Progress 12/20/10 to 12/19/15
Outputs
Progress Report Objectives (from AD-416): Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416): To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. Parasite forms were concentrated from fecal samples collected from dairy calves as part of a collaboration study with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS). Samples were examined as processed by Immunofluorescence Microscopy (IFA) for Cryptosporidium and Giardia to determine prevalence of infection. Overall, presence of both parasites appears to be very common in dairy calves. DNA was extracted from all samples to do future molecular characterization to identify species and genotypes and investigate public health impact of the species/genotypes found in dairy calves in this study. Initiated collaboration with scientists from Brazil to conduct molecular characterization of E. bieneusi in sheep and chickens for the first time. This information will help better identify if they could play a role in the transmission of this parasite to humans in Brazil. This is the final report for the project 8042-32000-089-00D which terminated in December 19, 2015. Key findings of the project were: 1) Blastocystis tropism in the pig intestine was studied using monoclonal and polyclonal antibodies to this parasite; 2) development of a new subtyping technique to characterize an emerging zoonotic pathogen, Cryptosporidium ubiquitum, that allows showing associations between human and animal cases of C. ubiquitum infections; 3) conducted a study to determine the presence of potential human pathogens in white-tailed deer in Maryland that provided an advance in understanding the epidemiology of these parasites in wildlife; 4) first molecular identification and characterization of E. bieneusi in pigs and cattle from South America indicated that this parasite is widespread in swine and cattle and that they could play an important role in the transmission of this parasite to humans; 5) development of a new assay to measure levels of the Cryptosporidium viral symbiont Cryspovirus during in vitro development of C. parvum to assist understanding the role of the virus in Cryptosporidium; and 6) antibodies were produced against two unique proteins of parasite Giardia duodenalis that were labeled with a flurence dye to use in microscopic diagnosis in clinical and environmental samples (U.S Patent number 8852596). Accomplishments 01 Enterocytozoon bieneusi genotypes from cattle. Microsporidia are widely recognized as important human pathogens with E. bieneusi as the most common species infecting humans and animals, including cattle. Although Brazil has the second largest cattle herd in the world and it is the largest exporter of beef there are no data on the presence or impact of E. bieneusi on this important population. To fill this knowledge gap, fecal specimens were collected from 452 cattle from pre- weaned calves to adult cattle in the state of Rio de Janeiro. In this first report of E. bieneusi in Brazilian cattle, a significantly higher prevalence was found in dairy cattle than in beef cattle, and calves less than 2 months of age and those 3-8 months of age versus heifers and adults. Molecular characterization of the internal transcribed spacer (ITS) revealed 12 genotypes; five previously reported in cattle (BEB4, BEB8, D, EbpA and I), and seven novel genotypes (BEB11 to BEB17). The finding of zoonotic genotypes highlights the risk of human contamination with E. bieneusi spores through direct contact, especially with young dairy cattle, and environmental contamination affecting water and plants.
