Progress 02/01/16 to 01/27/21
Outputs
Target Audience:Our efforts are targeted to help large animals and large animal farmers (sheep, cattle, horses, pigs, alpacas, llamas, poultry) deal with ubiquitous and damaging intestinal nematode parasites, especially those with existing and/or increasing resistance to current drugs, which are becoming pervasive. Our efforts are also targeted to help owners of companion animals (e.g., dogs) that are also infected by these parasites (e.g., dog hookworm), which are also showing signs of multi-drug resistance to current drugs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?UMMS: Two technicians working on this project have since moved on the PhD programs. These two technicians have been replaced by newly graduated undergraduates who are now getting first rate research experience. One researcher has been promoted to Senior Scientist position. All members present biweekly at lab meetings and every researcher has presented at an outside meeting at least once per year. We have presented yearly at CRWAD and AAVP meetings. University of Kentucky, the project has offered training activities for the following: Holli Gravatte, Laboratory technician Haley Anderson, Undergraduate student Ashley Steuer, Graduate student Virginia Tech, the project has offered training activities for the following: Emily Suess, Veterinary student Catherine Pouliot, Veterinary student Brittany Weicht, Veterinary student Olivia Stover, Undergraduate student Rebecca Wilson, Undergraduate student John Sanders, Graduate student How have the results been disseminated to communities of interest?The findings were presented at five meetings annually (except for 2020 due to COVID pandemic): the Society for Invertebrate Pathology International Meeting, The American Association for Veterinary Parasitology (AAVP), The International C. elegans Meeting, The Conference of Research Workersin Animal Diseases (CRWAD), and the American Society for Tropical Medicine and Hygiene (ASTMH). In 2020, the research was presented in remote conferences for CRWAD, ASTMH, and AAVP. In addition, we reached out to stake holders in the equine health industry (Kentucky, Florida, New York, Massachusetts) to let them know about our progress and get their feedback. There was universal agreement that this research would have a profound impact on equine (foal) health. We reached out to stake holders in canine health, with the conclusion that there is a tremendous need for our product in greyhounds and dog shelters. We reached out to stake holders in the exotic camelid health industry (alpacas, llamas), again with agreement that this product would have a valuable niche market. With regards to our published sheep work (IJPDDR 2020), we have participated in 4 interviews with the press and have heard from sheep groups around the country as the need and desire to have Cry5B IBaCC introduced into the market. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Intestinal roundworms are the most common parasites in the United States of horses, cattle, sheep, goats, alpacas, llamas, pigs, poultry, dogs, and cats. The parasites 1) reduce reproduction of the animals, 2) decrease overall health, energy, and performance, 3) decrease animal productivity, 4) make animals more susceptible to disease, and 5) can kill the animals. Some of these parasites can pass (infect) from animals to humans. They cause billions in lost economics in the US yearly (e.g., $2,500,000,000 losses to the cattle industry alone). Although we have drugs to treat these infections, parasite resistance to these drugs is growing enormously for all animals with multidrug resistance common in sheep, goats, and dogs and growing in all others. For example, Howell et al. 2008 reported that 48% of sheep and goat farms in the southeastern U.S. had intestinal worm parasites simultaneously resistant to all three of the main deworming drugs. Thus new approaches are absolutely critical. Unfortunately, the biotechnology industry has been unwilling to devote significant resources to developing new therapeutics. Fortunately, our laboratory has discovered a new, organic solution to this challenging situation, the Cry5B protein made naturally by the soil bacterium Bacillus thuringiensis, which is harmless to higher animals but toxic to parasitic worms. The goal of this USDA/NIFA grant is to test the efficacy of this new therapeutic against key veterinary parasites of horses and sheep, with additional testing being done against cattle, dog, and pig parasites. Our research will most immediately help animals infected with parasitic roundworms and people who work/live with these animals (e.g., the livelihood of animal farmers, the owners of cats/dogs). Objective 1: Optimize Cry5B sequence to specifically target cyathstomins and Haemonchus contortus and test spectrum of activity. Progress: We succeeded in using the COPAS biosorter to measure the length of parasitic larvae. Using positive and negative controls, we showed that the Z factor of our assay is 0.53, which is excellent. We therefore have a highly quantitative parasite larval assay for Haemonchus (sheep parasite) and cyathostomins (horse parasites). We also succeeded in building a worminator box for measuring the intoxication of parasitic adults in a highly quantitative manner. We tested the five natural Cry5B amino acid variants. Variant 105 (T64M) and variant 104 (N172I,E248K), showed increased antiparasitic activity relative to our canonical Cry5B amino acid sequence against parasitic larvae. We deconvolved variant 104 and showed that E248K alone shows increased activity against parasitic larvae. Objective 2. Making a production-quality strain of Cry5B and optimizing production. We produced Cry5B at 350 liter scale with success for nine consecutive runs. We have also collaborated with a Bacillus production company to optimize expression at >5 g/Cry5B per liter of culture, with Cry5B representing up to 35% total protein by weight. This is phenomenal productivity and achieves commercializable productivity goals. We are also optimizing the genetic background of the Bacillus strain to allow for broader Crystal protein expression as we find that spo0A- strains are limited in making crystal proteins other than Cry5B. SigmaK- cells are superior for some crystal proteins apart from Cry5B. Objective 3. In vivo testing and formulation. We set up two in vivo tests in 2019, one in sheep and one in horses. Both tests were set up using IBaCC (inactivated Bacilli with cytosoloic crystals), our lead API (deliverable form) that has never been tested in vivo in these systems before. 