Molecular markers and sentinel organisms for environmental monitoring.

Molecular methods are useful for both to monitor anthropogenic viral, bacterial, and protozoan enteropathogens, and to track pathogen specific markers in a complex environment in order to reveal sources of these pathogens. Molecular genetic markers for fecal viruses, bacteria, and protozoans hold promise for monitoring environmental pollution and water quality. The demand for microbiologically safe waters grows exponentially due to the global demographic rise of the human population. Economically important shellfish, such as oysters, which are harvested commercially and preferentially consumed raw can be of public health importance if contaminated with human waterborne pathogens. However, feral molluscan shellfish which do not have an apparent economic value serve as indicators in monitoring aquatic environments for pollution with human waterborne pathogens and for sanitary assessment of water quality. Current technology allows for multiplexed species-specific identification, genotyping, enumeration, viability assessment, and source-tracking of human enteropathogens which considerably enhances the pathogen source-tracking efforts.

Sparganosis in wild-caught baboons (Papio cynocephalus anubis).

BACKGROUND: Sparganosis is the infection of a paratenic host with the plerocercoid metacestode of Spirometra spp. A 12-year-old captive, pregnant, wild-caught baboon from Tanzania had multiple subcutaneous nodules. METHODS: Examination of the biopsied nodules revealed the presence of viable metacestodes. The histological morphology of the metacestodes was consistent with the genus Spirometra and other pseudophyllidean cestodes. Since species of Spirometra produce growth hormones that are active in mammals, we measured fetal and placental growth and hormone levels. Blood samples were taken from the mother and the cesarean-derived fetus for hematological, biochemical, and hormonal analyses and to test for the presence of antispargana antibodies. RESULTS: Baboon placental weight and fetal hematological, biochemical, and morphometric parameters were within normal ranges. Antibody titers to spargana did not differ significantly between mother (1.08 OD(405)) and fetus (0.91 OD(405)). Baboon maternal insulin-like growth factor and growth hormone values were also within the normal range. Estradiol and progesterone analysis in four of these animals (antibody titers ranged from 0.71 to 1.7 OD(405)) showed no statistically significant difference with age- or phase-matched cycle parameters compared with antibody-negative females. CONCLUSIONS: Based on the results that have been obtained, sparganosis did not appear to affect the endocrinological profile of pregnant and cycling female baboons.

Accumulation of human waterborne parasites by zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea).

Zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea) are nonindigenous invasive bivalves present in North American fresh waters that are frequently contaminated with human enteric parasites, Cryptosporidium parvum and Giardia lamblia. Six-week laboratory exposure of D. polymorpha and Corbicula fluminea to both parasites seeded daily at concentrations reported from surface waters demonstrated efficient removal of Cryptosporidium parvum oocysts and G. lamblia cysts by both bivalve species. The number of parasites in mollusk tissue progressively increased in relation to the concentration of waterborne contamination, and decreased after cessation of the contamination. Oocysts outnumbered cysts in the tissue of both bivalves, and more parasites were identified in D. polymorpha than in Corbicula fluminea; overall 35.0% and 16.3% of the parasites seeded, respectively. Because C. fluminea and D. polymorpha can accumulate human waterborne parasites in proportion to ambient concentrations, these species of bivalves can be effective bioindicators of contamination of freshwater habitats with Cryptosporidium and Giardia.

Evaluation of the recovery of waterborne Giardia cysts by freshwater clams and cyst detection in clam tissue.

The Asian freshwater clam, Corbicula fluminea, inhabits environments recognized to be contaminated with waterborne Giardia cysts. Sixty-four tissue samples of Giardia-free clams were spiked with various numbers of Giardia duodenalis cysts within the range of 50-700 cysts. Regression analysis showed that paired numbers of spiked (x) versus recovered (y) cysts regressed significantly (P < 0.01) according to the equation y = 42.57 +/- 1.81x (+/- 64.3). The cyst detection threshold was 43 cysts/clam, the coefficient of determination was 77%, and the overall sensitivity of cyst detection was 42.9%. All 20 values of cyst numbers in clam tissue samples that were processed blind were located within the 95% prediction limits of the linear regression equation. The cyst retention rate of 160 clams kept in an aquarium with 38 l of water spiked with 1.00 x 10(5) G. duodenalis cysts was approximately 1.3 x 10(3) cysts/clam. No waterborne cysts were detected by the membrane filtration method 90 min after spiking the aquarium water. G. duodenalis cysts were detected in clam tissue up to 3 weeks post-exposure. Filtration of water by clams substantially depleted the aquarium water of its particulate matter. The sampling program demonstrated that the population of 160 clams examined during the study could be accurately assessed for exposure to waterborne Giardia cysts by random sampling of 86 (54%) clams. The results indicate that C. fluminea clams can be used for biological monitoring of contamination with Giardia.

Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Corbicula fluminea).

Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectious Cryptosporidium parvum oocysts (1.00 x 10(6) oocysts/liter; approximately 1.9 x 10(5) oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used--acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit).

