Project : USDA ARS

ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #432459

Research Project: Molecular Approaches to Control Intestinal Parasites that Affect the Microbiome in Swine and Small Ruminants

Location: Animal Parasitic Diseases Laboratory

2018 Annual Report

Objectives
Objective 1. Determine the change in the intestinal metabolome and microbiome during parasitic nematode infection and after anti-parasitic clearance. Sub-objective #1. Characterize parasite-induced molecular mechanisms that modulate intestinal inflammation. Sub-objective #2. Evaluate the potential impact of Cry5B on the native and parasitized gut microbiome. Objective 2. Identify pan-nematode secretome products with immune modulating activity that along with nutritional supplements eliminate parasites and enhance enteric health. Sub-objective #1. Use antibodies from parasite infected pigs and goats to select for immunogenic cloned parasite products that have been computationally identified as vaccine targets. Sub-objective #2: Test for cloned parasite products that induce innate immune responses at the mucosal surface of explanted intestinal tissues from pigs and goats.

Approach
Objective 1. Determine the change in the intestinal metabolome and microbiome during parasitic nematode infection and after anti-parasitic clearance. Sub-objective #1. Characterize parasite-induced molecular mechanisms that modulate intestinal inflammation. Hypothesis #1: Parasitic infections alter the relative abundance of butyrate-producing bacteria in the gut and change the composition and concentration of total short-chain fatty acids (SCFA) as well as anti-inflammatory butyrate, which in turn modulates intestinal inflammation and host immunity. Sub-objective #2. Evaluate the potential impact of Cry5B on the native and parasitized gut microbiome. Hypothesis #2: The administration of the Cry5B anthelmintic will have minimal effects on the native microbial community in the gut due to its transient nature and invertebrate gut targets. Hypothesis #3: Parasite-induced changes in the microbiome will be restored to the native structure and function after treatment with Cry5B that reduces worm burden. Experimental design: Quantifying changes in the intestinal metabolome and gut microbiome induced by parasitic infection, and characterizing the abilities of anti-parasitic treatments to restore altered gut microbiota, are important in dissecting mechanisms of host pathophysiology and immunity. We will conduct an in-depth comparison of the gut metabolome and microbiome between animals randomly assigned to two conditions (naive and infected) and exposed to Cry5B in an optimally determined delivery system. Objective 2. Identify pan-nematode secretome products with immune modulating activity that along with nutritional supplements eliminate parasites and enhance enteric health. Sub-objective #1. Use antibodies from parasite infected pigs and goats to select for immunogenic cloned parasite products that have been computationally identified as vaccine targets. Hypothesis #1: Immunization of target host species with computationally selected immunogenic cloned parasite products will disrupt parasitism and prevent infections. Sub-objective #2: Test for cloned parasite products that induce innate immune responses at the mucosal surface of explanted intestinal tissues from pigs and goats. Hypothesis #2: Selected cloned immunogens that also have innate immune features defined by responses in intestinal explants will enhance vaccine efficacy and disrupt parasitism. Experimental design: Powerful new technologies to characterize the transcriptomes from multiple life stages of parasitic nematodes (Heizer et al., 2013) can be used to predict secreted peptides common to the pan-secretome. Combining this bioinformatics approach with antibody detection systems of immune peptides and innate responses of intestinal explanted tissues from pigs and goats will be used to identify vaccine candidates for immunization in the target host species of interest.

