Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: A. Cryptosporidium parvum. Our laboratory has characterized a subset of long-chain polyunsaturated fatty acids (L-PUFFA), isolated from bovine colostrum, which block Cryptosporidium parvum and Toxoplasma gondii host cell infectivity in vitro as well as both Toxoplasma gondii and Plasmodium gallinaceum infectivity in vivo. Our results indicate the mechanism of L-PUFFA-mediated inhibiton is due to blockage of parasite microneme secretion. Molecular genetic and chemical biology studies reveal calcium-dependent protein kinase 1 (CDPK1) regulates microneme secretion and that inhibition of this kinase leads to profound defects in parasite motility, invasion, and egress from host cells. Thus, CDPK is a potential target for development of new drugs for the treatment of not only cryptosporidiosis but also other serious apicomplexan diseases. To test this hypothesis we has leveraged NC-1041 funding by partnering with Dr. David Sibley, Washington University School of Medicine, on an NIH-funded project (beginning 7/1/12) entitled Designing Selective Inhibitors of Calcium-Dependent Kinases in Parasites. The University of Illinois portion of this project is to evaluate the ability of small molecule inhibitors of CDPK to block Cryptosporidium infectivity and growth in mammalian host cells. B. Rotavirus. Our research has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. We demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding to and infectivity of host cells in vitro. The production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic nutriceutical in swine, however, is cost prohibitive. To circumvent this problem, a sialyllactose-containing neoglycolipid (SLPE) was synthesized and assayed for its ability to inhibit rotavirus binding and infectivity of host cells. Using concentrations comparable to GM3, SLPE was able to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE decreased rotavirus infection of host cells by over 90%. Finally, the results of in vivo animal challenge studies using newborn pigs in their natural environment demonstrated SLPE afforded complete protection from rotavirus disease. We have also demonstrated selected natural human milk oligosaccharides (HMO), the third most abundant component in human milk, reduce the duration of diarrhea in piglets, possibly in part by promoting immunoglobulin response to rotavirus infection and modulating the gut microbiota. PARTICIPANTS: A new collaboration with Dr. David Sibley, Washington University School of Medicine, is ongoing to evaluate the effect of small molecular weight inhibitors of CDPK on infectivity and growth of Cryptosporidium parvum in mammalian host cells. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. The phylum, Apicomplexa, contains some of the most significant pathogens infecting humans and animals. Of the more than 4,000 species of apicomplexan parasites, malaria parasites, Toxoplasma gondii, and Cryptosporidia are the most important pathogens of humans, causing death or disability for millions of people each year. The goal of our research in this area is to define the early mechanisms of Apicomplexa-host interactions and to identify new drug candidates that can block these interactions. We have developed a battery of complementary in vitro and in vivo assays that allow us to quantitfy Cryptosporidium, Toxoplasma, and Plasmodium microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity/growth of these parasites is inhibited by selected inhibitors of parasite microneme secretion. These studies are now culminating in screening novel small molecular weight inhibitors of CDPK for the development of new drugs that show broad efficacy for treatment of apicomplexan parasitic diseases. B. Rotavirus. Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of both animals and people. They are of prime agricultural importance since they cause serious neonatal diarrhea of many animal species, most importantly neonatal and post-weaning pigs and calves. Extensive efforts to produce an effective commercial animal vaccine, including the use of reassortants, attenuated live strains and vector expression of viral capsid proteins have yet to be successful. Accordingly, the prophylaxis or treatment of rotavirus diarrheal disease by nutritional intervention, through the use of easily deliverable receptor mimetics and nutriceuticals, such as milk derived oligosaccharides, neoglycoconjugate receptor mimetics (SLPE), and bioactive free fatty acids are potentially of great value for the control of this disease in both agricultural and human medicine settings.
Publications
- Andres, A., Donovan, S.M., Kuhlenschmidt, T.B. and Kuhlenschmidt, M.S. 2007. Isoflavones at concentrations present in soy infant formula inhibit rotavirus infection in vitro., J. Nutr. 137: 2068 - 2073.
- Schmidt, J. and Kuhlenschmidt, M.S. 2008. Microbial adhesion of cryptosporidium parvum: Identification of a colostrum-derived inhibitory lipid. Molecular and Biochemical Parasitology 162:32-9.
- Liu, Y., Janjaroen, D., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2009. Deposition of cryptosporidium parvum oocysts on natural organic matter surfaces: Microscopic evidence for secondary minimum deposition in a radial stagnation point flow cell. Langmuir 25:1594-1605.
- Pineda, M.F., Chan, L.L., Kuhlenschmidt, T., Kuhlenschmidt, M.S. and Cunningham, B.T. 2009. Rapid label-free selective detection of porcine rotavirus using photonic crystal biosensors. Sensors Journal, IEEE 9: 470-477.
- Andres A, Donovan, S.M. and Kuhlenschmidt, M.S. 2009. Isoflavones and virus infections. J. Nutritional Biochemistry 20:563-569. (Invited Review).
- Donovan, S.M., Andres, A., Mathai, R.A., Kuhlenschmidt, T.B. and Kuhlenschmidt, M.S. 2009. Soy formula and isoflavones and the developing intestine. Nutritional Reviews 67(suppl 2) S192-S200.
- Janjaroen, D., Liu, Y., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2010. Role of divalent cations on deposition of cyrptosporidum parvum oocysts on natural organic matter surfaces. Environ. Sci. Technol. 44:4519-24.
- Liu, Y., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2011. Characterization of cryptosporidium parvum oocyst wall macromolecules and adhesion kinetics of oocysts on quartz surfaces. Biomacromolecules. 11:2109-15.
- Bergner, D.W., Kuhlenschmidt, T.B., Firkins, L.D. and Kuhlenschmidt, M.S. 2011. Synthesis and characterization of a neoglycolipid that blocks porcine group A rotavirus infectivity. Nutrients 3:228-244.
- Li, M., Bauer, L.L., Chen, X., Wang, M., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S., Fahey, G.C. and Donovan, S.M. 2012. Microbial composition and in vitro oligosaccharides and prebiotics differ between formula-fed and sow-reared piglets. J. Nutrition 142:1-10.
- Fuller, K.L., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S., Jimenez-Flores, R. and Donovan, S.M. 2012. Milk fat globule membrane isolated from buttermilk or cheese whey and their lipid component inhibit infectivity of rotavirus in vitro. J. Dairy Science (In Press).
- Gutierrez, L., Li, X., Wang, J., Nangmenyi, G., Economy, J., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Nguyen, T.H. 2009. Adsorption of rotavirus and bacteriophage MS2 using glass fiber coated with hematite nanoparticles. Water Res 43:5198-208.
- Li, M., Monaco, M.H., Wang, M., Comstock, S.S., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Donovan, S.M. 2013. Human milk oligosaccharides and prebiotics reduce the duration of rotavirus-induced diarrhea in piglets. JPGN (In Press).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: A. Cryptosporidium parvum. Our laboratory has discovered and characterized a subset of long-chain polyunsaturated fatty acids (L-PUFFA), originally isolated from bovine colostrum, which block Cryptosporidium parvum and Toxoplasma gondii host cell infectivity in vitro as well as both Toxoplasma gondii and Plasmodium gallinaceum infectivity in vivo. Our current studies address the mechanism of L-PUFFA-mediated inhibition of infectivity. Our results have led to the hypothesis that L-PUFFA block a conserved apicomplexan pathway that regulates parasite motility. This pathway involves a unique process of microneme secretion that regulates calcium mobilization and an actin-myosin motor that is critical for parasite motility. Our results suggest microneme secretion is a potential target that could be exploited for development of new drugs for the treatment of not only cryptosporidiosis but also other serious apicomplexan diseases such as toxoplasmosis and malaria. To probe this hypothesis I have leveraged NC-1041 funding by partnering with Dr. David Sibley, Washington University School of Medicine and submitted a NIH R01 application entitled 'Designing Selective Inhibitors of Calcium-Dependent Kinases in Parasites'. The goal of the University of Illinois Consortium portion of this application is to evaluate small molecule inhibitors of parasite-specific kinases belonging to the calcium-dependent protein kinase (CDPK) family. Recent studies using molecular genetic and chemical biology approaches reveal that CDPK1 controls microneme secretion