Impacts
(N/A)
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416): To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. In 2015, progress was made in elucidating the epidemiology of zoonotic parasites Cryptosporidium and Giardia in collaboration with the National Animal Health Monitoring Service of APHIS. 2,600 samples were collected from calves on more than 100 farms in 11 of the top dairy states and were examined by immunofluorescence microscopy (IFA) for Cryptosporidium and Giardia. Overall, presence of both parasites was very common with almost half of the calves infected with Cryptosporidium and more than one third infected with Giardia. At present, we are conducting the molecular characterization of the positive specimens to identify species and genotypes critical to estimate zoonotic potential of C. parvum cycling in bovine populations as several morphologically similar non C. parvum species of unproven clinical and public health impact are found in cattle. Findings will aid to understand sources and routes of transmission necessary to improve strategies for the control and prevention of cryptosporidiosis and giardiasis in bovines. Additionally, we performed PCRs to detect the presence in those calves of zoonotic parasites E. bieneusi and Blastocystis to later molecularly characterize to the genotypes and subtypes level to determine public heath impact. In collaboration with scientists from Brazil, the first molecular characterization of E. bieneusi in pigs and cattle from Brazil was conducted. Findings suggest that E. bieneusi is widespread in Brazilian swine and cattle and because zoonotic genotypes were identified they could play an important role in the transmission of this parasite to humans in Brazil. To understand the role of viral symbiont Cryspovirus in C. parvum, a new assay was developed to measure the level of Cryspovirus during in vitro development of C. parvum. This improved the cell culture system and molecular assays for tracking the virus and will be invaluable to future studies to discern the role of Cryspovirus in Cryptosporidium. It is possible that by inhibiting replication of the virus, one can alter the pathogenicity of C. parvum and C. hominis, and thus diminish the clinical effects in humans and animals. Accomplishments 01 Enterocytozoon bieneusi genotypes from pigs and their prevalence in pigs in Brazil. Enterocytozoon bieneusi is the most frequently diagnosed Microsporidia species in humans worldwide, mainly associated with chronic diarrhea and wasting syndrome. It has emerged as an important zoonotic species that is of public health concern because of its wide geographic distribution and broad host range that includes humans as well as domestic and wild animals. Despite Brazil being the world�s fourth largest producer and exporter of pork, there is no information on E. bieneusi in pigs in Brazil. ARS scientists in Beltsville, Maryland, in collaboration with scientists in the State of Rio de Janeiro, Brazil conducted the first identification and molecular characterization of E. bieneusi in pigs in Brazil. The prevalence was high and a wide genetic diversity was found with 21 E. bieneusi genotypes identified. The high prevalence, the identification of two known zoonotic genotypes (EbpA and O), and the report of 17 new genotypes represents an important advancement in the study of the wide genetic diversity of this organism. The presence of two known zoonotic genotypes raises concern on the role of pigs on the zoonotic transmission of E. bieneusi and points to pigs as a source of E. bieneusi transmission to humans through direct human contact with infected animals or by contamination of water and food. 02 Development of assays to measure the level of Cryspovirus during in vitro development of Cryptosporidium parvum. Intracellular viral symbionts have been found in a variety of protozoa. ARS scientists in Beltsville, Maryland, developed a method for culturing C. parvum, and a molecular technique for studying the replication of the viral symbiont Cryspovirus. The culture system allowed for expansion of the parasite, which will be useful in studying therapies to prevent cryptosporidiosis. The molecular assay directed to Cryspovirus revealed that viral particles are released into the culture medium during parasite development. This assay will allow scientists to study the role of Cryspovirus in C. parvum replication and possibly point to ways of preventing cryptosporidiosis.
Impacts
(N/A)
Publications
- Fayer, R., Esposito, D., Dubey, J.P. 2015. Sarcocystis in Humans. Clinical Microbiological Reviews. 28(2):295-311.
- Fiuza, V.D., Oliviera, F.S., Fayer, R., Santin, M. 2015. First report of Enterocytozoon bieneusi in pigs in Brazil. Parasitology International. 64:18-23.
- Santin, M., Fayer, R. 2014. Enterocytozoon bieneusi, Giardia and Cryptosporidium infecting white-tailed deer. Journal of Eukaryotic Microbiology. 62:34-43.
- Jenkins, M.C., Obrien, C.N., Santin, M., Fayer, R. 2015. Changes in the Levels of Cryspovirus During In Vitro Development of Cryptosporidium parvum. Parasitology Research. 114:2063-2068.
- Checkley, W., White, A.C., Jaganath, D., Arrowood, M., Chalmers, R., Fayer, R. 2015. Cryptosporidiosis: Global burden, novel diagnostics, therapeutics and vaccine targets. Lancet Infectious Diseases. 15:85-94.
