Behavioral analysis of Microphallus turgidus cercariae in relation to microhabitat of two host grass shrimp species (Palaemonetes spp.).

The behavior of Microphallus turgidus cercariae was examined and compared to microhabitat selection of the second intermediate hosts of the parasite, Palaemonetes spp. grass shrimp. Cercariae were tested for photokinetic and geotactic responses, and a behavioral ethogram was established for cercariae in control and grass shrimp-conditioned brackish water. Photokinesis trials were performed using a half-covered Petri dish, and geotaxis trials used a graduated cylinder. Both photokinesis and geotaxis trials were performed in lighted and unlighted conditions. In 9 of 10 photokinesis experiments, over half of the cercariae swam horizontally under the covered half of a Petri dish in both the lighted and the unlighted trials. However, movement of the cercariae to the covered half of the dish was highest (81.4%) when the parasites were exposed to light. In the geotaxis study, most cercariae were found in the bottom third of a graduated cylinder water column in both the lighted and unlighted trials. The most frequently observed activity of individual cercariae in a lighted Petri dish was swimming on the bottom of the dish. Activity patterns of the cercariae were not affected by shrimp-conditioned water. Movement of the cercariae away from light into dark, active swimming at or near the bottom of the water column, and a lack of response to host odors suggest that the cercariae utilize search patterns that place the parasite in the preferred microhabitat of the principle second intermediate host, the grass shrimp P. pugio.

In Vitro Insemination of the Microphallid Digenean Gynaecotyla adunca.

In vitro cultivation of adult digeneans can benefit research on their biology and contribute to the development of new drugs and vaccines. Successful in vitro growth of excysted metacercariae into adults capable of producing embryonated eggs typically requires that the worms be inseminated. The goal of the study was to develop an in vitro insemination procedure for the progenetic microphallid digenean Gynaecotyla adunca. To do so, we determined the length of time needed for in vitro sperm development in excysted metacercariae and whether the adult worms could self-inseminate in the absence of conspecifics. We also examined the effect of different culture vessels, worm densities, incubation temperatures, length of time incubated with conspecifics, and different pH levels on the percentage of worms inseminated. We found that sperm maturation time for G. adunca was 8-10 hr postexcystment. In the absence of conspecifics, the parasite did not self-inseminate. We observed the highest percentage of inseminated worms when 50 excysted metacercariae were incubated at 37 C for 48 hr in 15-ml conical-bottom tubes containing pH 7 Hank's balanced salt solution. Furthermore, freshly excysted worms incubated in these conditions and then transferred to culture in Dulbecco's modified Eagle medium/F-12 medium and horse serum deposited normal-shaped, embryonated eggs. Our findings provide the basis for a straightforward, reproducible procedure that permits the in vitro insemination of the parasite G. adunca and should be applicable to other progenetic digeneans.

Optimization of Conditions for In Vitro Culture of the Microphallid Digenean Gynaecotyla adunca.

In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode, Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs, Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco's Modified Eagle medium/F-12 (DME/F-12) than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails, Ilyanassa obsoleta. None of these snails produced G. adunca cercariae.

Optimization of culture conditions for in vitro fertilization and reproduction of Microphallus turgidus (Trematoda: Microphallidae).

Most attempts to culture adult digeneans in vitro are unsuccessful. Even progenetic digeneans typically fail to produce infective eggs in axenic culture. However, metacercariae of Microphallus turgidus grown in vitro mature into adults and release eggs infective to the hydrobiid snail Spurwinkia salsa. The objectives of the present study were to verify the reproducibility of the M. turgidus culture protocol, to define optimal culture conditions for M. turgidus further, and to investigate why the parasite can be grown successfully in the absence of the definitive host. In the original cultivation protocol, excysted M. turgidus metacercariae from grass shrimp (Palaemonetes pugio) were incubated overnight in a conical-bottom centrifuge tube containing Hank's balanced salt solution and then cultivated in flat-bottom culture plate wells containing RPMI-1640 plus 20% horse serum. The gas phase was air. Worms cultured under this protocol consistently deposited eggs infective to snails. Worms grown in anaerobic conditions deposited few eggs, and those cultured in a gas phase of 5% CO(2) survived longer and produced more eggs than those cultured in air. However, snails were less likely to become infected when fed eggs deposited by worms cultured in 5% CO(2). Additionally, worms incubated with conspecifics in conical-bottom tubes prior to cultivation were more likely to be inseminated than worms incubated in flat-bottom culture wells; the highest percentages of inseminated worms occurred when metacercariae were incubated 24 hr in conical-bottom tubes at a density of 50 worms/tube and at a temperature of 37 C. Worms incubated in the absence of conspecifics were not fertilized and failed to produce infective eggs.

In vitro cultivation of Microphallus turgidus (Trematoda: Microphallidae) from metacercaria to ovigerous adult with continuation of the life cycle in the laboratory.

In vitro cultivation of trematodes would aid studies on the basic biology of the parasites and the development of chemotherapies and vaccines. Our goal was to measure the in vitro survival and maturation of metacercariae of Microphallus turgidus under different culture conditions. Metacercariae of M. turgidus from grass shrimp (Palaemonetes pugio) were excysted and cultured in humidified air at 37 degrees C in RPMI-1640 medium supplemented with 20% calf, chicken, or horse serum. Deposition of eggs was greatest in media containing horse or calf serum. Worms survived longest at 37 C, but did not produce greater numbers of eggs than worms cultured in RPMI-1640-supplemented horse serum at 42 degrees C. Most eggs deposited in vitro (>80%) were normal in shape and, after incubation for 10 days at 30 degrees C in brackish water, approximately 30% of them contained miracidia. Eighteen percent of hydrobiid snails (Spurwinkia salsa) fed these eggs shed cercariae 5-6 wk later. The cercariae were infective to grass shrimp (Palaemonetes vulgaris) and developed into metacercariae. This study is significant because it is the second instance in which a digenean, and the first time that a microphallid, has been demonstrated to develop in vitro from metacercariae into adult worms capable of producing infective eggs.