10 foals in the herd were identified as naturally infected with the parasite Parascaris equorum. All had fecal egg counts from between 180-2121 eggs per gram of feces. They were randomly split into two groups of 6 (treated orally with 30 mg/kg Cry5B IBaCC) and 4 (untreated; water only). Of the 6 treated, all had their fecal egg counts drop to zero within 7 days and remain so for up to 28 days. Of the 4 untreated, all retained positive egg counts for up to 14 days; by 28 days 1 had lost the infection but three retained it. These data demonstrate that Cry5B IBaCC is highly efficacious against this parasite of paramount importance to the horse industry. Amazingly, there is no drug on the market today that has this strong an impact. The second in vivo study was done in sheep. Sheep were experimentally infected with Haemonchus contortus parasites and, after confirmation of strong infection, treated orally with 60 mg/kg Cry5B three days in a row or water control (6 sheep per group). Fecal egg counts were taken daily and the animals were euthanized on day 8 for worm counts. A 95% drop in fecal egg counts were seen and a 72% drop in total parasitic worms. Most strikingly, there was a 96% drop in female parasitic worms and a 60% drop in male parasitic worms (all differences were statistically significant). In 2020, one addition study in both sheep and horses was conducted. Ten miniature ponies naturally infected with cyathostomin parasites were enrolled. Five were given Cry5B IBaCC at 10 mg/kg and five were left untreated. Fecal egg counts were taken days 7 and 14 post-treatment. No differences were seen. Thus, Cry5B IBaCC at this dose is not effective against this parasite, which lives deeper in the GI tract than Parascaris. In sheep, a dose-ranging study was carried out. Instead of 3x60mg/kg IBaCC doses, sheep were split into three groups: no treatment, 1x40 mg/kg IBaCC, 1x10 mg/kg IBaCC. Within 48 hrs of dosing there was a 58% and 89% reduction in FEC by 48 hours and 67% and 92% FEC reduction by the conclusion of the study for lambs in the 10mg/kg BW and 40mg/kg BW Cry5b IBACC treatment groups respectively. There was a 40% reduction in worm burden in lambs dosed with 10 mg/kg BW of Cry5B IBACC (976 ± 249 worm, mean ± SEM, p = 0.014 versus Control) and a 82% reduction in worm burden in lambs dosed with 40 mg/kg BW (296 ± 115 worms, p < 0.0001 versus control lambs). Taken together, our data indicate that single dose Cry5B IBaCC is highly effective against Parascaris infections in horses and Haemonchus infections in sheep, with further formulation work needed for cyathostomin parasites in horses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans. Li H, Abraham A, Gazzola D, Hu Y, Beamer G, Flanagan K, Soto E, Rus F, Mirza Z, Draper A, Vakalapudi S, Stockman C, Bain P, Urban JF Jr, Ostroff GR, Aroian RV. Antimicrob Agents Chemother. 2020 Dec 14:AAC.01469-20. doi: 10.1128/AAC.01469-20.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep. Sanders J, Xie Y, Gazzola D, Li H, Abraham A, Flanagan K, Rus F, Miller M, Hu Y, Guynn S, Draper A, Vakalapudi S, Petersson KH, Zarlenga D, Li RW, Urban JF Jr, Ostroff GR, Zajac A, Aroian RV.Int J Parasitol Drugs Drug Resist. 2020 Dec;14:230-236. doi: 10.1016/j.ijpddr.2020.11.004. Epub 2020 Nov 19.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Anthelmintic Activity of Yeast Particle-Encapsulated Terpenes.
Mirza Z, Soto ER, Hu Y, Nguyen TT, Koch D, Aroian RV, Ostroff GR.
Molecules. 2020 Jun 27;25(13):2958. doi: 10.3390/molecules25132958.
PMID: 32605043
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gut microbial signatures associated with moxidectin treatment efficacy of Haemonchus contortus in infected goats.
Liu F, Xie Y, Zajac AM, Hu Y, Aroian RV, Urban JF Jr, Li RW.
Vet Microbiol. 2020 Mar;242:108607. doi: 10.1016/j.vetmic.2020.108607. Epub 2020 Feb 8.
PMID: 32122611
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
An inactivated bacterium (paraprobiotic) expressing Bacillus thuringiensis Cry5B as a therapeutic for Ascaris and Parascaris spp. infections in large animals. Joseph F. Urban, Jr.*#1,2, Martin K. Nielsen*3, David Gazzola4, Yue Xie1,2,5, Ethiopia Beshah1,2, Yan Hu4!, Hanchen Li4, Florentina Rus4, Kelly Flanagan4, Austin Draper6, Sridhar Vakalapudi6, Robert W. Li1, Gary R. Ostroff4, and Raffi V. Aroian#4. One Health, Elsevier.
|
Progress 02/01/19 to 01/31/20
Outputs
Target Audience:Our efforts are targeted to help large animals and large animal farmers (sheep, cattle, horses, pigs, alpacas, llamas, poultry) deal with ubiquitous and damaging intestinal nematode parasites, especially those with existing and/or increasing resistance to current drugs, which are becoming pervasive. Our efforts are also targeted to help owners of companion animals (e.g., dogs) that are also infected by these parasites (e.g., dog hookworm), which are also showing signs of multi-drug resistance to current drugs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?UMASS Medical School: Ambily Abraham: continued postdoctoral training; co-first author publication in preparation; presentation at international meeting in July Hanchen Li: continues development towards independent position; co-first author publication in preparation; two presentations, one at international meeting in June, one at campus wide retreat in September Tasia Kellogg: allowed her to gain valuable research experience and strong letters of recommendation for admittance into graduate school; now matriculating as a PhD student David Gazzola: co-first author publication in preparation; presentation at international meeting in November; continued development as a research associate; promotion to next level (from RA0 - RA1); helped him choose a career in science and applying to graduate school this year Raffi Aroian, Gary Ostroff: faculty; presentations at several international meetings relevant to this research, including CRWAD University of Kentucky, the project has offered training activities for the following: Holli Gravatte, Laboratory technician Haley Anderson, Undergraduate student Ashley Steuer, Graduate student Virginia Tech, the project has offered training activities for the following: Emily Suess, Veterinary student Catherine Pouliot, Veterinary student Brittany Weicht, Veterinary student Olivia Stover, Undergraduate student Rebecca Wilson, Undergraduate student John Sanders, Graduate student How have the results been disseminated to communities of interest?The findings were presented at four meeting this year, the Society for Invertebrate Pathology International Meeting, The American Association for Veterinary Parasitology, The International C. elegans Meeting, and the American Society for Tropical Medicine and Hygiene. In addition, we reached out to stake holders in the equine health industry (Kentucky, Florida, New York, Massachusetts) to let them know about our progress and get their feedback. There was universal agreement that this research would have a profound impact on equine (foal) health. We reached out to stake holders in canine health, with the conclusion that there is a tremendous need for our product in greyhounds and dog shelters. We reached out to stake holders in the exotic camelid health industry (alpacas, llamas), again with agreement that this product would have a valuable niche market. Three publications were published. What do you plan to do during the next reporting period to accomplish the goals?We will continue sequence optimizaiton of Cry5B against these parasites using larval development assays. We will introduce more studies on other Cry proteins, to compare to Cry5B and development next set of Cry proteins for the anthelmintic (drug) pipeline. One set of sheep in vivo experiments and one set of foal (equine) in vivo experiments are planned to perform dose ranging studies: what is the minimal dose needed to maintain a strong therapeutic effect.
