximal tubule cell death.

The role of altered mitochondrial function in citrinin-induced toxicity to rat renal proximal tubule suspensions

Citrinin (CTN), a mycotoxin produced by several species of Penicillium and Aspergillus, causes renal proximal tubule (RPT) cell injury and death by an unknown mechanism of action. Using suspensions of rat RPT, the cellular events preceding CTN-induced cytotoxicity were investigated. Tubule viability decreased in a concentration- and time-dependent manner after CTN exposure, with cell death beginning 1, 2, and 4 hr after exposure to 500, 125-250, and 63 microM, respectively. Basal oxygen consumption (QO2) of RPT increased from 41 to 53 nmol O2.mg protein-1.min-1 30 min after exposure to 250 microM CTN and returned to control values 1 hr after exposure. A similar concentration- and time-dependent transitory rise in basal QO2 occurred at all concentrations of CTN tested (63-500 microM). Nystatin-stimulated QO2, an indirect measure of mitochondrial state 3 respiration in RPT, decreased 11% at 0.5 and 1 hr after exposure to 500 and 250 microM CTN, respectively, but was not affected after exposure to 63 and 125 microM CTN. Adenosine triphosphate content declined 22% to 48% in RPT at 0.5 and 1.5 hr after exposure to 500 and 125-250 microM CTN, respectively. Although lipid peroxidation occurred concurrently with RPT cell death, iron-mediated oxidative stress was not a causative factor in the development of toxicity since pretreatment with 1 mM deferoxamine prevented iron-mediated lipid peroxidation but did not protect RPT from CTN-induced cell death. Further studies using RPT and isolated renal cortical mitochondria (RCM) showed that CTN had multiple effects on mitochondrial function. Direct probing of mitochondrial function within RPT showed that a 1-hr exposure to 250 microM CTN increased spontaneous respiration 55% in RPT respiring on the site I respiratory substrates glutamate/malate while state 3 respiration decreased 34%. CTN also decreased succinate supported respiration but had no effect on cytochrome c-cytochrome oxidase. With isolated RCM, a 3-min exposure to 125 and 250 microM CTN increased state 4 respiration in the absence of a phosphate acceptor 27 and 67%, respectively, while 250 microM CTN decreased state 3 respiration 23%. Respiration in the presence of a known uncoupler was reduced after CTN exposure (63-250 microM) in a concentration-dependent manner. These results indicate that CTN has multiple effects on mitochondrial function in RPT and isolated RCM which may contribute to the development of cell death in rat RPT.

Intestinal absorption of L-methionine and glucose in chickens with aflatoxicosis.

Abstract Graded doses of dietary aflatoxin (1.25, 2.5 and 5 μg/g) fed to broiler chicks for 3 weeks did not affect subsequent in vitro absorption of methionine and glucose by the intestine, compared with contol birds receiving no aflatoxin. A dietary level of 10 μg/g produced a significant increase in the absorption rate of both nutrients. Inhibition studies showed that both the mediated and diffusion components of absorption were increased with 10 μg/g. A prolonged exposure to aflatoxin was necessary for the effect since birds fed toxin for 1 week only (2 to 3 weeks of age) absorbed methionine and glucose at the same rate as controls receiving no aflatoxin.

The Effect of Seston on Mortality of Simulium vittatum (Diptera: Simuliidae) from Insecticidal Proteins Produced by Bacillus thuringiensis subsp. israelensis

ABSTRACT Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65–52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.

