Gayle Pittman Noblet

Bioaccumulation of polychlorinated biphenyls in ranid frogs and northern water snakes from a hazardous waste site and a contaminated watershed

Livers of bullfrogs (Rana catesbeiana) from a polychlorinated biphenyl (PCB) contaminated watershed and hazardous waste site located in Pickens County, South Carolina, contained significantly higher concentrations of PCBs (2.33 and 2.26 ppm, respectively) than those from a reference site (0.05 ppm). Green frogs (R. clamitans) from the two contaminated sites also accumulated higher levels of PCBs (2.37 and 3.88 ppm, respectively) than those from the reference site (0.02 ppm). No temporal variation was observed in PCB concentrations of bullfrogs or green frogs from the contaminated sites between 1992 and 1993. Levels of PCBs in the livers of northern water snakes (Nerodia sipedon) were significantly higher in snakes from the contaminated watershed (13.70 ppm) than in those from the waste site (2.29 ppm) and two reference sites (2.50 and 1.23 ppm). When compared to frogs, significantly higher bioaccumulation occurred in water snakes from the contaminated watershed. No significant differences in PCB levels were found with respect to sex or body size (snout-vent length (SVL) or body mass) for frogs or snakes. PCBs were detected also in eggs of both frogs and snakes. Results of this study provide baseline data and document the bioaccumulation of PCB residues in frog and snake tissues; however, the significance of these tissue residues to reproduction, survival, growth/development, and population dynamics in contaminated habitats is unknown.

COMPARISON OF SEROLOGICAL METHODS AND BLOOD CULTURE FOR DETECTION OF TRYPANOSOMA CRUZI INFECTION IN RACCOONS (PROCYON LOTOR)

The indirect immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were compared with blood culture for the detection of Trypanosoma cruzi infection in 83 raccoons (Procyon lotor) trapped in 4 counties of southeast Georgia. Both IFAT and ELISA detected 24 of 25 culture-positive samples (96% sensitivity). Cultures from 25 raccoons (30%) were positive for epimastigotes, whereas a total of 50 raccoons (60%) was seropositive by either the IFAT or ELISA. Forty-five of 83 serum samples (54%) were positive for anti-T. cruzi antibodies with the ELISA, and 47 were IFAT positive (57%). Forty-two of the 50 seropositive raccoons (84%) were seropositive by both tests. Endpoint titers of IFAT-positive samples were determined by testing doubling dilutions from 1:40 to 1:1280. High titers of 640 and 320 were observed for 4 raccoons trapped in 1 county (St. Catherines Island, Liberty County) and titers of 160 for 1–2 raccoons from each of the 4 counties sampled. IFAT titers and ELISA optical density values were positively correlated. Both serological tests have a high sensitivity and should be excellent tools for studying the prevalence of T. cruzi in wildlife populations.

Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from South Carolina

Biological and molecular characteristics of a raccoon isolate of Trypanosoma cruzi (R36) were compared with those of a known virulent strain (Brazil). Included in the characterization were growth rate in liver infusion tryptose medium, infectivity for murine fibroblasts, intracellular amastigote replication and trypomastigote release rates, polymerase chain reaction (PCR) profiling of the mini-exon gene, isoenzyme and random amplified polymorphic DNA (RAPD) profiles, and in vivo virulence for C3H/HeJ mice. Similar growth curves were noted for both strains; however, infectivity and rates of intracellular amastigote replication and trypomastigote release were significantly lower for the R36 isolate than for the Brazil strain. To determine virulence, C3H/HeJ mice were exposed intraperitoneally to the R36 isolate. No parasite was observed in blood by direct examination or in tissues by histology; however, T. cruzi was detected by PCR in tissues (quadriceps and spleen) at 21 days postinfection. Analyses of the mini-exon gene, isoenzyme, and RAPD profiles indicate that R36 is in the T. cruzi II group and the Brazil strain is in the T. cruzi I group. Although infectivity and virulence of the raccoon isolate were lower than those for the Brazil strain, autochthonous infections in the United States have been reported, which suggests the need for further study of local T. cruzi isolates.