Impacts
What was accomplished under these goals?
Intestinal roundworms are the most common parasites in the United States of horses, cattle, sheep, goats, alpacas, llamas, pigs, poultry, dogs, and cats. The parasites 1) reduce reproduction of the animals, 2) decrease overall health, energy, and performance, 3) decrease animal productivity, 4) make animals more susceptible to disease, and 5) can kill the animals. Some of these parasites can pass (infect) from animals to humans. They cause billions in lost economics in the US yearly (e.g., $2,500,000,000 losses to the cattle industry alone). Although we have drugs to treat these infections, parasite resistance to these drugs is growing enormously for all animals with multidrug resistance common in sheep, goats, and dogs and growing in all others. For example, Howell et al. 2008 reported that 48% of sheep and goat farms in the southeastern U.S. had intestinal worm parasites simultaneously resistant to all three of the main deworming drugs. Thus new approaches are absolutely critical. Unfortunately, the biotechnology industry has been unwilling to devote significant resources to developing new therapeutics. Fortunately, our laboratory has discovered a new, organic solution to this challenging situation, the Cry5B protein made naturally by the soil bacterium Bacillus thuringiensis, which is harmless to higher animals but toxic to parasitic worms. The goal of this USDA/NIFA grant is to test the efficacy of this new therapeutic against key veterinary parasites of horses and sheep, with additional testing being done against cattle, dog, and pig parasites. Our research will most immediately help animals infected with parasitic roundworms and people who work/live with these animals (e.g., the livelihood of animal farmers, the owners of cats/dogs). Objective 1: Optimize Cry5B sequence to specifically target cyathstomins and Haemonchus contortus and test spectrum of activity. Progress: We succeeded in using the COPAS biosorter to measure the length of parasitic larvae. Using positive and negative controls, we showed that the Z factor of our assay is 0.53, which is excellent. We therefore have a highly quantitative parasite larval assay for Haemonchus (sheep parasite) and cyathostomins (horse parasites). We also succeeded in building a worminator box for measuring the intoxication of parasitic adults in a highly quantitative manner. We tested the five natural Cry5B amino acid variants discussed in last year's update. Variant 105 (T64M) and variant 104 (N172I, E248K), showed increased antiparasitic activity relative to our canonical Cry5B amino acid sequence against parasitic larvae. We deconvolved variant 104 and showed that E248K alone shows increased activity against parasitic larvae. We are currently constructing NNK libraries and have successfully incorporated >17 amino acid variants for C177 and E248. Next year we will test these (and S407 variants) to identify which substitions give rise to the highest activity. Objective 2. Making a production-quality strain of Cry5B and optimizing production. We continued to produce at 350 liter scale with success. We have also collaborated with a Bacillus production company to optimize expression at >5 g/Cry5B per liter of culture, with Cry5B representing ~35% total protein by weight. This is phenomenal productivity and, if we can continue this development, achieves commercializable productivity goals. We are also optimizing the genetic background of the Bacillus strain to allow for broader Crystal protein expression as we find that spo0A- strains are limited in making crystal proteins other than Cry5B. We are experimenting with sigmaK- cells. Objective 3. In vivo testing and formulation. We set up two in vivo tests this year, one in sheep and one in horses. Both tests were set up using IBaCC (inactivated Bacilli with cytosoloic crystals), our lead API (deliverable form) that has never been tested in vivo in these systems before. 10 foals in the herd were identified as naturally infected with the parasite Parascaris equorum. All had fecal egg counts from between 180-2121 eggs per gram of feces. They were randomly split into two groups of 6 (treated orally with 30 mg/kg Cry5B IBaCC) and 4 (untreated; water only). Of the 6 treated, all had their fecal egg counts drop to zero within 7 days and remain so for up to 28 days. Of the 4 untreated, all retained positive egg counts for up to 14 days; by 28 days 1 had lost the infection but three retained it. These data demonstrate that Cry5B IBaCC is highly efficacious against this parasite of paramount importance to the horse industry. Amazingly, there is no drug on the market today that has this strong an impact. The second in vivo study was done in sheep. Sheep were experimentally infected with Haemonchus contortus parasites and, after confirmation of strong infection, treated orally with 40 mg/kg Cry5B three days in a row or water control (6 sheep per group). Fecal egg counts were taken daily and the animals were euthanized on day 8 for worm counts. A 95% drop in fecal egg counts were seen and a 72% drop in total parasitic worms. Most strikingly, there was a 96% drop in female parasitic worms and a 60% drop in male parasitic worms (all differences were statistically significant). Taken together, these amazing data indicate that our new therapeutic, Cry5B IBaCC, works in the field to effect significant cure of two of the most important parasitic worm infections in US farm animals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro. Charuchaibovorn S, Sanprasert V, Sutthanont N, Hu Y, Abraham A, Ostroff GR, Aroian RV, Jaleta TG, Lok JB, Nuchprayoon S., Am J Trop Med Hyg. 2019 Nov;101(5):1177-1182. doi: 10.4269/ajtmh.19-0083.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.
Elfawal MA, Savinov SN, Aroian RV.
Sci Rep. 2019 Aug 26;9(1):12347. doi: 10.1038/s41598-019-48720-1.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics.
Tyagi R, Elfawal MA, Wildman SA, Helander J, Bulman CA, Sakanari J, Rosa BA, Brindley PJ, Janetka JW, Aroian RV, Mitreva M.
Sci Rep. 2019 Jun 24;9(1):9085. doi: 10.1038/s41598-019-45548-7.