Recovery of waterborne oocysts of Cyclospora cayetanensis by Asian freshwater clams (Corbicula fluminea).

Asian freshwater clams (Corbicula fluminea) were exposed for 24 hr in 38 liters of water contaminated with 1.0 x 10(5) Cyclospora cayetanensis oocysts (2.6 x 10(3) oocysts/L). The hemolyph and gill smears of 30 clams were examined by acid-fast stain on days 1, 3, 5, 7, 10, 13, and 18 postexposure (PE). Since no oocysts were detected in the water 24 hr after contamination by the membrane filter-dissolution method, the oocyst retention rate was 4.6 X 10(2) oocysts/clam. The prevalence of oocyst-positive clams significantly decreased (P < 0.01) from 93% to 47% during 13 days PE. None of the clams contained oocysts on day 18 PE; no oocysts were detected in the clam feces. The numbers of oocysts recovered from six clam size classes varied and significantly decreased with smaller clam size (P < 0.01). The lowest prevalence values of oocyst-positive clams, 45% and 34%, were observed in the two lowest size classes: 12.1-14.0 mm and 14.1-16.0 mm, respectively. The prevalence values in the remaining four classes ranged from 84% to 100%. The sampling program demonstrated that the population of 180 clams examined during the study up to 13 day PE could be assessed for C. cayetanensis positivity by random testing of a minimum of 75 clams (42%). When the two lowest clam size classes are eliminated, the population of 114 clams could be assessed by sampling a minimum of 32 clams (28%). The results demonstrate that Corbicula fluminea can recover waterborne oocysts of C. cayetanensis, and could be used as biological indicators of contamination of water with C. cayetanensis oocysts.

In Vitro Interactions of Asian Freshwater Clam (Corbicula fluminea) Hemocytes and Cryptosporidium parvum Oocysts.

Corbicula fluminea hemocytes phagocytosed infectious oocysts of Cryptosporidium parvum in vitro. After 15, 30, 60, 90, and 120 min of incubation, averages of 35.8, 58.0, 69.7, 77.7, and 81.6% of the oocysts were phagocytosed by 24.3, 70.0, 78.5, 87.3, and 93.0% of the hemocytes, respectively. A single clam can retain by phagocytosis an average of 1.84 x 10(sup6) oocysts per ml of hemolymph. C. fluminea bivalves can serve as biological indicators of contamination of wastewaters and agricultural drainages with Cryptosporidium.

In vitro phagocytosis of Giardia duodenalis cysts by hemocytes of the Asian freshwater clam Corbicula fluminea.

Hemocytes of the Asian freshwater clam Corbicula fluminea, phagocytosed in vitro infectious Giardia duodenalis cysts. After 15, 30, 60, 90, and 120 min of incubation an average of 22%, 32%, 43%, 54%, and 72% of the cysts were phagocytosed by 22%, 55%, 63%, 81%, and 86% of the hemocytes, respectively. The number of hemocytes showing phagocytosis and the mean number of cysts ingested per hemocyte increased significantly over time (P < 0.01); the numbers of nonphagocytosed cysts significantly decreased (P < 0.02). Extrapolation reveals that C. fluminea can retain by phagocytosis an average of 1.6 x 10(6) G. duodenalis cysts/ml hemolymph. The phagocytic capacity of C. fluminea hemocytes indicates the applicability of this freshwater benthic bivalve for bioindication of contamination of waste waters and agricultural drainage with Giardia cysts.

Chaetogaster limnaei (annelida: oligochaeta) as a parasite of the zebra mussel Dreissena polymorpha, and the quagga mussel Dreissena bugensis (mollusca: bivalvia).

Dreissenid mussels, Dreissena polymorpha and D. bugensis, were found to be infected by the naidid oligochaete Chaetogaster limnaei at four sites in the St. Lawrence River. This is the first report of this species infecting dreissenids anywhere in the world. Most worms inhabited the mantle cavity, where they caused erosion of the mantle and gill epithelia as determined by histopathological examination. Others penetrated various tissues; one had invaded the ovary and was feeding on oocytes and ovarian tissues. Of 606 mussels examined, 166 (27.4%) harbored at least 1 C. limnaei. The prevalence varied between 1% and 80%, depending on the collection site and date. The worms were slightly but significantly more prevalent in D. bugensis than in D. polymorpha. The intensity ranged from 1 to 18 worms per infected host. Variations in prevalence and intensity were not related to the size or sex of the host, but the data did suggest some seasonality.

Experimental infection of zebra mussels Dreissena polymorpha (Mollusca:Bivalvia) by metacercariae of Echinoparyphium sp. (Platyhelminthes:Trematoda).