and that inhibition of this kinase leads to profound defects in parasite motility, invasion, and egress from host cells. B. Rotavirus. Previous research in this laboratory has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding to and infectivity of host cells in vitro. The production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic nutriceutical in swine, however, is cost prohibitive. To circumvent this problem, a sialyllactose-containing neoglycolipid was synthesized and assayed for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination and the product purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with sialyllactosyl-DPPE (SLPE). Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, the results of in vivo animal challenge studies using newborn pigs in their natural environment demonstrated SLPE afforded complete protection from rotavirus disease. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Cryptosporidium. The phylum, Apicomplexa, contains some of the most significant pathogens infecting humans and animals. Of the more than 4,000 species of apicomplexan parasites, malaria parasites, Toxoplasma gondii, and Cryptosporidia are the most important pathogens of humans, causing death or disability for millions of people each year. The goal of our research in this area is to define the early mechanisms of Apicomplexa-host interactions and to identify new drug candidates that can block these interactions. We have developed a battery of complementary in vitro and in vivo assays that allow us to quantitfy Cryptosporidium, Toxoplasma, and Plasmodium microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity of these parasites is inhibited by L-PUFFA. Our results provide hope that small molecule natural products, such as L-PUFFA or CDPK inhibitors, will be valuable for the development of new drugs that show broad efficacy for treatment of apicomplexan parasitic diseases. B. Rotavirus. Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of both animals and people. They are of prime agricultural importance since they cause serious neonatal diarrhea of many animal species, most importantly neonatal and post-weaning pigs and calves. Extensive efforts to produce an effective commercial animal vaccine, including the use of reassortants, attenuated live strains and vector expression of viral capsid proteins have yet to be successful. Accordingly, the prophylaxis or treatment of rotavirus diarrheal disease by nutritional intervention, through the use of easily deliverable receptor mimetics and nutriceuticals, is potentially of great value for the control of this disease in both agricultural and human medicine arenas.
Publications
- Kowalewski, M.M., Salzer, J.S., Deutsch, J.C., Rano, M., Kuhlenschmidt, M.S. and Gillespie, T.R. 2011. Black and gold howler monkeys as sentinels of ecosystem health: Patterns of zoonotic protozoa infection relative to degree of human primate contact. Am. J. Primatology 73:75-83.
- Fuller, K.L., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S., Jimenez-Flores, R. and Donovan, S.M. 2011. Milk fat globule membrane isolated from buttermilk or cheese whey and their lipid component inhibit infectivity of rotavirus in vitro. Nutrients (In Press).
- Perez, V.G., Jacobs, C.M., Barnes, J., Jenkins, S.N., Kuhlenschmidt, M.S., Fahey, G.C. and Pettigrew, J. 2011. Effect of corn distillers dried grains with solubles MC and Eimeria acervulina infection on growth performance and the intestinal microbiota of young chicks. J. Poultry Sci. 9:959-964,
- Bhattrai, R., Kalita, P., Trask, J. and Kuhlenschmidt, M.S. 2011. Development of a physically-based model for transport of Cryptosporidium parvum in overland flow. Environ. Mod. SoftWare 26:1289-1287.
- Liu, Y., Zhang, C., Hilper, M., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2011. Transport of Cryptosporidium parvum oocysts in a silicon micromodel. Environ. Sci. Tech. (Submitted).
- Liu, Y., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2011. Characterization of cryptos-poridium parvum oocyst wall macromolecules and adhesion kinetics of oocysts on quartz surface. Biomacromolecules. 11:2109-15.