Prevalence of antibodies to Trypanosoma cruzi in raccoons (Procyon lotor) from an urban area of northern Virginia.

Raccoons (Procyon lotor) are reservoir hosts for Trypanosoma cruzi. A 3-yr-long serological survey was conducted to determine the prevalence of antibodies to this zoonotic parasite in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 464 raccoons were examined for T. cruzi antibodies at a 1:40 dilution with an indirect fluorescent antibody test (IFAT) and Brazil strain T. cruzi amastigotes and trypomastigotes as antigen. A positive IFAT test was found in 154 (33%) of the 464 samples. The yearly prevalence was 49 of 132 (37%) in 2000; 19 of 120 (16%) in 2001; and 86 of 212 (41%) in 2002. Our study indicates that raccoons in this area of Virginia are frequently exposed to T. cruzi.

Effects of Microphallus turgidus (Trematoda: Microphallidae) on the predation, behavior, and swimming stamina of the grass shrimp Palaemonetes pugio.

The effect of the trematode Microphallus turgidus on its second intermediate host, the grass shrimp, Palaemonetes pugio, was tested. To do so, we measured the susceptibility of infected and uninfected shrimp to predation by the mummichog, Fundulus heteroclitus. Shrimp behavior was compared in the presence and absence of a fish predator, and the swimming stamina and backthrust escape responses of infected and uninfected shrimp were measured. Infected shrimp were more likely to be eaten by a predator than uninfected shrimp, had lower swimming stamina, and spent more time swimming and less time motionless in the presence of a predator. There was no difference between backthrust distances traveled in response to a stimulus by either infected or uninfected shrimp. Thus, M. turgidus may increase the predation of P. pugio in the wild, possibly by affecting the swimming stamina and predator avoidance responses of the shrimp.

In vitro excystation of metacercariae of Microphallus turgidus under different physicochemical conditions.

This study examined the excystation of metacercarial cysts of Microphallus turgidus under varying conditions of temperature, NaCl concentration, and pH. Metacercarial cysts were dissected from abdominal muscle tissue of grass shrimp Palaemonetes pugio, and immediately incubated for 24 h under one of the following abiotic conditions: temperature (22, 26, 30, 34, 38, and 42 degrees C), NaCl concentrations (0.5, 0.6, 0.7, 0.8, and 0.9%), pH (2, 3, 4, 5, 6, 7, and 8), and percent excystation was recorded. The optimum in vitro excystation of metacercarial cysts occurred in 0.7% saline at 38 degrees C at pH 7; 92% of the cysts excysted under these conditions. Our results indicate that such a combination of extrinsic factors is provided by the definitive host for successful excystation of M. turgidus.

Amplification of Trypanosoma cruzi-specific DNA sequences in formalin-fixed raccoon tissues using polymerase chain reaction.

This investigation applied polymerase chain reaction (PCR) using 3 sets of Trypanosoma cruzi-specific primers to amplify DNA from 31 archived formalin-fixed and fresh-frozen raccoon hearts. PCR successfully amplified T. cruzi-specific sequences, with at least 1 primer set, from multiple sites within the myocardium of formalin-fixed and fresh-frozen raccoon hearts that had previously tested positive using enzyme-linked immunosorbent assay and indirect immunofluorescent antibody titer in the absence of positive hemoculture results. Trypanosoma cruzi DNA was most frequently amplified from the interventricular septum, right ventricle, and left atrium. In addition, T. cruzi DNA was amplified with all 3 primers in at least I raccoon that was hemoculture positive and 2 animals that were borderline negative for the T. cruzi antibody and hemoculture negative. The amplification of T. cruzi-specific DNA sequences in the presence of an elevated antibody titer and negative culture results suggests good sensitivity of this method for detecting the presence of the parasite in archival tissues.

Prevalence, geographic distribution, and fitness effects of Microphallus turgidus (trematoda: Microphallidae) in grass shrimp (Palaemonetes spp.) from coastal Georgia.

Grass shrimp, Palaemonetes pugio Holthuis and P. vilgaris (Say), were collected at 11 localities along the coast of Georgia and surveyed for digenetic trematode metacercariae. The effect of trematode infection on grass shrimp fitness was also examined. Microphallus turgidas (Leigh) was the only trematode observed. The prevalence of metacercarial cysts of this parasite in P. pugio (75%) was higher than in P. vulgaris (24%), as were the mean intensity, abundance, and population density (no. cysts/cm host body length) of the parasite. Infected shrimp were found at every collection locality and parasite prevalence and density were greatest in P. pugio from higher salinity localities (> or = 20 parts per thousand [ppt]). There was no relation between host body size and parasite density in P. vulgaris, and parasite density increased with host body size in P. pugio, suggesting that the parasite does not affect host survival. There was no relation between parasite density and shrimp egg mass, but nonovigerous female P. pugio were more heavily infected than ovigerous ones. In addition, 1 metacercaria of M. turgidus in each of 2 specimens of P. vulgaris was parasitized by the haplosporidian Urosporidium crescens De Turk. This represents a new shrimp host record for this hyperparasite.