- Ryan, U., Fayer, R., Xiao, L. 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology. 141:1667- 1685.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416): To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. As part of Objective 1, Blastocystis tropism in the pig intestine was studied using monoclonal and polyclonal antibodies to this parasite. Tissue sections showed that Blastocystis subtype 5 was found primarily in the intestinal lumen in close proximity or adhere to epithelium, but no stages were found to penetrate epithelium or lamina propria. In collaboration with CDC scientists a new subtyping technique targeting the 60-kDa glycoprotein (GP60) gene was developed to characterize C. ubiquitum, an emerging zoonotic pathogen, using specimens from humans, various animals, and drinking source water. As part of Objective 2, progress was made to demonstrate that CP33, a protein that plays a role in invasion host cells, differ phenotypically between C. parvum and C. hominis; this may reflect a role of this protein in host specificity. This protein was found to be localized in the cytoplasm, and concentrated primarily at the anterior end of C. parvum sporozoites, suggesting its implication in cell invasion. Additionally, lyophilized Bifidobacterium lactis (Bb12) and Lactobacillus paracasei strains, as well as a placebo, were provided by an ARS Collaborator to evaluate the interaction of these probiotics strains and infection with protozoans Cryptosporidium and Giardia in rodent models. Experiments will proceed once the animal protocol submitted to the Beltsville Area Animal Care and Use Committee has been approved. As part of Objective 3, progress was made in elucidating the epidemiology of zoonotic parasites, Cryptosporidium, Giardia, and E. bieneusi by determining prevalence in white-tailed deer in Maryland; strains were characterized molecularly. This study represents the most comprehensive study for these protists in deer to date and provides an advance in understanding the epidemiology of these parasites in wildlife. It demonstrates that white-tailed deer contribute to environmental contamination with parasites of unknown pathogenicity, as well as zoonotic parasites (G. duodenalis Assemblage A and E. bieneusi genotypes I, J, and LW1). In collaboration with scientists from Argentina, the first molecular characterization of C. parvum and E. bieneusi in cattle from Argentina was conducted. Findings suggest that cattle could play an important role in the transmission of those parasites to humans in Argentina. In collaboration with scientists from Brazil, we also conducted the first identification and molecular characterization of E. bieneusi in pigs in Brazil. The prevalence of E. bieneusi in pigs was high and 17 new genotypes were found which represents an important advance in the study of the wide genetic diversity of this organism. Two known zoonotic genotypes, O and EbpA, were also identified raising concern on the role of pigs on the zoonotic transmission of E. bieneusi. Additionally, collaborators at the National Animal Health Monitoring Service of APHIS started collecting specimens from preweaned dairy calves; those specimens are being processed in our laboratory to detect and characterize to the species, genotypes, and subtypes level Cryptosporidium, Giardia, Blastocystis, and E. bieneusi. Accomplishments 01 New subtyping technique for Cryptosporidium ubiquitum. Cryptosporidium ubiquitum has emerged as a major Cryptosporidium zoonotic species that is of public health concern because of its wide geographic distribution and broad host range that includes humans, domestic and wild ruminants, rodents, carnivores, and primates. It has also been found in drinking source water, storm water runoff, stream sediment, and wastewater. Thus far, showing an association between human and animal cases of C. ubiquitum infection has not been possible because of the lack of suitable genetic markets for subtyping. An ARS scientist in Beltsville, MD in collaboration with CDC scientist in Atlanta, GA identified the 60- kDa glycoprotein (GP60) gene of C. ubiquitum by whole-genome sequencing and used it to develop a new subtyping technique to characterize C. ubiquitum specimens from humans, various animals, and drinking source water. The application of this new tool has shown the existence of host adaptation in C. ubiquitum infections. The findings indicate sheep and rodents are a key source of C. ubiqitum transmission to humans through direct human contact with infected animals or by contamination of drinking source water. 02 Zoonotic protists infecting white-tailed deer. Despite a white-tailed deer population in the United States of about 32 million animals, there is a lack of information worldwide on the zoonotic protist parasites Cryptosporidium, G. duodenalis, and E. bieneusi in these animals. Because of this lack of data regarding the potential role of white- tailed deer in the transmission of waterborne zoonotic protists to humans, ARS scientists in Beltsville, MD conducted a study to determine the presence of potential human protist pathogens in white-tailed deer in Maryland. Fecal specimens from fawns to adults were examined by molecular methods to first detect and later characterize to species and genotype level Cryptosporidium, G. duodenalis, and E. bieneusi to determine if deer posed a public health risk. It is well known that those parasites are waterborne given that spores, oocysts, and cysts are commonly detected in water worldwide. The identification of zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that white-tailed deer could play a role in the transmission of those parasites to humans.