- Bergner, D.W., Kuhlenschmidt, T.B., Firkins, L.D. and Kuhlenschmidt, M.S. 2011. Synthesis and characterization of a neoglycolipid that blocks porcine group A rotavirus infectivity. Nutrients 3:228-244.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 1. Focus on effective interventions. Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents. A. Cryptosporidium parvum. Previously, we have shown that certain colostrum-derived, long-chain polyunsaturated fatty acids (LPUFA) inhibit C. parvum sporozoite-host-cell invasion. In current studies, we show linolenate, but not EA acid, the trans isomer of oleate, inhibits both C. parvum sporozoite and T. gondii tachyzoite infectivity in vitro. Similarly, LA also blocked in vivo infectivity of T. gondii tachyzoites and P. gallinaceum sporozoites. Preliminary mechanistic studies suggest LA or oleate, but not EA acid, block gliding motility of T. gondii tachyzoites and C. parvum sporozoites. While these data suggest that certain LPUFA represent possible therapeutic or prophylactic nutriceuticals, they clearly serve as useful reagents to probe what may be a common mechanism of host-cell interaction among these apicomplexan parasites. B. Rotavirus. Milk fat is encapsulated in a milk fat globule membrane (MFGM) that contains bioactive glycoproteins, carbohydrates and lipids. The MFGM inhibits infectivity of rotavirus (RV) and this activity had been attributed to its glycoprotein and carbohydrate components. Herein, the anti-RV activity of an organic extract of MFGM was tested. MFGM enriched in polar lipids was prepared from buttermilk (BM) and cheese whey (CW) by microfiltration and supercritical fluid extraction. MFGM lipid was extracted using single and dual phase extraction methods. Whole MFGM and organic extracts were screened in MA-104 cells for anti-infective activity against neuraminic acid-sensitive and -insensitive RV strains using a focus forming unit assay (FFU). Dose-dependent inhibition was observed for whole BM and CW against the OSU RV, but not Wa, strain of RV (p<0.05), with greater inhibitory activity observed against neuraminic acid-sensitive RV. In general, BM MFGM exhibited greater RV % inhibition than CW (p<0.05). Organic-soluble anti-RV compounds were identified in bovine MFGM. The most active fraction, isolated by dual phase extraction and Iatrobead chromatography, was free of proteins and highly non-polar. Further separation of this fraction in a less polar solvent (30:1 Chloroform:MeOH) resolved at least five lipid-containing compounds, which likely contribute to the anti-RV activity associated with bovine MFGM. In follow up animal challenge experiments evaluating the use of SLPE, a synthetic receptor mimetic, for protection of pigs from rotavirus disease we demonstrated SLPE was shown to decrease rotavirus infection of host cells by over 90% and afforded complete protection from rotavirus disease when fed to piglets twice daily while maintained with the sow. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bovine milk appears to be a rich source of lipids of anti-infective properties. Lipid components associated with MFGM appear to contribute in large part to the anti-RV activity associated with the bovine MFGM although further research is required to define the specific lipids responsible for this activity, as well as the mechanism of action. In addition, given the large quantities of whey generated from buttermilk and cheese production, it is warranted to explore value-added uses for MFGM isolated from these sources. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large-scale production capabilities make SLPE a promising candidate for further exploration as a possible prophylactic or therapeutic nutriceutical for combating rotavirus disease in animals. Most importantly, the results presented here for MFGM and SLPE provide proof of concept that the nutriceutical approach of providing natural or synthetic dietary receptor mimetics for protection against gastrointestinal virus infectious disease in all species is plausible.
Publications
- Chen, X., Li, M., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Donovan, S.M. 2011. Effect of human milk oligosaccharides on rotavirus infectivity in MA104 cells. Experimental Biology, April 2011.
- Li, Y., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Donovan, S.M. 2011. Isoflavones reduce rotavirus infectivity in MA104 cells through inhibition of protein kinases in the JNK and p70 signaling pathways. Experimental Biology, April 2011.
- Janjaroen, D., Liu, Y., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2010. Role of divalent cations on deposition of Cyrptosporidum parvum Oocysts on natural organic matter surfaces. Environ. Sci. Technol. 44:4519-24).
- Y, L., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2011. Characterization of Cryptosporidium parvum Oocyst wall macromolecules and adhesion kinetics of Oocysts on quartz surface. Biomacromolecules. 11:2109-15.
- Perez, V.G., Jacobs, C.M., Barnes, J., Jenkins, M.C., Kuhlenschmidt, M.S., Fahey Jr., G.C. and Pettigrew, J. 2010. Effect of corn distillers dried grains with solubles and Eimeria acervulina infec-tion on growth performance and the intestinal microbiota of young chicks. J. Poultry Science (In Press).
- Bergner, D.W., Kuhlenschmidt, T.B., Firkins, L.D. and Kuhlenschmidt, M.S. 2010. Synthesis and characterization of a neoglycolipid that blocks porcine group A rotavirus infectivity. Nutrients (Submitted).