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:Our efforts are targeted to help large animals and large animal farmers deal with ubiquitous and damaging intestinal nematode parasites, especially those with existing and/or increasing resistance to current drugs, which are becoming pervasive. Our efforts are also targeted to help owners of companion animals (e.g., dogs) that are also infected by these parasites (e.g., dog hookworm), which are also showing signs of emerging resistance to current drugs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?For UMASS Medical School, the project has offered training activities for the following: Ambily Abraham, postdoctoral scholar Raffi Aroian, faculty David Gazzola, Research Associate. This experience has given this talented young man the opportunity to learn how to do research for the first time. Michelle, Haigbea, High school student. This experience has been an amazing opportunity for this young high school student who comes from a high school that aims to increase educational opportunities to promising students from disadvantaged backgrounds. Yan Hu, Research Professor. This experience was the springboard for this talented scientist to successfully attain her own faculty position in August. Tasia Kellogg, Research Technician. This experience has given this budding young scientist the training and background to get into graduate school and continue her scientific training towards a PhD. Hanchen Li, Research Associate. Gary Ostroff, faculty. For University of Kentucky, the project has offered training activities for the following: Ashley Steuer, PhD Student Holli Gravatte, Laboratory technician Haley Anderson, undergraduate student Alyssa Carpenter, undergraduate student John Hines, undergraduate student Avery Martin, undergraduate student DeAnna Muscarello, undergraduate student For Virginia Tech, the project has offered training activities for the following students: Emily Suess (Veterinary) Catherine Pouliot (Veterinary) Emily Siegel (Veterinary) Brittany Weicht (Veterinary) Olivia Stover (Undergraduate) Carolyn Martin (Undergraduate) John Saders (Masters) How have the results been disseminated to communities of interest?Dr. Aroian has presented the findings from this research to the Veterinary School at Tufts University in Grafton Massachusetts. Two presentations were made to the American Association for Veterinary Parasitologists in Denver Colorado in July 2018. Three manuscripts have been published or submitted. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to optimize the Cry5B sequence against these parasites and to develop highly quantitative in vitro assays with the parasites. Sheep/Haemonchus studies planned: Sheep were born in January. The plan is to infect in May and treat in June with IBaCC. Up to four groups. Group A will be controls. Group B will be maximum single dose of IBaCC. Group C maximum dose of IBaCC 1/3 split over 3 days. Group D BaCC. Expected weight of sheep 50 killigrams. Would also be good to perform in vitro assays with adult H.contortus compare male vs. female PCC vs. IBaCC. Horse/Parascaris studies planned: Twenty foals about to be born. Plan rolling enrollment. They will be placed in fields where infection occurs. The goal is to repeat the 10 mg/kg study with omeprazole but using IBaCC instead of PCC and larger group size. Estimated weight of horses 250 killigrams times ten horses equls 25 grams of IBaCC. Screen in August. Pig studies planned: March, dose ranging study against Ascaris with IBaCC 30, 10, 3 mg/kg. Hidden antigen study in May with IBaCC and natural infections. Microbiome study in August with IBaCC to look for impacts of treatment on microbiome.
Impacts
What was accomplished under these goals?
Intestinal roundworms are amongst the important parasites of farm and companion animals in the United States. These roundworms are critical parasites of, for example, horses, cattle, sheep, pigs, dogs, and cats. The parasites are capable of 1) reducing reproduction of the animals, 2) decreasing their overall health, energy, and performance, 3) decreasing their productivity, 4) making animals more susceptible to disease, and 5) killing the animals. Some of these parasites are also zoonotic and can pass (infect) from animals to humans. They are thus a major health problem in the United States and cause billions in lost economics (e.g., $2,500,000,000 lost per year to the cattle industry alone). Although we have dru1gs to treat these infections, the parasites readily develop resistance to these drugs. Thus for sheep, horses, cattle, and dogs there are already significant and alarming instances of the parasites being resistant to one or even several deworming drugs. The situation is particularly dire for sheep. For example, a 2008 report (Howell et al.) reported that 48% of sheep and goat farms in the southeastern U.S. had intestinal worm parasites simultaneously resistant to all three of the main deworming drugs. Thus new approaches are absolutely critical. Unfortunately, the biotechnology industry has been unwilling to devote significant resources to developing new therapeutics. Fortunately, our laboratory has discovered a new, organic solution to this challenging situation, the Cry5B protein made naturally by the soil bacterium Bacillus thuringiensis, which is harmless to higher animals but toxic to parasitic worms. The goal of this USDA/NIFA grant is to test the efficacy of this new therapeutic against key veterinary parasites of horses and sheep, with additional testing being done against cattle, dog, and pig parasites. Our research will most immediately help animals infected with parasitic roundworms and people who work/live with these animals (e.g., the livelihood of animal farmers, the owners of cats/dogs). Objective 1: Optimize Cry5B sequence to specifically target cyathstomins and Haemonchus contortus and test spectrum of activity. Progress: Last year we reported on developing a new and improved assay for measuring the impacts of drugs on these parasites and on identification of three amino acid changes that give improved activity against both target parasites. This year, we have continued our assay development work. We have learned how to isolate large quantities of parasite larvae and automatically sort them in defined numbers into 96-well format using a COPAS Biosort apparatus. Thus, we can now, for example, place exactly 60 first staged H. contortus larvae in each well fo a 96-well plate. We have begun to use the aspiration function of the COPAS Biosort to measure the length of the larvae that develop so that we can automate the scoring of the assay. In this way, if successful, we will have the first high-throughput highly quantitative assay for gastrointestinal parasitic nematode research. We have also identified 6 natural protein variants of Cry5B (from soil bacteria) and have successfully sythesized and expresed 5/6 of these. These will be tested against the parasites for improved activity relative to the canonical sequence. We have also begun the process of substituting all 20 amino acids at the C177 position of the protein, identified last year as a leading amino acid variant for improved activity. Objective 2. Making a production-quality strain of Cry5B and optimizing production. Last year we described a breakthrough in engineering a production-quality called iBaCC for inactivated Bacillus with Cytosolic Crystals and PCC for Purified Cry5B Crystals. PCC is bioactive against H. contortus in the E2L assay and against hookworms in hamsters. We have continued our work to improve our industrial-scale production of both. We have now