Detection of Aeromonas caviae in the common housefly Musca domestica by culture and polymerase chain reaction.

Aeromonas caviae has been implicated in diarrhoeal disease of livestock and humans. The potential role of houseflies in the epidemiology of this pathogen was investigated by examining the prevalence of A. caviae in houseflies collected from two South Carolina farms and one restaurant. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific PCR using novel primers (APW-PCR). All isolates cultured from houseflies were identified as A. caviae by biochemical characteristics and direct sequencing approximately 800 bp of the 16S rRNA gene. Aeromonas caviae was detected in 78% (272/349) dairy farm flies, 55% (54/99) pig farm flies and 39% (77/200) restaurant flies. Faeces from cows and pigs at the farms also were positive for A. caviae (58% and 100%, respectively). The APW PCR method provided a rapid, convenient way to identify A. caviae from faeces and houseflies that contained hundreds of bacterial species.

Fumonisin B1 affects viability and alters nitric oxide production of a murine macrophage cell line

Fumonisin B1 (FB1), the major toxin produced by Fusarium verticillioides contaminating corn, is known to elicit many organ- and species-specific toxicities in animals. In the present study, exposure to FB1 decreased viability of a murine macrophage cell line (RAW264.7) in a dose-dependent manner (1-100 microM). Further, when cells exposed to FB1 were stimulated with lipopolysaccharide (LPS), a dose-dependent increase in production of nitric oxide (NO) was observed, but only at FB1 concentrations (10-50 microM) that induced significant cytotoxicity. Stimulation of cells with phorbol myristate acetate (PMA) resulted in increased NO production at 50 microM FB1, but induced a variable NO response at 1-10 microM FB1. Results suggest that FB1 affected cell viability and altered inducible NO production by RAW macrophages in a manner that was dependent on the pathway of stimulation.

Gametocytogenesis of Leucocytozoon Smithi

ABSTRACT. Development of young gamelocytes of Leucocytozoon smithi into morphologically mature forms was studied using electron microscopy. Gametocytogenesis began on day seven post inoculation when merozoites, released from ruptured hepatic schizonts, developed into gametocytes within mononuclear phagocytes or leukocytes (monocytes or lymphocytes). No gametocytes were observed in any erythrocytes or polymorphonuclear leukocytes. Two gametocyte forms, round and elongate, were observed. Immature round gametocytes occurred on days 7‐10 post inoculation in the deep vasculature of liver, lung and spleen. Mature elongate gametocytes were observed beginning on day 12 post inoculation in both the deep tissue vasculature and peripheral circulation of the turkey host. Growth and elongation of the gametocyte resulted in distortion of the host cell and its nucleus. the host cell nucleus initially was elongated and displaced to one side or indented by the growing parasite. Eventually, the nucleus was laterally compressed or split into two or three fragments. the compressed host cell cytoplasm was displaced longitudinally and stretched over the parasite to form hornlike cytoplasmic extensions from each end. the potential role of microtubules in the elongation of the gametocyte and its host cell, and possibly in the indentation and splitting of the host cell nucleus, is discussed.

Occurrence of Metacercariae (Trematoda: Gymnophallidae) on Amphitrite ornata (Annelida: Terebellidae)

gotes can catabolize glucose and proline. Thymidine and uridine are also taken up and incorporated into TCA precipitable products (Fig. 2). Thus no metabolic lesion is apparent as measured by the incorporation of glucose, proline, uridine and thymidine into TCA precipitable products. These data coupled with our earlier demonstration of all enzymes of Embden-Meyerhof pathway, tricarboxylic acid cycle and pentose shunt in the amastigote form (Meade et al., 1984, loc. cit.) show that the cytozoic stage of the parasite is qualitatively comparable to the culture form. Metabolic lesions or limitations may, however, be present in other pathways which could not be detected by the types of ex(I)

Gametogenesis, Fertilization and Ookinete Differentiation of Leucocytozoon smithi

Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1–2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the zygote elongated. Development of sausage-shaped ookinetes was completed within 6–8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double-layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.ABSTRACT. Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1–2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the xygote elongated. Development of sausage‐shaped ookinetes was completed within 6–8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double‐layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.