- Li, M., Bauer, L.L., Chen, X., Kuhlenschmidt, T., Kuhlenschmidt, M.S., Fahey Jr., G.C. and Donovan, S.M. 2010. In vitro fermentation of human milk oligosaccharides by ascending colon microbiota from formula-fed and sow-reared piglets. Experimental Biology, April 2010.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1. Focus on effective interventions. Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents. A. Cryptosporidium parvum. Our laboratory has discovered and characterized a subset of long-chain unsaturated fatty acids (L-UFFA), originally isolated from bovine colostrum, which block in vitro Cryptosporidium parvum and Toxoplasma gondii host cell infectivity as well as both Toxoplasma gondii and Plasmodium gallinaceum infectivity in vivo. The results of in vitro host cell invasion assays indicate multiple L-UFFA can inhibit the invasion of MDBK cells by C. parvum sporozoites; however, there is a remarkable structural specificity requirement. To display inhibitory activity the fatty acids must be unsaturated, have a free carboxyl end, and have at least one bend (unsaturation) along the carbon chain. Elaidic acid, the trans isomer of oleic acid, which differs only in conformation (straight versus bended hydrocarbon chain, respectively) showed no inhibitory activity. In addition, L-UFFA must be no shorter than sixteen carbons and no more than twenty carbons in length, with maximal activity between eighteen and twenty carbons. Of the fatty acids tested within these specifications, polyunsaturated fatty acids were more effective inhibitors than monounsaturated fatty acids. Preliminary results suggest L-UFFA are inhibiting parasite-host cell invasion by inhibiting sporozoite microneme secretion and gliding motility. B. Control of Microbial Contamination of Agriculture Watersheds. Our outputs in this area are essentially the same as last year since conducting and analyzing overland transport experiments and measuring long term pathogen (rotavirus and cyrptosporidium) survival using native soil beds requires nearly two years to complete. In this continuing study, rotavirus survival was investigated in three different soil fractions and at three different temperatures (4, 25, and 37C). A rotavirus suspension was mixed with whole soil, sand, and clay and allowed to incubate for up to 30 days. Samples were collected daily to investigate virus survival over time, which was quantified using a tissue-culture infectivity assay. In summary, our preliminary results indicate, in the absence of any soil particles, rotavirus survival is highest at 4C, with survival decreasing as temperature increases. These data also indicate whole soil has some protective effect, allowing rotavirus to survive better in soil for the entire range of temperatures and for more than a week at 37C. The results also show that sand fractions are the most effective media for reducing rotavirus survival at all temperature conditions tested. There is little or no infective rotavirus extracted from sand fractions. Although the mechanism responsible for the low recovery of infective virus from sand is unknown, this finding strongly supports the use of sand as a filtering material to remove rotavirus from both point and nonpoint sources of water pollution. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Preventing pathogens from entering drinking water supplies is of great importance worldwide. Eighty-eight percent of diarrheal disease in humans is attributed to unsafe water supply, inadequate sanitation and hygiene. Approximately 39% of this diarrheal disease in humans is caused by rotavirus. Rotaviruses can be transmitted to both humans and animals in various ways, including the use of manure as a fertilizer for food crops and by stormwater runoff that contains manure. Therefore, it is necessary to determine the survival and fate of viruses after the application of wastewater on land since secondary treatment of sewage does not remove all viruses present in domestic sewage. The objective of this study is to understand the fate and transport of rotavirus in the environment. Since the survival of rotavirus in soil and soil components has not been previously studied, this research focused on developing the necessary methods for extracting and analyzing rotavirus in the soil-water environment. Our investigation focused on the kinetics of survival of infective rotavirus in clay, sand, and intact soil at three temperatures ranging from 4 to 37C. The results of this research are important because they contribute (1) to an improved understanding of the risks of rotavirus transmission due to environmental contamination and survival, (2) to the design of systems to control its overland transport in natural animal agricultural environments, and (3) to providing sustainable best management practices for animal producers. The phylum, Apicomplexa, contains some of the most significant pathogens infecting humans and animals. Of the more than 4,000 species of apicomplexan parasites, malaria parasites, Toxoplasma gondii, and Cryptosporidia are the most important pathogens of humans, causing death or disability for millions of people each year. The goal of our research in this area is to define the early mechanisms of Apicomplexa-host interactions and to identify new drug candidates that can block these interactions. We have developed a battery of complementary in vitro and in vivo assays that allow us to quantitfy Cryptosporidium, Toxoplasma, and Plasmodium microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity of these parasites is inhibited by L-UFFA. Our results thus far provide hope that small molecule natural products, such as L-UFFA or their derivatives, will be valuable for the development of new drugs that show broad efficacy for treatment of apicomplexan parasitic diseases. As far as we are aware, we are the only investigators who have observed inhibition of parasite-host cell infectivity across divergent species of Apicomplexa by a single class of compounds, namely L-UFFA or type A free fatty acids. Accordingly, L-UFFA represent a unique opportunity to identify a common mechanism used by the Apicomplexa to infect host cells and thus a potential "Achilles heel" target for the development of new anti-parasitic drugs.