scaled up to 350 liters at Utah State Fermentation Facility and have now completed 6 runs. These materials were used for all the studies in Objective 3 below. We are also working on optimizing production yields of IBaCC, testing 10 different conditions for better yields. This work is on-going. We have also improved the PCC process and can, at times, get >85% pure Cry5B from these preparations. Objective 3. In vivo testing and formulation We set up an in vivo study in sheep using our new IBaCC formulation. As previously reported, treating sheep to cure Haemonchus contortus parasites with Cry5B material by direct injectino into the abomasum failed, likely due to technical challenges. We therefore took a step back and tested the stability of Cry5B PCC in rumen (from cattle). Amazingly Cry5B PCC (purified protein) was completely stable in cattle rumen for 24 hr at 37C and 50% stable (intact) after 48 hr at 37 C. This result gave us confidence that direct gavage of Cry5B might be administered by direct oral gavage and survive delivery to the abomasum. We therefore set up an in vivo study. Six female and 6 castrated male lambs aged approximately 7-8 months were removed from pasture and confined for the duration of the study. All were dewormed to remove existing trichostrongyle infection. Sheep were subsequently each orally administered 10,000 third stage H. contortus. Following patency of infection, the 10 sheep with the highest FEC were divided into two groups with similar mean FEC (determined by the Modified McMaster test, detection limit 25 epg). Treatment group sheep were orally administered 200 mL of Cry5B IBaCC daily for 4 days, each Cry5B dose corresponding to 40 mg/kg. Control group sheep were given 200 mL of water. FEC were determined daily for 7 days post infection and thereafter every two days until necropsy at 14 days PI. After 24 hours, mean FEC of treated animals was reduced by 78% compared to controls and reduced by 94% after 72 hours, eventually showing >99% reduction over time. Of the few eggs produced by treated animals, only 27% developed to the L3 compared to >90% of eggs from control lambs. These exciting data indicate that our new Cry5B API is a highly effective new therapeutic for H. contortus, the most damaging and pervasive parasite of small ruminents and one that is multidrug resistant to all classes of commonly used anthelmtintics. This is a major finding directly a consequence of NIFA funding. We also set up an in vivo study in pigs to test the efficacy of Cry5B IBaCC against Ascaris suum parasites. To date, there have been no studies of Cry5B IBaCC against Ascarids in a large animal. For this study, small pigs (14 - 30 kg in weight) were inoculated with 10,000 A. suum eggs (6 pigs/group). Twelve days post inoculation, pigs received a single dose of Cry5B IBaCC by oral gavage translating to a single 30 mg/kg dose. Four days later, pigs were euthanized and total L4 Ascaris counts were taken. Average Ascaris counts in control (water treatment) was 2358.3. Average Ascaris counts in treated group were 88.5, representing >96% reduction. Thus, Cry5B IBaCC is highly effective against ascarids in a large animal. We also continued our plot study in which we used native Bt Cry5B strain to see if we could provide environmental control of cyathostome parasites. Feces from horses were mixed with Cry5B Bt spore crystal lysate (10^8 CFU/gram feces) and placed in the field covered with cheese cloth to prevent scavenger activity. Control included Bt spore lysates (no crystals). Each group contained 10 replicates. After seven days, the average number of larvae of various stages were counted. Whereas L3i (infectious staged L3s) were found in the control fecal plots, barely any were found in the treated fecal plots. These data are the first to suggest that Bt spore-crystal lysates could be useful for environmental control of parasitic nematodes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure.
Hu Y, Nguyen TT, Lee ACY, Urban JF Jr, Miller MM, Zhan B, Koch DJ, Noon JB, Abraham A, Fujiwara RT, Bowman DD, Ostroff GR, Aroian RV. Int J Parasitol Drugs Drug Resist. 2018 Aug;8(2):287-294. doi:10.1016/j.ijpddr.2018.05.001. Epub 2018 May 4.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
A highly expressed intestinal cysteine protease of Ancylostoma ceylanicum protects vaccinated hamsters from hookworm infection
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Cognitive and Microbiome Impacts of Experimental Ancylostoma ceylanicum
Hookworm Infections in Hamsters
|
Progress 02/01/17 to 01/31/18
Outputs
Target Audience:Our efforts are targeted to help large animals and large animal farmers deal with ubiquitous and damaging intestinal nematode parasites, especially those with existing and/or increasing resistance to current drugs, which are becoming pervasive. Our efforts are also targeted to help owners of companion animals (e.g., dogs) that are also infected by these parasites (e.g., dog hookworm), which are also showing signs of emerging resistance to current drugs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?For UMASS Medical School, Aroian Lab: The project offered training to two researchers who recently received their bachelors (David Gazzola and Tasia Kellogg), giving them their first in depth scientific research trainign. In additon Prof. Yan Hu, paid off ths award, was able to give a seminar at an international meeting inWashington DC on our NIFA/USDA research as was Dr. Hanchen Li (Senior Scientist) who presented this work at the American Associationfor Veterinary Parasitologistsin Indianapolis, IN July 2017. For both of these scientists, this award allowed them to professionally grow by giving these talks in front of large audiences. The research also supported a postdoctoral scholar Anand Sitaram and advancing his career towards a more independent position. For UMASS Medical School, Ostroff Lab: Four members of the Ostroff Lab have contributed to the Cry5B NIFA grant and have benefited greatly at a number of levels. Most of our development-minded collaborators are off campus and Dr. Ostroff's staff is insulated from the frenetic nature of preclinical development work because it is primarily done in industry, not academia. Through frequent team meetings they got to see what life was like on the development side. This had both good and bad impacts in that 2 of the team members left to take jobs in industry based on the experience they got working on the NIFA grant - good for them, bad for us. Also a key training benefit to the staff was to participate in producing 30g of Cry5b protein at the 100L scale for large animal studies. This required substantial training and provided invaluable experience as the team worked to improve the Cry5b manufacturing process to a scalable, reproducible, cost-effective method for commercialization. They have also been introduced to the role of raw material, bulk and finished product QC and documentation as a way to ensure a robust manufacturing process. For University of Kentucky:The project has offered training activities for the following students: Ashley Steuer, PhD student Kelly Thomas, Undergraduate student Samantha Naughton, Undergraduate student Elizabeth Bennett, Undergraduate student For Viginia Tech University: The project has offered training activities for the following veterinary students: Emily Suess Catherine Pouliot Lauren Page Emily Siegel How have the results been disseminated to communities of interest?An article mentioning our NIFA/USDA research waswritten by Dr. Nielsen and appeared in Vol 26 No 2 in Equine Disease Quarterly, reaching out to the horse owners those with broadinterest in the equine community. In addition, we presented this work at the American Associationfor Veterinary Parasitologistsin Indianapolis, IN July 2017. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Take the three Cry5B mutants shown to have increased activity against cyathstomins and H. contortus, place every amino acid in their position (currently only alanine), and see which amino acid substition gives the greatest efficacy at each of the three positions. From then, the double and triple mutants will be made. Repeat testing of the 15 Bt strains to identify top 3-5 strains. Test all of these improved Cry5B variants and Bt strains against adult parasites as they become available. Incorporate cattle parasites, Ostertagia ostertagii, into the screening protocol. Work to further automate the E2L screening system to increase throughput. Objective 2: Continue to produce large amounts of API for in vitro and in vivo testing. Potentially production of IBaCC and PCC using fermentation optimization and promoter swapping. For formulation, we plan a multi-pronged approach utilizing both in-house and commercial resources with large ruminant animal drug formulation experience. The general in-house approach will focus on adapting our recently developed oral, room temperature vaccine formulation technology to Cry5B IBaCC. This technology is based on encapsulating proteins inside yeast cell wall ghosts and then carrying out an ensilication reaction, immobilizing the protein in a biodegradable glass matrix and stabilize the protein against thermal and protease degradation. Such ensilicated antigen particle formulations are highly effective as vaccines and are non-toxic and stable in accelerated stability studies. Cry5B IBaCC can be thought of as a bacterial cell wall ghost containing a Cry5B crystal(s), analogous to our yeast cell wall based technology. We propose to prepare ensilicated Cry5B IBaCC and test for in vitro thermal and protease stability in rumen fluid and anti-STH activity in vitro and in vivo. Stable formulations will be scaled up and used in sheep and goat studies planned for next year. If ensilication alone is insufficient to produce a more effective Cry5B product, then we propose to prepare rumen-tolerant coated, ensilicated formulations for testing as described above. A complementary approach will be taken for horses (monogastrics). Objective 3. Continue plot studies in the spring, early summer to avoid difficulties with weather seen with late fall studies. Attempt pass-through studies in which spores are passed through the large animal host to deposit spores into feces and eliminate parasite reproduction. For in vivo studies, repeat successfull Parascaris studies in foals using experiment (instead of natural) infections with increased sample size. For sheep, take top 3 Bt strains and gavage at higher doses (~40mg/kg) for three straight days to see if any effect can be seen with this aproach.
Impacts
What was accomplished under these goals?
Intestinal roundworms are amongst the important parasites of farm and companion animals in the United States. These roundworms are critical parasites of, for example, horses, cattle, sheep, pigs, dogs, and cats. The parasites are capable of 1) reducing reproduction of the animals, 2) decreasing their overall health, energy, and performance, 3) decreasing their productivity, 4) making animals more susceptible to disease, and 5) killing the animals. Some of these parasites are also zoonotic and can pass (infect) from animals to humans. They are thus a major health problem in the United States and cause billions in lost economics (e.g., $2,500,000,000 lost per year to the cattle industry alone). Although we have drugs to treat these infections, the parasites readily develop resistance to these drugs. Thus for sheep, horses, cattle, and dogs there are already significant and alarming instances of the parasites being resistant to one or even several deworming drugs. The situation is particularly dire for sheep. For example, a 2008 report (Howell et al.) reported that 48% of sheep and goat farms in the southeastern U.S. had intestinal worm parasites simultaneously resistant to all three of the main deworming drugs. Thus new approaches are absolutely critical. Unfortunately, the biotechnology industry has been unwilling to devote significant resources to developing new therapeutics. Fortunately, our laboratory has discovered a new, organic solution to this challenging situation, the Cry5B protein made naturally by the soil bacterium Bacillus thuringiensis, which is harmless to higher animals but toxic to parasitic worms. The goal of this USDA/NIFA grant is to test the efficacy of this new therapeutic against key veterinary parasites of horses and sheep, with additional testing being done against cattle, dog, and pig parasites. Our research will most immediately help animals infected with parasitic roundworms and people who work/live with these animals (e.g., the livelihood of animal farmers, the owners of cats/dogs). Objective 1: Optimize Cry5B sequence to specifically target cyathstomins and Haemonchus contortus and test spectrum of activity. Progress: Last year we reported on 11 amino acid changes that make Cry5B more active against human hookworm larvae and stated that we need to develop better larval development assays for testing against cyathostomin and H. contortus, the two main intestinal parasitic nematodes of this grant (major parasites of horses and sheep respectively). Here we report on improvement of those assays and identification of three amino acid changes that give improved activity against BOTH target parasites. To improve the assay, we incorporated Lugol's staining and sodium thiosulfate destaining steps. These result in a slight but noticeable and specific red staining of the L3i, which allows for easier identification and counting in the well. This method, which we call the egg-to-larval or E2L assay. We then set out to study Cry5B point mutations using this improved assay. The first point mutation we studied is S407C. S407C has noticeable improvement of activity relative to wild-type Cry5B against both cyathostomin and H. contortus parasites. We next expanded this study. E248A and C177A but not other mutants show improved activity against both parasites. Objective 2. Making a production-quality strain of Cry5B and optimizing production. Last year we described a breakthrough in engineering a production-quality called iBaCC for inactivated BaCC. To turn this dead bacterial product into a drug-like product, after significant effort (cell biological and biochemical, bioassays with parasites), we have established a protocol for purifying Cry5B crystals from IBaCC strain. We call these PCC for Purified Cry5B Crystals. PCC is bioactive against H. contortus in the E2L assay and against hookworms in hamsters. We next scaled-up production. We contracted with a bioproduction facility at a nearby university, Worcester Polytechnic Institute (WPI), to scale up production to 100 liters. For 6-8 weeks during the summer, the entire team at UMMS worked closely with WPI personnel to run three different 100 liter fermentor runs and purify PCC from 300 liters of fermentation broth. The total yield from these runs was ~30 g of PCC protein. Our efforts on our NIFA award have now yielded two new and validated deliverable forms of Cry5B--IBaCC and PCC. Objective 3. In vivo testing and formulation. We set up two in vivo tests this year, one in sheep and one in horses. Twelve sheep with infected with H. contortus larvae in June. The infections were allowed to mature until August, as confirmed