Impacts
(N/A)
Publications
- Li, N., Xiao, L., Alderisio, K., Elwin, K., Chalmers, R., Santin, M., Fayer, R., Kvac, M., Ryan, U. 2014. Subtyping Cryptosporidium ubiquitum, an emerging zoonotic pathogen in humans. Emerging Infectious Diseases. 20(2)217-224.
- Del Coco, V.F., Cordoba, M.A., Bilbao, G., Pinto De Almeida, A., Basualdo, J.A., Santin, M. 2014. First report of Enterocytozoon bieneusi from dairy cattle in South America. Veterinary Parasitology. 199(2014):112-115.
- Del Coco, V.F., Cordoba, M.A., Billbao, G., Pinto De Almeida, A., Basualdo, J.A., Fayer, R., Santin, M. 2014. Cryptosporidium parvum GP60 subtypes in dairy cattle from Buenos Aires, Argentina. Research in Veterinary Science. 96(2)311-314.
- Fayer, R., Elsasser, T.H., Gould, R., Solano Aguilar, G., Santin, M., Urban Jr, J.F. 2014. Blastocystis tropism in the pig intestine. Parasitology Research. 113:1465-1472.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416): To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. New polyclonal and monoclonal antibodies were tested that identified Blastocystis in tissues and feces by immunohistochemistry and fluorescence microscopy as part of the goal associated with objective 1. These antibodies were extremely helpful in clearly identifying the parasite in the intestinal tract of pigs and elucidating its role in invasion of host tissue. In addition, a new approach to detect foodborne parasites using transparent adhesive tape to capture organisms combined with molecular and microscopy detection tools, was developed for use on fresh produce and food preparation surfaces. As part of objective 2, progress was made to test anti-viral drugs against Cryptovirus by developing a reproducible cell culture assay for C. parvum with conditions that allowed parasite and Cryptovirus replication. A reproducible in vitro assay that could measure C. parvum development from 24�72 hr using quantitative and semi-quantitative PCR was develped. An increase in Cryptovirus over time in the cell culture media was observed, indicating reproduction of virus in combination with parasite development, suggesting a possible target for parasite control. With confidence in this test system, we will now go back and re-test viral inhibitory drugs for inhibition of parasite development. As part of objective 3, progress was made to elucidate the epidemiology of zoonotic parasites by determining the prevalence and molecularly characterizing Blastocystis subtypes in wildlife, cattle, pigs, and humans. Using DNA primers developed in our laboratory, new variations of the ST 5 subtype of Blastocystis were identified; this may be helpful in epidemiologic traceback studies. Further progress was made to determine the prevalence of Cryptosporidium, Giardia, Microsporidium, and Blastocystis in food animals by developing and submitting plans for collaboration with epidemiologists at the National Animal Health Monitoring Service of APHIS to collect specimens from 1500 preweaned dairy calves in 13 states beginning in FY2014. Additionally, in collaboration with scientist from Argentina, the first identification and molecular characterization of Enterocytozoon bieneusi in cattle in South America was carried out with the identification of a novel genotype. Accomplishments 01 Giardia duodenalis detected in horses. Giardiasis, caused by G. duodenalis, is a gastrointestinal disease with worldwide distribution. In the United States, it is the most common intestinal parasitic disease causing diarrhea in humans. Giardia duodenalis is not only a widespread parasite in humans but also infects animals that can spread the disease to humans. Because of a lack of data regarding the potential role of horses in the transmission of giardiasis to humans, ARS scientists in Beltsville, MD in collaboration with scientists in Colombia conducted a molecular study of G. duodenalis from horses to determine if horse manure used for crop production posed a public health risk. Using a multilocus molecular approach to characterize isolates from 195 horses, G. duodenalis Assemblages A and B (both infectious for humans) were detected in horses. These findings indicate the potential risk of these companion animals as reservoir of G. duodenalis with the potential to cause disease in humans through direct contact or by contamination of food and water supplies. 02 New method to detect Cryptosporidium. Cryptosporidium is a prevalent and widespread protozoan parasite that causes severe diarrheal disease in humans and livestock. Infection with this parasite is common in children and can be life threatening in immunocompromised persons. Fresh produce contaminated with Cryptosporidium oocysts has been the source of sporadic cases and outbreaks of foodborne infection. Although Cryptosporidium has been detected on the surface of various fresh fruits and vegetables worldwide, detection has been problematic because the parasite adheres tenaciously to surfaces and cannot be grown like bacteria into easily detectable populations on Petri dishes or in tubes. Many current detection methods lack sensitivity and specificity to detect this parasite and others require highly trained personnel and equipment found only in specialized laboratories. ARS scientists in Beltsville, MD developed an adhesive tape recovery method, combined with molecular and microscopic tools, for detection of Cryptosporidium oocysts on fresh produce and on food preparation surfaces. This method is rapid, inexpensive, and can be applied to many products, surfaces, and locations.