Publications
- Donovan, S.M., Andres, A., Mathai, R.A., Kuhlenschmidt, T.B. and Kuhlenschmidt, M.S. 2009. Soy formula and isoflavones and the developing intestine. Nutritional Reviews (In Press).
- McLaughlin, S.J., Kuhlenschmidt, T.B., Kalita, P.K. and Kuhlenschmidt, M.S. 2009. Interaction of cryptosporidium parvum oocysts with soil particles and vegetated filter strips (Submitted).
- Pineda, M.F., Chan, L.L., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Cunningham, B.T. 2009. Rapid label-free selective detection of porcine rotavirus using photonic crystal biosensors for groundwater monitoring. IEEE Sensors Journal 9(4):470-477.
- Andres, A., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S., Mathai, R.A., Monaco, M. and Donovan, S.M. 2008. Time course of the gastrointestinal responses to rotavirus infection in the neonatal piglet. (Submitted).
- Andres, A., Donovan, S.M. and Kuhlenschmidt, M.S. 2009. Isoflavones and virus infections (Invited Review), J. Nutritional Biochemistry (20(8):563-9).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: 1. Development of novel methods for detection of waterborne pathogens (Cryptosporidium and rotavirus). We have developed and are in the process of optimizing new technology for the sensitive detection of environmental microbial pathogens using a label-free photonic crystal biosensor. Using this new sensor technology, we have demonstrated that porcine rotavirus can be specifically detected in environmental water samples as sensitively as conventional ELISA but without the need for secondary label processing. 2. Focus on effective interventions - Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents. A. Porcine Group A Rotavirus: Receptor Therapeutic Approaches for Porcine Rotavirus Disease. We have synthesized asialyl-lactosylphosphatidyl-ethanolamine (SLPE) neoglycolipid that displays a potent ability to inhibit both virus binding and infectivity in vitro. In field trials this inhibitor blocked infection, virus shedding, and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. We are also investigating the synergistic effect of SLPE and specific porcine and human milk oligosaccharides, as well as the soybean derived isoflavone, genistein, on inhibition of rotavirus in vitro and in vivo infectivity. B. Cryptosporidium parvum. We have purified a bovine colostrum-derived sporozoite adhesion inhibitory lipid (SIL) which we have characterized as oleic acid. A comparison of commercial oleic acid and SIL demonstrates oleic acid effects a dose-dependent inhibition of host cell adhesion in vitro, with an IC50=5 micromolar. These results suggest oleic acid, and possibly other long-chain unsaturated fatty acids (L-PUFA) derived from bovine colostrum, is a natural, non-immune, anti-cryptosporidial component and may have utility not only as a reagent useful for investigating the mechanism of fatty acid mediated-inhibition of sporozoite-host cell adhesion but also as a possible dietary treatment of human and veterinary cryptosporidiosis. Suppressive subtractive hybridization experiments indicate the mechanism responsible for L-PUFA-mediated inhibition of host cell invasion occurs without the necessity for lipid-induced differential gene expression. Preliminary results suggest both oleic and linolenic acid are inhibiting parasite-host cell invasion by inhibiting sporozoite microneme secretion and gliding motility. C. Control of Microbial Contamination of Agriculture Watersheds. We have determined the cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil that occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil. Preliminary studies measuring the interaction of rotavirus with various soil particles indicate rotavirus infectivity is more quickly neutralized upon exposure to sand particles as compared to clay, silt or intact soil. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of both animals and people. They are of prime agricultural importance since they cause serious neonatal diarrhea of many animal species, most importantly neonatal and post-weaning pigs and calves. Extensive efforts to produce a deliverable commercial vaccine, including the use of reassortants, attenuated live strains, and vector expression of viral capsid proteins have yet to be successful. Furthermore, despite the likelihood that efficacious rotavirus vaccines for humans will be available in the near future, it is not certain these vaccines will be available or deliverable to many of the people in developing countries who are most affected by the disease. Accordingly, the prophylaxis or treatment of rotavirus diarrheal disease by nutritional intervention, through the use of easily deliverable nutriceuticals, is potentially of great significance for the control of both agricultural and human cryptosporidiosis. Cryptosporidium parvum causes a debilitating diarrhea of livestock either alone or in concert with other enteropathogens. This agricultural problem is compounded by the public health concern of contamination of municipal water supplies by domestic and wild animal feces such as occurred in Milwaukee. In addition, cryptosporidiosis is part of the AIDS related disease complex. Our approach to the control of enteric diseases of livestock has been non-traditional in the sense that it is not aimed towards vaccine production or immune regulation. Instead, we reason that if we develop assay systems that are biologically relevant and can precisely reproduce, in vitro, the interactions between the infectious agent and the host cell, then we can use these assays to identify natural products (e.g. the inhibitory lipid above) or synthetic derivatives that interfere with parasite-host cell interactions required for infection. Such molecules, particularly natural products, could be utilized as nutriceutical feed additives to inactivate parasites and thus prevent or reduce parasite load and limit the severity of disease. Such an approach could benefit not only animal health but would reduce the likelihood of zoonotic spread of Cryptosporidium parvum through contamination of the water supply from domestic livestock operations.
Publications
- Davidson, P., Koch, D., McLaughlin, S.J., Kuhlenschmidt, T.B., Kalita, P.K. and Kuhlenschmidt, M.S. 2009. Interaction of rotavirus and cryptosporidium parvum oocysts with soil particles and vegetative filter strips. USDA-CSREES National Water Conference Abstract/Oral Presentation, St. Louis, MO. February 8-12.
- Schmidt, J. and Kuhlenschmidt, M.S. 2008. Microbial adhesion of cryptosporidium parvum: Identification of a colostrum-derived inhibitory lipid. Molecular and Biochemical. Parasitology 162:32-9.
- Pineda, M.F., Chan, L.L., Kuhlenschmidt, T., Kuhlenschmidt, M. and Cunningham, B.T. 2008. Rapid label-free selective detection of porcine rotavirus using photonic crystal biosensors. IEEE Sensors Journal (In Press).
- Liu, Y., Janjaroen, D., Kuhlenschmidt, M.S., Kuhlenschmidt, T.B. and Nguyen, T.H. 2009. Deposition of cryptosporidium parvum oocysts on natural organic matter surfaces: Microscopic evidence for secondary minimum deposition in a radial stagnation point flow cell. Langmuir (Under Revision).