by presence of parasite eggs in the feces. Because we are in the process of developing formulations and given the challenge of delivering Cry5B through three stomachs (rumen, reticulum, omasum) before reaching the true fourth stomach (abomasum) where the parasites reside, we decided to inject Cry5B (in spore-crystal lysates) directly into the abomasum via surgery. This was done in 6 sheep on August 24, with the other six sheep receiving placebo. The effective Cry5B dose was 10 mg/kg (~0.5-0.6 g/sheep). Fecal egg counts were followed for up to two weeks after treatment. There was no significant change in fecal egg counts with Cry5B treatment relative to controls, suggesting that the Cry5B had no effect (based on this one measure of efficacy). Although no effect was seen, we believe this may be actually due to a technical challenge rather than efficacy indicator. More importantly, it was clear that manipulation of the abomasum caused significant peristaltic contractions that likely resulted in the protein being expelled from the abomasum before having time to act. In horses (foals), we used PCC since we had less concern about getting it through 3 stomachs. However, to maximize the efficacy, all foals were pretreated with omeprazole to neutralize stomach acid and allow more Cry5B to pass into the small intestine. Sixteen hours post-treatment with omeprazole, five foals received Cry5B via nasogastric tube at a Cry5B dose of 10 mg/kg (~2.5 g/horse). As a control, five foals received placebo. Two treated foals had the Ascarid parasite Parascaris equorum, a potentially deadly small intestinal nematode parasitic infection of foals and one that is becoming more difficult to treat due to multi-drug resistance. To our delight, both foals that were infected with P. equorum and treated with Cry5B were rapidly and completely cleared of the infection based on the complete elimination of parasite eggs in the feces (fecal egg counts). Based on these data, this NIFA award has yielded a new cure for Ascarid infections in horses. For both sheep and horse parasites, we also performed a plot study in which infected fecal samples from both animals were treated with Cry5B outdoors to test if Cry5B might have utility in controlling development of infectious stages in the field/pasture. Unfortunately, the timing of these tests proved to be a challenge. In the case of the sheep experiments, excessive (3X normal) rainfall destroyed the field samples. In the case of the horse experiments, the weather turned very cold, resulting in very slow development of the parasitic eggs. Based on these results, it was determined that such studies need to happen earlier in the year, preferably spring. We also have investigated whether other Bacillus thuringiensis crystal proteins with excellent anthelmintic properties in order to expand our studies and to see if, especially for sheep and for cyathostomes, if we can improve efficacy. Using our E2L assay, we therefore tested 15 Bt strains that we know or suspect have good nematicidal activity. Relative to our Cry5B strain (HY65), several strains emerge has potentially having excellent activity, including HY45, 46, 27,and 58 (negative control is BSA and/or E. coli). None of these strains express Cry5B. These data are promising and are to be repeated.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Recombinant subunit vaccines for soil-transmitted helminths. Parasitology 144(10): 1-26.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
In vivo and in vitro studies of Cry5B and nicotinic acetylcholine receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:Our efforts are targeted to help large animals and large animal farmers deal with ubiquitous and damaging intestinal nematode parasites, especially those with existing and/or increasing resistance to current drugs. Our efforts are also targeted to help owners of companion animals (e.g., dogs) that are also infected by these parasites (e.g., dog hookworm), which in some cases are showing signs of emerging resistance to current drugs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?UMASS Medical School: Research Assistant Prof. Yan Hu gave an oral presentation about our USDA/NIFA research at the annual American Association of Veterinary Parasitologists (AAVP) meeting in August 2016. This presentation is part of her growing independence running a small laboratory group. The grant also provided the opportunity for one recent graduate (bachelor's level) to explore and learn what scientific research is and how it is carried out. The grant also supports a more experienced Senior Research Associate with a background in human gastrointestinal disease who is now learning about veterinary diseases of the GI tract. All of these scientists have been meeting with me regularly (weekly or semi-weekly) one-on-one to further their training and career goals. All present regularly at our weekly lab meeting. Collaborative: The collaboration holds team phone calls every 6 weeks with all members of the consortium participating. The goal is for all team members to coordinate but also learn how collaborative research is carried out. This is an excellent regular, training opportunity for all trainees in the program. U Kentucky: Two graduate students and one undergraduate student are participating in the project and gaining valuable experience in veterinary parasitology and with developing and evaluating parasite in vitro assays. Virginia Tech: Five veterinary/graduate students are working on the project all of whom are involved in maintaining infected sheep and share in all technical tasks. All are getting experience with veterinary parasitology and learning about how to set up and organize research and research goals. How have the results been disseminated to communities of interest?Our research and collaboration was featured in an article in Equimanagement in 2016, describing how our work could have significant and positive impact on equine health. What do you plan to do during the next reporting period to accomplish the goals?We will maintain the current research plan, which is alignment with our approved grant proposal. With regards to aim 1, we will continue to investigate the 11 amino acid changes that we found might be more active. We are currently optimizing protocols with horse cyathostome parasites in order to better detect amino acid variants that have improved activity. Combinations of these 11 amino acid changes, as well as altered variants of these amino acid changes will be investigated. For example, C177A is cysteine 177 altered to an alanine. We will attempt other amino acid changes at this position as well, once our improved protocol is in place. With regards to spectrum of activity studies, a key additional parasite to test against this year is adult Parascaris equorum, an important parasite of horses. With regards to aim 2, we are making a production quality strain that lacks all antibiotic resistance cassettes. In addition, we are performing RNAseq to identify promoters with higher expression levels in our IBaCC background (spo0A-). Constructs will be made using these high expressing promoters to see if we can drive up the expression of the protein even more. With regards to aim 3, we are setting up to perform initial in vivo experiments with Cry5B in sheep and horses with enterically coated capsules. Furthermore, we are continuing to work on the THISTL concept by feeding SCL to animals, following spore release in the stools, and then assaying the bioactivity of spore-containing stool against larval stages of the parasites.
Impacts
What was accomplished under these goals?
