Risk of handling as a route of exposure to infectious waterborne Cryptosporidium parvum oocysts via Atlantic blue crabs (Callinectes sapidus)

Commercial Atlantic blue crabs (Callinectes sapidus) were exposed to 2.0x10(4) infectious waterborne oocysts of Cryptosporidium parvum. The study demonstrated that blue crabs can transfer C. parvum oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage.     

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 × 10⁶ oocysts/liter; approximately 1.9 × 10⁵ 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).     

High Connectivity among Blue Crab (Callinectes sapidus) Populations in the Western South Atlantic

Population connectivity in the blue crab Callinectes sapidus was evaluated along 740 km of the Western South Atlantic coast. Blue crabs are the most exploited portunid in Brazil. Despite their economic importance, few studies report their ecology or population structure. Here we sampled four estuarine areas in southern Brazil during winter 2013 and summer 2014 in order to evaluate diversity, gene flow and structure of these populations. Nine microsatellite markers were evaluated for 213 adult crabs, with identification of seven polymorphic loci and 183 alleles. Pairwise F_ST values indicated low population structure ranging from -0.00023 to 0.01755. A Mantel test revealed that the geographic distance does not influence genetic (r = -0.48), and structure/migration rates confirmed this, showing that even the populations located at the opposite extremities of our covered region presented low F_ST and exchanged migrants. These findings show that there is a significant amount of gene flow between blue crab populations in South Brazil, likely influenced by local current dynamics that allow the transport of a high number of larvae between estuaries. Considering the elevated gene flow, the populations can be considered a single genetic stock. However, further information on population size and dynamics, as well as fishery demands and impacts at different regions, are necessary for harvest management purposes.     

Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10³ oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.     

Survival of Infectious Cryptosporidium parvum Oocysts in Seawater and Eastern Oysters (Crassostrea virginica) in the Chesapeake Bay

Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.     

Giardia sp. Cysts and Infectious Cryptosporidium parvum Oocysts in the Feces of Migratory Canada Geese (Branta canadensis)

Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment.     

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.     

Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Water fluxes and urine production in blue crabs (Callinectes sapidus) as a function of environmental salinity

• 1.1. Water efflux and urine production rates were measured in blue crabs acclimated to several salinities.
• 2.2. In 100% seawater the mean rate of water efflux (31.3 ml/100g hr⁻¹) was significantly greater than that in 50% seawater (18.9 ml/100 g hr⁻¹.
• 3.3. Water efflux was directly related to body weight.
• 4.4. The mean urine production rate was significantly greater in crabs acclimated to 50% and 30% seawater (0.17 and 0.18 ml/100g hr⁻¹) than in animals conditioned to 100% seawater (0.09 ml/100 g hr⁻¹).
• 5.5. The difference between theoretical net water fluxes for crabs exposed to 100% seawater and 50% seawater was similar to the difference in urine output in the same salinities, demonstrating the importance of the antennal gland in volume regulation.

     

Synthesis of a high-density lipoprotein in the developing blue crab (Callinectes sapidus)

An important lipoprotein in the hemolymph of crustaceans is LpI. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the LpI peptide to demonstrate by ELISA, western blot and immunohistochemistry the appearance of LpI during development of Callinectes sapidus, the blue crab. LpI was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic immunoreactivity for LpI at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic resemblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for LpI.     

Immunomagnetic Separation (IMS)-Fluorescent Antibody Detection and IMS-PCR Detection of Seeded Cryptosporidium parvum Oocysts in Natural Waters and Their Limitations

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.     

Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.     

Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml⁻¹. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×10⁷ pfu fish⁻¹ hour⁻¹ one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d⁻¹ were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.     

Cardiac frequency compensation responses of adult blue crabs (Callinectes sapidus Rathbun) exposed to moderate temperature increases

• 1.1. Cardiac frequency patterns of Callincctes sapidus Rathbun were used to evaluate potential thermal stress after exposure to 5°C increases over a range of acclimation temperatures from 5° to 30°C.
• 2.2. An acclimated rate-temperature curve (R-T curve), acute R-T curves of the stabilized rates at the increased temperatures and Q₁₀ temperature coefficients were used to assess the significance of the changes in rate frequency.
• 3.3. The acclimated R-T curve showed that blue crabs go through a series of seasonal adaptation types characterized by a plateau of perfect adaptation for both cold and warm adapted organisms. Paradoxical adaptation occurred between the transition from cold to warm acclimation temperatures.
• 4.4. The acute R-T curves showed that cardiac frequency was highly responsive to a 5°C increase when the organisms were acclimated to low temperatures.
• 5.5. The Q₁₀'s of the acute R-T curves at the warm acclimation temperatures approximated those values derived for the acclimated R-T curve.
• 6.6. This suggests that the temperature increase had a negligible effect on the warm adapted crabs, that is, little or no thermal stress occurred.

     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

Comparison of Sensitivity of Immunofluorescent Microscopy to That of a Combination of Immunofluorescent Microscopy and Immunomagnetic Separation for Detection of Cryptosporidium parvum Oocysts in Adult Bovine Feces

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.