Impacts
(N/A)
Publications
- Fayer, R., Santin, M., Macarisin, D., Bauchan, G.R. 2013. Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce. Parasitology Research. 112(4):1567-1574.
- Santin, M., Cortes Vecino, J., Fayer, R. 2013. A large scale molecular study of Giardia duodenalis in horses. Veterinary Parasitology. DOI: 10. 1016/jvetpar.3013.02.006.
- Santin, M. 2012. Clinical and subclinical infections with Cryptosporidium in animals. New Zealand Veterinary Journal. DOI: 10.1080/00480169.2012. 731681.
- Del Coco, V., Cordoba, M., Sidoti, A., Santin, M., Drut, R., Basualdo, J. 2012. Experimental infection with Cryptosporidium parvum IIaA21G1R1 subtype in immunosuppressed mice. Veterinary Parasitology. 190(3-4):411- 417.
- Suresh, K., Toranzos, G., Fayer, R., Olveda, R., Ashbolt, N., Gannon, V. 2012. Chapter 2. Assessing the importance of zoonotic waterborne pathogens. In: Dufour, A., Bartram, J., editors. Animal Waste, Water Quality and Human Health. London, England: IWA Publishihg. p. 17-72.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416): To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. Under Objective 1a, significant progress was made in detecting Blastocystis, a widespread parasite of humans and animals. In collaboration with a small biotech company, a new diagnostic reagent was modified and tested that quickly and accurately identified this organism in livestock feces, demonstrating the practicality of its application for epidemiological studies planned in Objective 3, and for diagnosis in human and animal medicine. Under Objective 2a, progress was made by demonstrating in cell cultures that anti-viral drugs associated with reduction of cryptosporidiosis in AIDS patients do not affect the RNA virus harbored within stages of Cryptosporidium. Therefore, medication with these drugs for treatment of non HIV patients with cryptosporidiosis and for treatment of animals would impose potential health risk and cost without evidence of effective treatment results. Under Objective 3a, significant progress was made by demonstrating that asymptomatic dairy cattle can serve as carriers and sources of multiple concurrent infections with four species of protozoa of public health importance. This objective relates to the safety of farm workers and farm visitors exposed to cattle as well as contamination of milk and meat products. A manuscript describing these infections and the public health implications was published. Further progress was made in Objective 3a to determine the prevalence of Blastocystis in food animals by developing and submitting plans for collaboration with epidemiologists at the National Animal Health Monitoring Service of APHIS to collect specimens from 7500 market pigs in 13 states beginning later in FY2012. Accomplishments 01 Identified giardins in the attachment organelle of Giardia and developed fluorescence microscopy based diagnostic test for Giardia duodenalis in environmental and biological specimens. ARS scientists in Beltsville, M identified the location of two unique proteins in the intestinal stage o Giardia parasites in the ventral disc, the organelle used for attaching the parasite to the intestine of an infected host. Because these protein participate in controlling disc shape, essential for the parasite to attach to host cells, this finding provides a basis for developing metho of prevention and treatment of Giardia infections by utilizing anti- giardin antibodies, immunizations, or drugs to block attachment of the parasite to cell surfaces. Antibodies produced against these proteins labeled with a fluorescent dye were found useful for microscopic diagnos of infection of intestinal tissue and diagnosis of the parasite in feces of infected animals and humans, as well as for the presence of the parasite in the environment (water, soil, plant contamination). A paten application is in the process of being filed with the U.S. Patent Office
Impacts
(N/A)
Publications
- Santin, M., Dargatz, Fayer, R. 2012. Prevalence of Giardia duodenalis assemblages in weaned cattle on cow-calf operations in the United States. Veterinary Parasitology. 183:231-236.
