Lester Independent Research Grant Proposal for Fall 2016 Environmental Sciences

Transcription

1 Lester Independent Research Grant Proposal for Fall 2016 Environmental Sciences Project Title: Understanding and analyzing the distribution of genotypes of Giardia intestinalis in the black and gold howler monkeys, Alouatta caraya, of Corrientes, Argentina Project Supervisor: Dr. Thomas R. Gillespie, Department of Environmental Sciences, Emory College Statement of Problem and Proposed Research Question Human population and development expansion has led to increased contact between humans and other animals, giving rise to a frequent exchange of infectious diseases. The constant transmission of pathogens and parasites are a part of the challenges facing conservation of biodiversity and global health worldwide. The most common intestinal parasite in humans across the world is Giardia (Thompson 2004). This genus of protozoan flagellates live in the intestinal tract of a broad range of hosts (Molina 2011; Thompson 2004) and causes a diarrheal disease called Giardiasis. Although Giardia was first observed in 1681 by Anton von Leeuwenhoek in his own stool and then described by Vilem Dusan Lambl and Alfred Giard in 1859 and 1895, respectively, it was not deemed to be pathogenic until the late 1900s after a series of outbreaks occurred in developing regions with poor sanitation practices (Erlandsen and Meyer 2013). The most common mode of disease transmission occurs through water. Humans are most likely to become infected through ingestion of cysts from contaminated water. Symptoms include variations of diarrhea and gastroenteritis (Sahagun et al. 2007); however, these symptoms are highly variable and are not always present in all infected individuals (Feng and Xiao 2011). Humans can also become infected through contact with cysts from animals (Hunter and Thompson 2005), demonstrating evidence that Giardia has zoonotic potential. Zoonosis occurs when a disease or infection has a natural transmission from vertebrate animals to humans and vice versa. Previous research has demonstrated a high prevalence of Giardia sp. in black and gold howler monkeys (Alouatta caraya) in a variety of sites in the Corrientes province of Argentina (Kowalewski et al. 2011), suggesting that these wild primates may serve as sources or sentinels of this potentially zoonotic parasite.

2 However, molecular characterization of Giardia sp. isolates from these monkeys is needed to determine the genotypes and therefore, the zoonotic potential of individual genotypes in this region. Thus, this project s question is, what is the distribution of genotypes of Giardia intestinalis found in the black and gold howler monkeys populations of the Corrientes province of Argentina? During the Summer 2016, I successfully used SIRE/Lester funding to travel to Corrientes, Argentina and collected fecal samples from Alouatta caraya. I sampled 34 different howler monkey groups 8 village groups, 12 rural groups, and 14 remote groups and a total of 130 individual samples. During the initial phase of my work in Fall 2016, I will characterize the genotypes of Giardia intestinalis found in the monkeys through molecular analysis. It is important to determine which genotypes exist in howler monkeys, a known reservoir of Giardia, to assess distinct modes of transmission and the possibility of zoonosis. