Targeted and Stimulus-Responsive Delivery of Surfactant to the Oil-Water Interface for Applications in Oil Spill Remediation.

The use of chemical dispersants is a well-established approach to oil spill remediation where surfactants in an appropriate solvent are contacted with the oil to reduce the oil-water interfacial tension and create small oil droplets capable of being sustained in the water column. Dispersant formulations typically include organic solvents, and to minimize environmental impacts of dispersant use and avoid surfactant wastage it is beneficial to use water-based systems and target the oil-water interface. The approach here involves the tubular clay minerals known as halloysite nanotubes (HNTs) that serve as nanosized reservoir for surfactants. Such particles generate Pickering emulsions with oil, and the release of surfactant reduces the interfacial tension to extremely low values allowing small droplets to be formed that are colloidally stable in the water column. We report new findings on engineering the surfactant-loaded halloysite nanotubes to be stimuli responsive such that the release of surfactant is triggered by contact with oil. This is achieved by forming a thin coating of wax to stopper the nanotubes to prevent the premature release of surfactant. Surfactant release only occurs when the wax dissolves upon contact with oil. The system thus represents an environmentally benign approach where the wax coated HNTs are dispersed in an aqueous solvent and delivered to an oil spill whereupon they release surfactant to the oil-water interface upon contact with oil.

Antibodies to the ventral disc protein delta-giardin prevent in vitro binding of Giardia lamblia trophozoites.

A cDNA coding for detlaq-giardin was cloned from Giardia lamblia trophozoites to localize the protein and to study its function in mediating surface attachment. Recombinant delta-giardin antigen was expressed in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatography for production of antisera to delta-giardin. By immunoblotting analysis, antisera to recombinant delta-giardin antigen recognized a 31-kDa protein on G. lamblia trophozoites. Anti-recombinant delta-giardin was used to localize the native protein to the trophozoite ventral disk in both immunofluorescence and immunoelectron microscopy assays. Pre-treatment of G. lamblia trophozoites with anti-delta-giardin sera caused morphological changes in the parasite and inhibited trophozoite binding to the surface of cell culture slides. Binding of antibodies to delta-giardin may provide a means of inhibiting attachment of G. lamblia trophozoites to the intestinal epithelium and thereby prevent clinical giardiasis.

A longitudinal study of Giardia duodenalis genotypes in dairy cows from birth to 2 years of age.

Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence of Giardia duodenalis genotypes in cattle of different ages. Fecal samples were subjected to density gradient centrifugation to remove debris and concentrate cysts. Specimens were analyzed by immunofluorescence microscopy and polymerase chain reaction (PCR). All PCR positive specimens were sequenced using the SSU-rRNA gene of Giardia. All 30 calves shed G. duodenalis cysts at some time during the study. Of 990 specimens, 312 were positive for G. duodenalis (31.5%). The highest prevalence of infection occurred at weeks 4 and 5 of age with 25 out of 30 calves shedding cysts at those sampling times. Overall, pre-weaned calves (<8 weeks of age) exhibited the highest prevalence (60.8%), followed by post-weaned calves (3-12 months of age) (32.1%) and heifers (12-24 months of age) (11.4%). Sequence analysis of the 312 PCR-positive samples revealed the presence of both Assemblages A and E, G. duodenalis, with cumulative prevalences of 70% and 100%, respectively. Assemblage A was not detected in pre-weaned calves, but was detected in 6.9% and 4.7% of post-weaned calves and heifers, respectively. These data indicate not only that calves are infected with both Assemblages A and E simultaneously, but also that infections with zoonotic Assemblage A, G. duodenalis are more common than previously reported. Thus, calves appear to be a more significant reservoir of human infectious G. duodenalis than previous data have suggested.

Molecular and immunohistochemical detection of assemblage E, Giardia duodenalis in scouring North Dakota calves.

Despite many reports on the shedding of Giardia parasites by scouring calves, the role of Giardia as a cause of calf diarrhea is still controversial. To elucidate the role of Giardia duodenalis in calf scours, diagnostic samples from 189 scouring calves were tested by different assays during a 1-year-study period. Giardia antigens were detected in 22/189 scouring calves by a fecal-based enzyme-linked immunosorbent assay and 10 of these were positive for assemblage E, G. duodenalis by polymerase chain reaction. Giardia trophozoites were demonstrated by immunohistochemistry in intestinal sections from five calves in which the parasites were spatially distributed in areas of microscopically detectable enteritis. Our data suggest that under certain circumstances, Giardia may cause intestinal lesions leading to calf scours. Gnotobiotic calf-based infectivity studies are needed if the pathogenicity of Giardia in calves is to be definitively determined.