The zebra mussel Dreissena polymorpha a species recently introduced to North America from Europe, was studied to determine whether it could potentially serve as host for echinostomatid metacercariae. Forty adult mussels were collected from the St. Lawrence River and exposed to cercariae of Echinoparyphium sp. that was emerging from naturally infected Physa sp. collected from a native population in northern New York state. When necropsied 24-30 hr postexposure, 2 (5%) of the 40 harbored a metacercaria of Echinoparyphium sp. in the gonad. None of 200 control mussels collected from the same site was infected. This is the first report of any species or stage of echinostomatid from this host species worldwide. This is also the first report of an echinostomatid metacercaria from the gonad of any host species.

Ultrastructure of the gravid uterus of Hymenolepis diminuta (Platyhelminthes: Cestoda).

The fine structure of the uterus in gravid proglottids of Hymenolepis diminuta was examined by standard techniques for scanning and transmission electron microscopy. The uterus consisted of a syncytial uterine epithelium attached to the medullary parenchyma through a thin extracellular basal matrix. The epithelium contained prominent nuclei in the juxtalumenal cytoplasm. The cytoplasm was dominated by extensive granular endoplasmic reticulum, with dilated cisternae containing an electron-lucent material and widely scattered electron-dense spherical bodies. No Golgi body or other agranular endomembrane component was observed, but the epithelium contained numerous free ribosomes and a few mitochondria. The apical plasma membrane was folded into long microlamellae. Epithelial and epitheliomesenchymal folds and villi resulted in a compartmentalized uterine lumen, with each chamber containing 1 to several eggs. These data suggest a high level of synthetic activity within the uterine epithelium, but the chemical products and functional significance of this activity are not yet known.

Helminth parasites of unisexual and bisexual whiptail lizards (Teiidae) in North America. V. Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea) from four species of Cnemidophorus.

Two hundred and one whiptail lizards, Cnemidophorus spp., from Texas and Colorado (USA), were examined for Mesocestoides sp. tetrathyridia. Eleven (5%) were infected, including three of 58 (5%) C. dixoni, six of 70 (9%) C. gularis septemvittatus, one of 35 (3%) C. marmoratus, and one of 34 (3%) C. tesselatus; four C. inornatus heptagrammus were not infected. In addition, 41 non-cnemidophorine lizards from the same study area were not infected. Free tetrathyridia were found in the body cavity of lizards and encapsulated tetrathyridia were observed in the heart, liver, stomach, mesenteries, ovaries, intestines, and lungs. None of the Mesocestoides sp. exhibited any evidence of asexual proliferation such as multiple scoleces or buds. This note, the fifth in a series of reports on helminths of Cnemidophorus spp., represents the first time Mesocestoides sp. has been reported from these four taxa, and Colorado is a new geographic locality record for this parasite.

A new host and locality record for Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea), with a summary of the genus from snakes of the world.

A new host and distribution record is reported for tetrathyridia of Mesocestoides sp. One of 5 (20%) Namib tiger snakes, Telescopus beetzi, from South Africa was infected. Numerous tetrathyridia were found encapsulated in mesentery attached to the small intestine. Morphological examination of tetrathyridia revealed absence of buds, multiple scoleces, or any other evidence of asexual proliferation. A summary of the snakes of the world reported as hosts of tetrathyridia of Mesocestoides sp. is presented.

Occurrence of tetrathyridia of Mesocestoides sp. (Cestoidea: Cyclophyllidea) in North American anurans (Amphibia).

A new host and geographic locality record is reported for tetrathyridia of Mesocestoides sp. in two species of ranid frogs (Rana berlandieri and R. pipiens) from Texas and New York, respectively. Tetrathyridia were found encapsulated in liver and mesenteries of the hosts. Morphological examination and experimental inoculation of these tetrathyridia into mice demonstrated the absence of capacity for asexual proliferation. Overall prevalence of infection was low in anurans from Arkansas, Texas and New York, but intensities can be generally high. In addition, a summary of frogs and toads from North America reported as hosts of tetrathyridia of Mesocestoides sp. is presented.

The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (Trematoda) infecting the kidneys of the frogs Rana clamitans and Rana pipiens.

Light and scanning electron microscopy were used to examine the localization and pathogenicity of echinostomatid metacercariae infecting the kidneys of leopard frogs, Rana pipiens, and green frogs, Rana clamitans. Cysts occurred predominantly in the ventrolateral renal cortex, and at least some were confined to the lumen of the Bowman's capsules. Each vermiform metacercarial body was enclosed by a spherical cyst wall that had a uniform thickness. The wall was composed of a homogeneous material containing basic and keratinlike proteins, with sulfated acid mucopolysaccharides on the outer surface. Most cysts were enclosed by a fibrous capsule of host origin, or were surrounded by an inflammatory focus. Fibrosis was always focal, but its degree varied between individual hosts and between different cysts within the same host. Some heavily encapsulated cysts were darkened and contained disintegrating worms. In heavily infected kidneys, confluence of fibrotic or inflammatory foci resulted in the displacement of functional renal tissue. These data suggest that infection by echinostomatids may impair renal function and that the host's response affects parasite viability.