The Lack of Effect of Low Temperature and High Turbidity on Operational Bacillus thuringiensis Subsp. Israelensis Activity Against Larval Black Flies (Diptera: Simuliidae)

Abstract Black fly suppression programs are conducted across a wide range of environmental conditions, targeting a variety of pest species with diverse life histories. Operational applications of Vectobac® 12AS (Bacillus thuringiensis subsp. israelensis) were conducted during times characterized by water temperature and turbidity extremes. Applications were conducted in the Yellow River in central Wisconsin targeting Simulium annulus and S. johannseni when water temperatures were 1–2°C. Applications were conducted in the Green River in western North Carolina targeting the S. jenningsi group after a rain event, when portions of the treatment zone experienced turbidities of 276 nephelometric turbidity units. Excellent larvicidal activity was observed in both programs, with 97% mortality or greater being observed at distances over 5 km downstream of a treatment site. Mortality data for larval black flies in 2 operational suppression programs conducted in 2011 demonstrated a negligible effect of near-freezing water temperatures and exceptionally high turbidity on Bti activity.

Genetic variation of physiological response to aflatoxin in Gallus domesticus.

SummaryA pedigreed, commercial broiler population of 31 sire families was administered dietary aflatoxin at levels of either 0.0 or 5.0 μg of aflatoxin per g of diet from 7 to 21 days of age and their response assessed by various physiological parameters.Body weight, gain, packed red blood cell volume (PCV). plasma albumin, plasma protein and cholesterol responses were significantly reduced from control values by the 5.0 μg/g aflatoxin diet. Males had greater body weights and gains in both dietary regimes than females. Females had significantly higher PCV, protein, albumin and cholesterol values in the 5.0 μg/g aflatoxin group than their male counterparts. These differences resulted in significant sex × aflatoxin level interactions for these parameters. Coefficients of variation were increased for all parameters measured in the 5.0 μg/g aflatoxin treatment compared to values for the control group. This increase was greatest for plasma protein, albumin, and cholesterol responses. Heritabilities were calculated for all responses within both treatment groups and were found to be increased in all cases by the 5.0 μg/g aflatoxin diet. Highly significant phenotypic correlations were determined between body weight and gain and between plasma albumin and total plasma protein in both treatment groups. High phenotypic correlations among PCV, plasma cholesterol, plasma protein, and plasma albumin were noted in the 5.0 μg/g aflatoxin group. Significant genetic correlations were determined between body weight and gain and between plasma albumin and plasma protein in the control group. Body weight and gain and plasma protein, albumin, cholesterol and PCV were genetically correlated in the 5.0 μg/g aflatoxin group. Genetic correlations calculated across environments for the same traits were high for PCV, body weight and gain and much lower for plasma albumin, plasma protein, and plasma cholesterol.The results of this study demonstrate that genetic variability for resistance to aflatoxin exists in commercial broiler populations. Strong genetic and phenotypic relationships, and high heritabilities associated with plasma albumin and protein suggest their applicability as selection criteria for aflatoxin resistance. Genetic correlation for these traits across dietary environments indicate that responses for aflatoxin resistance should be measured during aflatoxin challenge and suggest that selection for growth and selection for aflatoxin resistance are not antagonistic.

Effects of phenobarbital and β-naphthoflavone on the in vivo toxicity and in vitro metabolism of aflatoxin in an aflatoxin-resistant and control line of chickens.

The in vivo toxicity of aflatoxin and the in vitro microsomal metabolism of aflatoxin B1 (AFB1) were investigated in a population of chickens previously selected for resistance to aflatoxin (AR line) and a corresponding control population (NS line) after in vivo pretreatment with saline, sodium phenobarbital (PB), or beta-naphthoflavone (BNF) solutions. PB pretreatment increased survival and BNF pretreatment increased mortality in both the NS and AR lines when a single oral dose of aflatoxin was administered. The rate of in vitro metabolism of AFB1 was greater with microsomes from saline pretreated AR chicks than with microsomes from similarly treated NS chicks. In vivo pretreatment with PB increased AFB1 metabolism by NS and AR microsomes. After BNF pretreatment of vivo, AR microsomes metabolized more AFB1 than NS microsomes, and there was a dramatic decrease in AFB1 metabolism in NS microsomes. AFB1-dihydrodiol was the major metabolite produced by both lines, with aflatoxin M1 and aflatoxin Q1 recovered in small quantities from BNF-pretreated AR microsomal incubations only. These data indicate that increased in vivo resistance of the AR line to acute aflatoxicosis may be related to increased hepatic AFB1 metabolism and that genetic selection has resulted in altered in vitro quantitative and qualitative metabolism of AFB1 in the AR line.