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Progress 01/01/07 to 12/31/07
Outputs
1. Development of novel methods for detection of waterborne pathogens (Cryptosporidium and rotavirus). We have begun collaborative studies to develop new technology, in collaboration with Dr. Brian Cunningham (University of Illinois Department of Electrical and Computing Engineering) for the sensitive detection of environmental microbial pathogens using label-free photonic crystal biosensor technology. Using this new sensor technology, we have demonstrated that porcine rotavirus can be specifically detected in aqueous samples as sensitively as conventional ELISA but without the need for secondary label processing. 2. Focus on effective interventions - Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents. A. Porcine Group A Rotavirus - Receptor Therapeutic Approaches for Porcine Rotavirus Disease We have synthesized a
sialyl-lactosylphosphatidylethanolamine (SLPE) neoglycolipid that displays a potent ability to inhibit both virus binding and infectivity in vitro. In field trials this inhibitor blocked infection, virus shedding and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. We are also investigating, in collaborative studies with Dr. Sharon Donovan, Department of Food Science and Human Nutrition, the synergistic effect of SLPE and specific porcine and human milk oligosaccharides, as well as soybean derived flavonoids, which exhibit anti-rotavirus activity. B. Cryptosporidium parvum - Using a cell-suspension sporozoite adhesion assay we have purified a lipid fraction that inhibits sporozoite-host cell invasion. Preliminary data suggest this lipid is inhibiting sporozoite microneme secretion and gliding motility. Suppressive subtractive hybridization experiments aimed at identifying specific sporozoite genes expressed in response to host cell
attachment or exposure to the inhibitory lipid indicate these processes occur without the necessity for attachment- or lipid-induced differential gene expression. C. Control of Microbial Contamination of Agriculture Watersheds - We have determined the cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil that occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil. These results have allowed us to leverage NC-1007 support to submit and receive a new USDA NRI grant 2006-35102-17344, CONTROL OF CRYPTOSPORIDIUM AND ROTAVIRUS CONTAMINATION IN AGRICULTURAL WATERSHEDS, PI: Kuhlenschmidt, M.S., Co-PI: Kalita, P., $400,000.00, 8/1/06 - 7/31/09, as well as a new submission to NSF entitled: Transport mechanism of Cryptosporidium parvum oocysts in overland and near-surface environment: A combined study of
experiments and modeling, PI: Nguyen, T.H.; Co-PIs: Prasanta K. Kalita and Mark S. Kuhlenschmidt, $472,000.00.
Impacts
Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of both animals and people. They are of prime agricultural importance since they cause serious neonatal diarrhea of many animal species, most importantly neonatal and post-weaning pigs and calves. Extensive efforts to produce a deliverable commercial vaccine, including the use of reassortants, attenuated live strains and vector expression of viral capsid proteins have yet to be successful. Furthermore, despite the likelihood that efficacious rotavirus vaccines for humans will be available in the near future, it is not certain these vaccines will be available or deliverable to many of the people in developing countries who are most affected by the disease. Accordingly, the prophylaxis or treatment of rotavirus diarrheal disease by nutritional intervention, through the use of easily deliverable nutriceuticals, is potentially of great significance for the control of this
disease in both agricultural and human medicine arenas. Cryptosporidium parvum causes a debilitating diarrhea of livestock either alone or in concert with other enteropathogens. This agricultural problem is compounded by the public health concern of contamination of municipal water supplies by domestic and wild animal feces such as occurred in Milwaukee. In addition, cryptosporidiosis is part of the AIDS related disease complex. Despite decades of research in a variety of animal models and utilizing varied technologies, effective prophylaxis or therapeutics for C. parvum infection or disease are not available. Our approach to the control of enteric diseases of livestock has been non-traditional in the sense that it is not aimed towards vaccine production or immune regulation. Instead, we reason that if we develop assay systems that are biologically relevant and can precisely reproduce, in vitro, the interactions between the infectious agent and the host cell, then we can use these
assays to identify natural products (e.g. the inhibitory lipid above) or synthetic derivatives that interfere with parasite-host cell interactions required for infection. Such molecules, particularly natural products, could be utilized as nutriceutical feed additives to inactivate parasites and thus prevent or reduce parasite load and limit the severity of disease. Such an approach could benefit not only animal health but would reduce the likelihood of zoonotic spread of Cryptosporidium parvum through contamination of the water supply from domestic livestock operations.
Publications
- Kuhlenschmidt, M.S., Kalita, P., Kuhlenschmidt, T.B. and Davidson, P. 2007. Control of Cryptosporidium and Rotavirus contamination in agricultural watersheds. Proceedings of the USDA-CSREES National Water Quality Conference, January 28 - February 1, 2007 Savannah, GA.
- Liu, Y., Kuhlenschmidt, M.S. and Nguyen, T.H. 2008. Role of ionic strength on deposition kinetics of Cryprosporidium Parvum oocysts to natural organic matter. Annual Meeting of the American Chemical Society, New Orleans, LA.
- Gutierrez, L.A., Li, X., Wang, J., Nangmenyi, G.N., Economy, J., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Nguyen, T.H. 2008. Study of sorption and inactivation of MS2 phage and rotavirus using iron oxide-coated glass fiber. Annual Meeting of the American Chemical Society, New Orleans, LA.
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