A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age.

Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1-8 weeks of age) (45.8%) than in post-weaned calves (3-12 months of age) (18.5%) and heifers (12-24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in pre-weaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1.

Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus).

A new species, Cryptosporidium ryanae, is described from cattle. Oocysts of C. ryanae, previously identified as the Cryptosporidium deer-like genotype and recorded as such in GenBank (AY587166, EU203216, DQ182597, AY741309, and DQ871345), are similar to those of Cryptosporidium parvum and Cryptosporidium bovis but smaller. This genotype has been reported to be prevalent in cattle worldwide. Oocysts obtained from a calf for the present study are the smallest Cryptosporidium oocysts reported in mammals, measuring 2.94-4.41micromx2.94-3.68microm (mean=3.16micromx3.73microm) with a length/width shape index of 1.18 (n=40). The pre-patent period for two Cryptosporidium-naïve calves fed C. ryanae oocysts was 11 days and the patent period was 15-17 days. Oocysts were not infectious for BALB/c mice or lambs. Fragments of the SSU-rDNA, HSP-70, and actin genes amplified by PCR were purified and PCR products were sequenced. Multi-locus analysis of the three unlinked loci demonstrated the new species to be distinct from all other species and also demonstrated a lack of recombination, providing further evidence of species status. Based on morphological, molecular and biological data, this geographically widespread parasite found only in Bos taurus calves is recognized as a new species and is named C. ryanae.

Detection of Assemblage A, Giardia duodenalis and Eimeria spp. in alpacas on two Maryland farms.

Sixty-one fecal samples were collected from adult alpacas and crias (ages 10 weeks to 10 years) on two farms in central Maryland. The farms raised both suri (silky-haired) and huacaya (crimpy-haired) breeds. Females and crias were housed together on pasture, whereas older/breeding males were maintained on separate pastures. Samples were subjected to a density gradient centrifugation protocol to concentrate parasites and remove fecal debris and were examined by immuno-fluorescent and differential interference contrast microscopy. Oocysts of Eimeria spp. were noted in 14 fecal samples, 6 on MD-1 and 8 on MD-2. Based on oocyst morphometrics two species of Eimeria were present: E. punoensis (19.2 microm x 16.5 microm) and E. alpacae (23.7 microm x 19.5 microm). Five animals shed exclusively E. punoensis, seven shed exclusively E. alpacae, and two had mixed infections. The Eimeria infections were not associated with obvious clinical signs. To determine the presence of Cryptosporidium and Giardia species and genotypes, DNA was extracted from feces and subjected to PCR utilizing specific primers for the ssu-rRNA gene for both parasites. All PCR positive samples were further analyzed by DNA sequencing to identify the species or genotypes that were present. Assemblage A, G. duodenalis was detected in fecal samples from two alpacas on MD-1 and in one alpaca on MD-2. Assemblage E, G. duodenalis and Cryptosporidium spp. were not detected on either farm. Although the prevalence on these two farms was low, alpacas can harbor zoonotic G. duodenalis, and this should be borne in mind by persons interacting with the animals.

Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein.

Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.

Prevalence of Giardia duodenalis genotypes in adult dairy cows.

The prevalence of Giardia duodenalis genotypes was determined in adult dairy cows. Fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. The prevalence of G. duodenalis infection, ranged from 3% to 64%, with an average prevalence of 27% (144 positive cows out of 541 examined). DNA sequence analysis of the 16S rRNA gene revealed the presence of both Assemblage A and Assemblage E, G. duodenalis. Overall, Assemblage E was present in 25% of all animals tested and Assemblage A was present in 2% of the animals. As a percentage of G. duodenalis isolates, Assemblage E represented 94% and Assemblage A represented 6%. Although, most of the cows were infected with a genotype that is not known to be infectious for humans, adult cows on five farms did harbor varying levels of zoonotic Assemblage A, G. duodenalis. Therefore, although adult cows do not appear to be a significant source of human infectious cysts in the environment, the risk from this age group should not entirely be discounted.

Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland.

In the United Kingdom and Australia sheep have been implicated as sources of Cryptosporidium and Giardia that infect humans, but no such studies have been conducted in North America. Therefore, a study was undertaken to investigate the prevalence of these parasites in sheep on a farm in Maryland. Feces were collected from 32 pregnant ewes 1, 2, and 3 days after parturition and from each of their lambs 7, 14, and 21 days after birth. The presence of Cryptosporidium oocysts and Giardia cysts was determined by both immunofluorescence microscopy and PCR/gene sequence analysis. PCR was consistently more sensitive than microscopy. The prevalence, by PCR, of Cryptosporidium in ewes and lambs was 25 and 77.4%, respectively. Three species/genotypes of Cryptosporidium were identified: C. parvum, a novel C. bovis-like genotype, and Cryptosporidium cervine genotype. Cryptosporidium parvum and the cervine genotype have been reported worldwide in human infections. The novel C. bovis-like genotype is reported here for the first time. The prevalence of Giardia in ewes and lambs was 12 and 4%, respectively. Most infections were Assemblage E which is not zoonotic; however, one ewe was infected with zoonotic Assemblage A. The identification of only two lambs infected with C. parvum and one ewe infected with G. duodenalis Assemblage A suggests a low prevalence of these zoonoses. However, the high prevalence of the zoonotic cervine genotype indicates that sheep should be considered a potential environmental source of this human pathogen.

Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations.

Feces collected from 541 milking cows on two dairy farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida were examined for the presence of Cryptosporidium oocysts. Oocysts were concentrated from 15 g of feces from each cow and DNA was extracted. A two-step nested PCR protocol was used to amplify an 830 base pair fragment of the SSUrRNA gene. PCR-positive products were purified and sequenced. PCR-positive findings were obtained from cows in all seven states and from 11 of 14 farms. Cryptosporidium parvum, Cryptosporidium bovis, and Cryptosporidium andersoni were found on 2, 6, and 8 farms, and infected 0.4, 1.7, and 3.7% of the 541 cows, respectively. The overall lower prevalence of Cryptosporidium in these cows was very highly significant (p< or =0.0001) compared with younger cattle and the relative prevalence of each species of Cryptosporidium also differed when compared with younger cattle previously examined on most of these same farms. The very low level of infection with C. parvum, the major species pathogenic to both cattle and humans, suggests that mature dairy cattle are a relatively low risk source of infection for humans.

Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates.

The prevalence of Cryptosporidium, Giardia, and Enterocytozoon bieneusi in cats from Bogota (Colombia) was determined from fecal specimens and scrapings of duodenal and ileal mucosa screened by PCR. All PCR-positive specimens were sequenced to determine the genotype(s) present. Of 46 cats, 6 (13%) were positive for Cryptosporidium, 5 (11%) were infected with C. felis and one (2%) with C. muris. Three (6.5%) cats were infected with Giardia duodenalis Assemblage F. Eight (17%) cats were infected with four genotypes of E. bieneusi: genotype D-like (9%), K (4%), Peru 10 (2%), and Peru 5 (2%). This is the first report on the presence of zoonotic species/genotypes of Cryptosporidium and E. bieneusi in cats in Colombia.

An improved electron microscopic technique for the immunolabeling of Cryptosporidium parvum oocysts.

A technique was developed for immunolabeling Cryptosporidium parvum oocysts for subsequent observation by transmission electron microscopy. This method was developed to maintain architectural integrity of the oocyst wall and improve fixation of internal contents. The improved fixation and embedding method permits efficient immunolabeling of both nonexcysted and excysted C. parvum oocysts and may be applicable to other oocyst- and cyst-forming protozoa.

Prevalence and genotypes of Giardia duodenalis in 1-2 year old dairy cattle.

To determine the prevalence of Giardia genotypes in 12-24 month old dairy heifers, fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. Prevalence of Giardia infection, ranged from 11% to 75% on 14 farms with an average prevalence of 36% (204 positive cattle out of 571 examined). DNA sequence analysis of the 16S rRNA gene revealed 91% of the 204 Giardia isolates were Assemblage E, and 9% were Assemblage A. The prevalence of these genotypes varied greatly from farm to farm, with four farms having exclusively Assemblage E Giardia. Overall, Assemblage E was present in 33% of all animals tested and Assemblage A was present in 3% of the animals. Thus, while many of the heifers were infected with a genotype that is not known to be infectious for humans, 1-2 year old heifers on 10 of 14 farms did harbor zoonotic Assemblage A Giardia. Therefore, heifers cannot be overlooked as a potential source of human infectious cysts in the environment, with some farms representing a much higher risk than others.

