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.

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.

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.

Gametocytogenesis of Leucocytozoon smithi.

Development of young gametocytes 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 horn-like 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.

Sporogonic development of Leucocytozoon smithi.

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.

Parasites of the Christmas turkey.

In the temperate West, the turkey remains popular fare for festive feasts. It is a large bird, amenable to intensive rearing, and now represents one of the cheapest forms of poultry meat available (Box 1). In the USA alone, nearly 100 million birds are raised annually - mainly in Minnesota and North Carolina. But intensive rearing can incur risks of epizootic parasitic diseases, often responsible for severe economic losses. Improved management and medication have reduced the impact of some, such as 'gapezvorm disease', histomoniasis and intestinal coccidiosis; leucocytozoonosis now presents less of a threat than in the past, but some 'newer' diseases such as cryptosporidiosis may yet present severe problems. In this article, Peter Long, William Current and Gayle Noblet review the main parasite challenges faced by the commercial turkey industry.

Seasonal distribution of thermotolerant free-living amoebae. II. Lake Issaqueena.

A quantitative study of the seasonal distribution of thermotolerant (37 degrees C and 45 degrees C), small free-living amoebae (FLA) was conducted in Lake Issaqueena, a warm, monomictic lake with steep, sloping banks and a maximum basin depth of 10 m in the Piedmont region of South Carolina. Naegleria and Vahlkampfia were the most frequently encountered FLA in littoral sediment and surface water samples whereas Acanthamoeba was most commonly isolated from profundal sediment, especially during late summer. In the water column, FLA populations were highest in a persistent detrital layer; however, few amoebae were isolated from a massive (approximately 1.5 m thick) layer of Oscillatoria. The only N. fowleri isolated in this study was from the detrital layer. Discussion of the influence of differences in watershed and basin morphology on variations in the size and generic composition of FLA populations for the aquatic ecosystems of Lake Issaqueena and Willard's Pond is included.

Seasonal distribution of thermotolerant free-living amoebae. I. Willard's Pond.

A quantitative study of the seasonal distribution of thermotolerant (37 degrees C and 45 degrees C), small free-living amoebae (FLA) was conducted in Willard's Pond, a warm, monomictic lake in the Piedmont region of South Carolina. Correlation of physical and chemical parameters with the seasonal distribution was facilitated by partitioning the aquatic ecosystem into benthic, planktonic, and neustonic habitats. Population densities of FLA peaked in late summer in each habitat; however, species composition varied between habitats. Littoral sediment appeared to be the major habitat for FLA, with peaks in populations of Acanthamoeba and Naegleria in August, Hartmannella in July, and Vahlkampfia in May. Populations in profundal sediment underwent dramatic seasonal shifts, apparently in response to the seasonal chemical changes in the hypolimnion. Acanthamoeba was most prevalent in late summer, representing as much as 82% of the FLA in profundal sediment. Distribution patterns and species composition of FLA from surface water were similar to those from littoral sediment; however, a greater percentage of Naegleria was found in surface water. Numerous FLA were isolated from the neustonic community (surface film), and the number of FLA isolated in the surface film at the deep water station was found to be significantly (P less than 0.05) greater than the number from subsurface (5-10 cm) samples. In the water column, FLA populations consistently were highest in the detrital layer, which persisted at a depth of 3.0-3.4 m throughout the summer period. The large percentage of Naegleria contributing to FLA in the detrital layer suggests that Naegleria amoeboflagellates sink through the layer, flagellate, and swim back up, such migrations possibly being triggered by a reduction of nutrients below the layer or by the presence of anoxic, reducing conditions in the hypolimnion. In addition, weather events were found to play a major role in the redistribution of FLA between various habitats in the aquatic ecosystem, with such changes probably due to resuspension of FLA from littoral sediment by wind action and input from the watershed via runoff.

Vertical distribution of potentially pathogenic free-living amoebae in freshwater lakes.

The vertical distribution of thermotolerant (37 degrees C and 45 degrees C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic particulate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by particulates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P less than 0.005) numbers of FLA were isolated from two particulate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe2+ state was predominant. In this midsummer period, 13 Naegleria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.