Progress Report
Feeding krill oil to pigs is anti-inflammatory in a model of whipworm (Trichuris suis)-induced chronic colitis. Krill oil is rich in Omega-3 polyunsaturated fatty acids (n-3 PUFAs such as eicosapentaenoic acid-EPA and docosahexaenoic acid-DHA), as well as the potent antioxidant astaxanthin, and it reaches relevant tissues more efficiently than other fish oils due to its phospholipid forms. Agricultural Research Service scientists in Beltsville, Maryland, investigated the potential health benefits of feeding krill oil to pigs. Whipworm infection elevated expression of pro-inflammatory cytokines, such as IL4 and IL13, and induced colon crypt hyperplasia which was significantly reduced by feeding pigs krill oil for 28 days. Compared to a control group of pigs fed soybean oil, krill oil induced a significant change in the relative abundance of dozens of gut microbial taxa, including the family Lachnospiraceae, especially in the infected pigs. Moreover, the results suggested that krill oil markedly increased indices of key gut microbial biodiversity. These findings support the use of krill oil as dietary supplement to improve intestinal health. Infection of pigs with the intestinal roundworm Ascaris suum modulates microbial composition and inflammation in the intestine. Agricultural Research Service scientists in Beltsville, Maryland along with colleagues at the University of Ghent, Belgium evaluated worm burden and the abundance of microbial species in the intestine of pigs infected with the common large roundworm, Ascaris suum. A group of control pigs without worms was compared to groups with high and low parasite burden. Previous studies showed that the Ascaris infection increased levels of fecal short-chain fatty acids that can support intestinal health and reduce harmful bacteria. The maximal effect of the infection on the gut microbiome was in the proximal colon with 16 bacterial phyla identified in the luminal contents, and the relative abundance of the phyla Spirochaetes and Planctomycetes was significantly changed. The abundance of approximately 29% of the genera, including Prevotella and Faecalibacterium, and 179 operational taxonomic units (OTUs or microbial species) was significantly altered by infection. Notably, roundworm infection significantly shifted the metabolic potential of the proximal colon microbiota and altered the abundance of at least 58 metabolic pathways, including those for carbohydrate and amino acid metabolism. The infection also significantly reduced several key indices of microbial biodiversity, and regulated mucosal inflammation and tissue repair; likely by altering the population of butyrate-producing bacteria. These findings reveal the pathophysiological consequence of parasitic infection in pigs that are often infected with roundworms. The Cry5B crystal protein is part of a large family of 3-domain crystal proteins made by Bacillus thuringiensis (Bt) that are non-toxic to vertebrates and used extensively and hazard-free in farming and insect control. The spore crystal lysate of Bt was modified by scientists at the University of Massachusetts to enhance expression the Cry5B gene and production of Cry5B crystal protein. This new modification, called BaCC, was developed as a recombinant strain of Bt that cannot sporulate (deletion in the sporulation gene). Therefore, the safety of this strain will be enhanced because it will have an even shorter life in the environment. This material was fed to pigs infected with the intestinal roundworm, Ascaris suum, by Agricultural Research Service scientists in Beltsville, Maryland resulting in the expulsion of the larval stage of the worm from the intestine. The adult stage of Ascaris suum, problematic because of the production of eggs that become infective, was significantly expelled after pigs were fed BaCC. This is the first step in the development of a new and safer formulation of the Cry5B protein as a novel anti-worm therapy. Identifying parasite antigens (proteins) capable of protecting the host against infection remains elusive to the industry. This is complicated because we are attempting to immunize large animals (pigs) against small animals (nematodes) rather than against single cell pathogens i.e., bacteria. Two groups of pigs were immunized 3 times over 3 months with five cloned proteins identified from parasitic worms by database mining; three were derived from Ascaris suum and two from Trichuris suis. The recombinant proteins were mixed with a Seppic-Montanide adjuvant by Agricultural Research Service scientists in Beltsville, Maryland. Two other control groups were immunized with a cloned protein from Toxoplasma gondii under the same conditions. Pigs were challenged with either infective eggs of Ascaris suum or Trichuris suis and the worm burdens determined after 28 or 36 days, respectively, to assess protection against worm infection in the intestine. There was no significant reduction in worm burden for either parasite in the pigs immunized with recombinant worm antigen compared to the control immunized pigs; however, there were detectable antibody responses to the proteins used for immunization. Thus, these antigens are representative of proteins released by the worm in the animal but they do not induce a protective immune response. A bioinformatic approach to select other vaccine candidates is supported by collaborative research with scientists at the Washington University Genome Center in St. Louis, Missouri. Web-based bioinformatics workflows for end-to-end RNA-seq data computation and analysis. Advances in Next Generation Sequencing (NGS) technologies, bioinformatics algorithms and computational technologies have significantly accelerated genomic research. However, complicated NGS data analysis remains as a major bottleneck. RNA-seq is one of the major area in the NGS field that focuses on transcribed RNA rather than genomic DNA and presents great challenges in data analysis. To address this problem, a web portal was developed by Agricultural Research Service scientists in Beltsville, Maryland that offers three integrated workflows that can perform end-to-end compute and analysis, including sequence quality control, read-mapping, transcriptome assembly, reconstruction and quantification, and differential analysis. These workflows support multiple and differential analyses between study groups in a single test. These tools support genomic research in chicken, cow, duck, goat, pig, horse, rabbit, sheep, turkey, as well as several other model organisms including yeast, nematodes, insects and human. Interleukin-17 receptor A (IL-17RA) is a central regulator of protective immunity. Host inflammation has been linked to controlling and abating parasite infections. The protein IL17A is a proinflammatory cytokine (immune response factor) produced by activated immune cells (T cells). Giardia is a protozoan parasite highly prevalent in the intestines of a wide range of hosts. Agricultural Research Service scientists in Beltsville, Maryland, and colleagues at the University of Ghent, Belgium showed that IL-17A is important in the development of a protective immune response to Giardia in mice, cattle and humans. A molecular technique used to examine changes in gene expression showed that infection with the strain Giardia muris in mice up-regulated a wide array of IL-17RA-dependent genes including various peptides that can kill parasites. The data also showed for the first time the involvement of the circadian clock in the host response following Giardia infection. The results support the use of signaling of the IL-17RA gene as a target to control this important protozoan parasite in both humans and livestock.

Accomplishments
1. A new cure for hookworm. Hookworms are intestinal nematode parasites that infect nearly half a billion people globally, causing iron-deficiency, anemia and disease burdens conservatively estimated at four million Disability-Adjusted Life Years and with productivity losses of up to $139 billion annually. In some places, available drugs have less than a 40% cure rate, increasing the threat posed by emerging drug resistance. Fortunately, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. ARS scientists at Beltsville, Maryland, broadened the application of Cry5B to dogs infected with Ancylostoma caninum, and hamsters infected with the dominant human hookworm, Necator americanus. This protein, Cry5B, was shown to be highly effective against all hookworm parasites tested, improving by the neutralizing effects of stomach acids. Efficacy did not depend on the host immune system and did not decline with repeated dosing. A pan-hookworm therapy with excellent properties for use in humans and other animals has thereby been discovered.

2. Flavanol-rich cocoa powder interacts with probiotic bacteria to alter the antibody response to a parasitic worm in pigs. Consumption of the probiotic bacteria Lactobacillus rhamnosus-GG (LGG) and flavanol-rich cocoa powder (CP) have purported immune modulating effects in humans and animals. Agricultural Research Service scientists in Beltsville, Maryland, fed pigs a standard growth diet supplemented with either or both of these prior to infecting them with the common intestinal roundworm parasite, Ascaris suum. Pigs fed cocoa powder produced fewer antibodies against Ascaris IgG2 if also fed LGG. Pigs fed LGG had significantly reduced expression of several pro-inflammatory genes. These results suggested a reduction in localized inflammation after infection, and showed that feeding bioactive supplements in the diet can have immune modulating and anti-inflammatory effects that may or may not act in synergy.

U.S. DEPARTMENT OF AGRICULTURE

Animal Parasitic Diseases Laboratory: Beltsville, MD