Intestinal roundworms are important parasites of farm and companion animals in the U.S. These roundworms are critical parasites of horses, cattle, sheep, pigs, dogs, and cats and are capable of 1) reducing reproduction of the animals, 2) decreasing their overall health, energy, and performance, 3) decreasing their productivity, 4) making animals more susceptible to disease, and 5) killing the animals. Some parasites are also zoonotic and can pass (infect) from animals to humans. They are a major health problem in the U.S. and cause billions in lost economics (e.g., $2.5 billion lost per year in the cattle industry alone). Although we have drugs to treat these infections, the parasites readily develop resistance to the drugs. For sheep, horses, cattle, and dogs there are significant and alarming instances of the parasites being resistant to one or even several deworming drugs. For example, a 2008 report (Howell et al.) reported that 48% of sheep and goat farms in the southeastern U.S. had intestinal worm parasites simultaneously resistant to all three of the main deworming drugs. Thus new approaches are absolutely critical. Fortunately, our laboratory has discovered a new, organic solution to this challenging situation, the Cry5B protein made naturally by the soil bacteriumBacillus thuringiensisor Bt, which is harmless to higher animals but toxic to parasitic worms. The goal of this USDA/NIFA grant is to test the efficacy of this new therapeutic against key veterinary parasites of horses and sheep, with additional testing being done against cattle, dog, and pig parasites. Our research will most immediately help animals infected with parasitic roundworms and people who work/live with these animals (e.g., the livelihood of animal farmers, the owners of cats/dogs). In objective 1, we aimed to 1) make our therapeutic better by modifying the Cry5B protein amino acids and 2) test it against a broader range of parasitic roundworms. We tested all 34 amino acid Cry5B variants mentioned in the grant application (at which point only 8 had been tested against parasites) against parasitic worms and looked at their expression levels (or how much Cry5B protein the bacteria could produce). Of these 34, 11 seemed to be more effective at killing worms than the normal (natural) variant of Cry5B in our tests and were produced by Bt in similar amounts as natural Cry5B. These 11 are: C177A, H277A, S407C, Q435A, H332A, G577A, S534A, I678A, G643A, E248A, and N656A. We are in the process of making all double combinations between these 11 amino acid variants. In the course of this work, we discovered that our tests need to be more sensitive in order to discern with statistical certainty than an effect seen with the variants is superior to the natural variant. For example, the variant C177A (cysteine #177 altered to alanine) is consistently but weakly more active against parasitic worms.We are developing tests using the parasitic worms that infect horses andsheep that give a statistically stronger results. With regards to spectrum of activity, we found that Cry5B is additionally active against the cattle parasite worm (brown stomach worm)Ostertagia ostertagiusing the egg to larval stages of this parasite. We have also now included data on the very important veterinary worm parasiteAncylostoma caninum(dog hookworm; see below). In objective 2, we are optimizing a Cry5B expressingBacillusstrain for high yields and bioactivity. The overall goal is to make the product as cheaply as possible and a simple way to do this is to have each and every bacterial cell make more of the protein so that the yields per liter of bacterial culture are higher. To begin with, we have newly engineered Bt to make our new therapy as cheap, easy, and approvable as possible. Bt is a great bacterium for production of Cry5B because it is the native bacterium from which the protein comes: Bt makes more Cry5B protein than any other bacterium that we have engineered to produce Cry5B, and makes it in a manner that is highly active against worms. However, Bt is not a bacterium that is approved for ingestion to treat internal parasites. To get around this issue, we have now engineered Bt (spo0A-) so that it cannot form spores (which are difficult to kill) but can still express Cry5B during normal bacterial growth phase (i.e., cry3A promoter). In this strain, called BaCC forBacilluswith Cytosolic Crystals, Cry5B crystals are made inside of Bt as normal but they are never released as the bacterium does not sporulate. The crystals stay within theBacillus. TheseBacilluscells, unlike spores, are readily killed by food grade substances, like carvacrol (oil of oregano). We therefore spent the past year making this strain, optimizing the carvacrol treatment, and the production of Cry5B from it. We have found that treatment of BaCC cells with 1 mg/mL of carvacrol for 15 minutes is sufficient to completely kill the Bacillus cells (0 colony forming units) while retaining bioactivity against relevant parasites. We call these cells IBaCC for Inactivated BaCC. For example, 500 ng/mL of Cry5B in IBaCC inhibits the development of 95% of cyathostome (horse) parasitic larvae in culture and 38 ng/mL of Cry5B in IBaCC inhibits the development of 100% of Haemonchus contortus (sheep) parasitic larvae in culture. Some optimization of fermentation studies has been carried out and we have discovered an extremely successful method to achieve excellent expression and bioactivity of Cry5B in BaCC or IBaCC. In objective 3, our goal is to test our product against parasitic wormsin vivo(in live infected animals) and formulate the product for so that it is not destroyed in the gastrointestinal (GI) tract and the protein arrives at the sites where the parasitic worms live (e.g., small intestine, large intestine, or stomach). We performed our firstin vivotesting using Cry5B spore crystal lysates and IBaCC against the dog hookworm parasite,Ancylostoma caninum. Studies were carried out using capsule endoscopy, which allows for counting of hookworm parasites in the small intestine of infected dogs both before and after treatment without any harm to the animals (the animals are adopted after the study). We found that two doses of Cry5B spore crystal lysates (specific dose, 0.2 µM of Cry5B/kg) essentially cured two dogs of hookworms: average of 95% reduction in hookworm burden and 92% reduction in fecal parasite egg counts. A preliminary study with IBaCC was also performed at half the dose (0.1 µM of Cry5B/kg). Although worm counts were not taken because of technical issues, fecal parasite egg counts dropped on average >68% in the two dogs tested. Thus, IBaCC is also active against parasitic wormsin vivo. We are also investigating a new idea that could bring a Cry5B product to the market that would also help prevent reinfection of parasites and control parasites in the pasture. We call this idea THISTL for Trojan Horse In Stool (from) Lysates. The hypothesis we are testing is that if we feed animals Cry5B spore-crystal lysates, some spores would survive the GI tract, germinate in the feces, and kill the parasitic larvae present in the stool. The bacterium would kill not only parasitic larvae shed from the animal fed but also parasitic larvae that were already developing in the soil. For this study, we are testing our non-recombinant Cry5B expressing strain called HY65. This strain came from the soil and expresses Cry5B. To begin with, we mixed Cry5B spore-crystal lysates from sheep stool containing high numbers ofHaemonchus contortuseggs. We find that 5 X 105, 5 X 106, and 5 X 107colony forming units of Cry5B Bt in stool respectively inhibit parasite development by 67%, 75%, and 92% respectively. Because we are using the native strain that came from the soil, we would be introducing back into the soil something live, natural, and non-recombinant. It would be compatible with all sorts of animal farming, including organic.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Recombinant subunit vaccines for soil-transmitted helminths
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