Effects of feeding schedules on diurnal periodicity of Leucocytozoon smithi gametocytes in the peripheral blood of domestic turkeys.

Eighteen domestic turkeys naturally infected with Leucocytozoon smithi Laveran & Lucet were maintained on restricted feeding schedules under conditions of either continuous light or natural light (light 13 h:darkness 11 h) photoperiods. Peripheral gametocyte numbers of L. smithi in all turkeys were determined every 2 h over a 36-h period. Peripheral gametocyte numbers of turkeys maintained under continuous light and restricted to either a 10-h feeding period (9:30 p.m. to 7:30 a.m.) once a day or a 2-h feeding period twice a day (7:30 a.m. to 9:30 p.m. and 7:30 p.m. to 9:30 p.m.) increased at or near the time of feed availability. Under natural photoperiod, gametocyte periodicity was not affected by restricting feed availability to the dark phase of the light-dark cycle. Mean parasite numbers were highest during the light phase when feed was not available, and lowest during the dark period when feed was accessible.

Tetrachlorvinphos as an ineffective feed additive in control of gastrointestinal nematodes of beef cattle.

Tetrachlorvinphos was evaluated for anthelmintic efficacy as a feed additive against gastrointestinal nematodes of cattle. Tetrachlorvinphos was added to the mineral mixture of medicated cattle (n = 13) at the rate of 1.6 mg/day/kg of body weight for a 29-week period beginning May 2, 1975. Data suggest that the level of parasitism, as estimated by the number of nematode eggs per gram of feces, was not reduced by the addition of tetrachlorvinphos to the mineral mixture of medicated cattle. Results obtained from identification and quantification of nematodes recovered at necropsy from 6 medicated and 6 nonmedicated control heifers indicated that tetrachlorvinphos was ineffective as an anthelmintic against gastrointestinal nematodes of cattle.

The effect of photoperiod on the deep body temperature of domestic turkeys and its relationship to the diurnal periodicity of Leucocytozoon smithi gametocytes in the peripheral blood of turkeys.

Eighteen turkeys naturally infected with Leucocytozoon smithi were separated into 3 groups of 6 each and were respectively exposed to conditions of natural, reversed, or continuous light. Turkey deep body temperature and peripheral gametocyte numbers of L. smithi in all three groups were determined every 2 hr over a 36-hr period. The peak period of both turkey body temperature and gametocyte numbers coincided with the midpoint of the light period for turkeys exposed to natural and reversed light conditions. The body temperature of turkeys exposed to continuous light remained relatively constant while L. smithi gametocytes exhibited asynchronous behavior.

Effect of photoperiod on the diurnal periodicity of Leucocytozoon smithi gametocytes in the peripheral blood of domestic turkeys.

The daily cyclical variation in Leucocytozoon smithi gametocyte numbers in the peripheral blood of domestic turkeys was shifted 12-hr. by exposure of hosts to a reversed photoperiod. Peak parasitemia coincided with the midpoint of the artificial light period (0100 hr.), while low parasitemia occurred between 1300 hr. and 1700 hr. Disruption of strict gametocyte periodicity was suggested by results of birds exposed to light varying in intensity from 700 lux during the day to 161 lux at night although marked fluctuations in gametocyte numbers occurred.

Periodicity of Leucocytozoon smithi gametocytes in the peripheral blood of domestic turkeys.

Gametocytes of Leucocytozoon smithi exhibited a cyclic activity both in cardiac and peripheral blood of domestic turkeys during a given 24-hr. period. Peak parasitemias occurred from 0800 to 2000 hr., and mean low parasitemias from 2200 to 0400. The most prominent feeding period of the vector Simulium slossonae coincided with the period of elevated parasitemia in the turkey host.

Survey of leucocytozoon in in South Carolina.

An extensive survey of birds for Leucocytozoon in South Carolina during the summer of 1972 revealed that Blue Jays, Purple Grackles and domestic chickens were commonly infected. No alternate hosts were found for Leucocytozoon smithi of turkeys.