Pathological and clinical effects of Eimeria tenella in partially immune chickens

Abstract Chickens were made partially resistant to Eimeria tenella by giving them several small doses of oocysts. Such chickens had severe lesions after challenge infection and had similar packed cell volumes and prothrombin times, but their body weight was only marginally affected compared with chickens having their first infection. Extracts were prepared from the caeca of partially immune and susceptible birds; the caeca used all had similar severe (grade 3) lesions. When these extracts were injected intravenously (i.v.) into coccidia-free chickens, sudden death occurred. However, the amount of “toxic” substances present in the caeca of chickens having their first infection with E. tenella was greater than that of caecal extract prepared from chickens previously exposed to the parasite. Extracts prepared from the caeca of chickens partially resistant to E. tenella also had lower thromboplastin activity. Chickens made resistant to E. tenella by repeated immunizing doses were not protected from the lethal effects of the caecal extract from E. tenella -infected chickens given i.v. Chickens inoculated intra-abdominally with caecal extract from E. tenella -infected chickens were slightly protected against the lethal effects of caecal extract i.v. and the pathogenic effects caused by the inoculation of oocysts.

Chironomus calligraphus (Diptera: Chironomidae), a New Pest Species in Georgia

Abstract Chironomid midges are ubiquitous and ecologically important aquatic insects. However, some species can become pests when they occur in extremely high numbers, particularly those that colonize man-made habitats. Chironomus calligraphus is a Neotropical, pan-American species that has recently been found in the Nearctic region. This paper represents the 1st reported occurrence of C. calligraphus in Georgia. Extensive larval populations were found in the leaf sheaths and root masses of cattails and in the firm sandy substrates of a wastewater lake at an industrial site in coastal Georgia. Chironomus calligraphus was causing a significant economic impact at this site.

INFLUENCE OF SELECTED ANTIBIOTICS ON THE RESPONSE OF BLACK FLY (SIMULIUM VITTATUM) LARVAE TO INSECTICIDAL PROTEINS PRODUCED BY BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS

A controlled current toxicity test (CCTT) was developed to evaluate the response of black fly (Simulium vittatum) larvae to insecticidal proteins following exposure to various antibiotics. The bacterium, Bacillus thuringiensis subsp. israelensis (Bti), produces proteins that are toxic to Nemotoceran Diptera, such as black flies and mosquitoes, when ingested. These insecticidal crystalline proteins (ICPs) are highly efficacious in controlling black flies; however, speculation has arisen regarding the potential for antibiotic contamination of waterways to mitigate the toxicity of these proteins. A series of experiments was conducted with the CCTT in which black fly larvae were exposed to enrofloxacin, tylosin, sulfamethoxazole, and trimethoprim followed by exposure to Bti ICPs. These antibiotics were selected based on their use in agricultural and documented anthropogenic contamination of rivers. Anthropogenic concentrations of a mixture of these four antibiotics did not affect the response of the larvae to Bti ICPs. Subsequent experiments were conducted with antibiotic concentrations 10,000 to 80,000 times higher than those found in contaminated rivers. Exposure of black fly larvae to high levels of enrofloxacin (0.5 mg/L) had no effect upon the susceptibility to Bti ICPs; however, exposure to high levels of tylosin (8 mg/L) resulted in a significant increase in the susceptibility of the larvae to Bti ICPs at 72 h of exposure, but not at 48 h. Exposure of black fly larvae to high concentrations of a mixture of sulfamethoxazole and trimethoprim resulted in a significant increase in the efficacy of the larvicide after 48 and 72 h of exposure. These results suggest that impairment of the efficacy of Bti ICPs to black fly larvae is not due to antibiotic contamination of the larval environment.