Detection of Cryptosporidium felis and Giardia duodenalis Assemblage F in a cat colony.

Eighteen cats, 3-6 months of age, bred and housed in a closed colony, were transferred from that colony and placed in separate stainless steel cages in a building designed for housing animals. At daily intervals, feces were collected from the litter pans in each cage, pans and cages were cleaned, and fresh food and water were provided. Beginning 4 weeks after the transfer, oocysts of Cryptosporidium were detected in the feces of two cats by brightfield microscopy. For the following 21 days, with minor exceptions, feces from each cat were collected daily and examined by immunofluorescence microscopy and by molecular methods that included DNA extraction, 18S rDNA gene amplification, and DNA sequence analysis. Within those 22 days, every cat was found to be infected with Cryptosporidium felis and excreted oocysts for 6-18 days. Eight of these 18 cats also excreted cysts of Giardia duodenalis Assemblage F, a genotype found only in cats. Six Giardia infections were concurrent during part of the patency with C. felis infections. Neither diarrhea nor other signs of illness were observed in any of the cats during this time. Because C. felis is zoonotic these findings suggest that care should be taken by veterinary health care providers and others in close contact with cats, even when cats appear healthy and asymptomatic.

Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States.

The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.

Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans).

Feces and duodenal scrapings were collected from 22 coyotes (Canis latrans) killed in managed hunts in northeastern Pennsylvania. Polymerase chain reaction (PCR) methods were used to detect Giardia and Cryptosporidium spp. PCR-amplified fragments of Giardia and Cryptosporidium spp. SSU-rRNA genes were subjected to DNA sequence analysis for species/genotype determination. Seven coyotes (32%) were positive for G. duodenalis: three assemblage C, three assemblage D, and one assemblage B. Six coyotes (27%) were positive for Cryptosporidium spp. One isolate shared 99.7% homology with C. muris, whereas five others (23%) shared 100% homology with C. canis, coyote genotype. This is the first report on multiple genotypes of Giardia spp. in coyotes and on the prevalence of Cryptosporidium spp. genotypes in coyotes.

Prevalence of Microsporidia, Cryptosporidium spp., and Giardia spp. in beavers (Castor canadensis) in Massachusetts.

Feces from 62 beavers (Castor canadensis) in Massachusetts were examined by fluorescence microscopy (IFA) and polymerase chain reaction (PCR) for Microsporidia species, Cryptosporidium spp., and Giardia spp. between January 2002 and December 2004. PCR-positive specimens were further examined by gene sequencing. Protist parasites were detected in 6.4% of the beavers. All were subadults and kits. Microsporidia species were not detected. Giardia spp. was detected by IFA from four beavers; Cryptosporidium spp. was also detected by IFA from two of these beavers. However, gene sequence data for the ssrRNA gene from these two Cryptosporidium spp.-positive beavers were inconclusive in identifying the species. Nucleotide sequences of the TPI, ssrRNA, and beta-giardin genes for Giardia spp. (deposited in GenBank) indicated that the four beavers were excreting Giardia duodenalis Assemblage B, the zoonotic genotype representing a potential source of waterborne Giardia spp. cysts.

Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves.

To determine the prevalence of Giardia genotypes in post-weaned dairy calves, fecal specimens were collected from 3 to 11-month-old dairy calves per farm on two farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and DNA sequence analysis. Overall, PCR provided more sensitive detection than IFA. Prevalence of Giardia infection, as detected by PCR ranged from 20% on NC-2 to 81% on VT-2, with an overall prevalence of 52% (237 positive samples out of 456 total). DNA sequence analysis of the 16S rRNA gene revealed 87% of the 237 Giardia isolates were Assemblage E, and 13% were Assemblage A although the prevalence of these genotypes varied greatly from farm to farm, with five farms having no Assemblage A Giardia. Therefore, Assemblage E was present in 45% of all animals tested and Assemblage A was present in 7% of the animals. Thus, while many of the calves were infected with a genotype that is not known to be infectious for humans, post-weaned calves on nine of 14 farms did harbor Assemblage A Giardia. Therefore calves should be considered as a potential source of human infectious cysts in the environment, with some farms representing a much higher risk than others.

Genotypes of Cryptosporidium species infecting fur-bearing mammals differ from those of species infecting humans.

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.