- Macarisin, D., Obrien, C.N., Bauchan, G.R., Fayer, R., Jenkins, M.C. 2012. Immunolocalization of Delta-Giardin within the Ventral Disc in of Trophozoites and in cysts of Giardia duodenalis using the Multiplex Laser Scanning Confocal Microscopy. Parasitology Research. 111:241-248.
- Lindsay, D., Dubey, J.P., Santin, M., Fayer, R. 2012. Coccidia and Other Protozoa. In: Zimmerman, J., Karriker, L., editors. Diseases of swine. 10th edition. San Francisco, CA: Wiley and Sons, Inc. p. 895-907.
- Santin, M., Dargatz, D., Fayer, R. 2011. Prevalence and genotypes of enterocytozoon bieneusi in weaned beef calves on cow-calf operations in the United States. Parasitology Research. 110(5):2033-2041.
- Fayer, R., Santin, M., Macarisin, D. 2012. Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitology Research. 111(3):1349-1355.
- Gomez-Munoz, M., Camara-Badenes, C., Martinez-Herrero, M., Dea-Ayuela, M., Perez-Gracia, M., Fernandez-Barredo, S., Santin, M., Fayer, R. 2012. Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high hetrogeneity. Research in Veterinary Science. 93(2):836-842.
- Jenkins, M.C., Obrien, C.N., Macarisin, D., Miska, K.B., Fetterer, R.H., Fayer, R. 2012. Analysis of Giardin expression during encystation of Giardia lamblia. Journal of Parasitology. DOI: 10.1645/GE-2970.1.
- Pomares, C., Santin, M., Miegeville, M., Espern, A., Albano, L., Marty, P., Morio, F. 2012. A new and highly divergent Enterocytozoon bieneusi genotype isolated from a renal transplant recipient. Journal of Clinical Microbiology. 50(6):2176-2178.
- Xiao, L., Ryan, U., Fayer, R., Bowman, D., Zhang, L. 2012. Cryptosporidium tyzzeri and Cryptosporidium pestis: which name is valid?. Experimental Parasitology. 130:308-309.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields. Approach (from AD-416) To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA- dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas. Contacted potential collaborators at Cornell University and the University of Maryland who have access to orchards where pond water is used for spraying fruit. They agreed to contact farmers who would allow us to collect and test water samples for the presence of Cryptosporidium, Giardia, and Microsporidia. Antiviral drugs are being tested in vitro against the virus found inside Cryptosporidium parvum stages to determine if they can inhibit virus and parasite replication. A DNA based study was initiated to determine the presence of Microsporidia in feces from beef cattle on cow-calf operations on 49 farms in 20 states. Another DNA based study in progress is determining the subtypes of Blastocystis in 30 dairy cattle followed from birth the 2 years of age. ARS collaborator in Nebraska provided samples of runoff from beef cattle feedlots that were filtered through a vegetative treatment system; Cryptosporidium and microsporidia were found.
Impacts
(N/A)
Publications
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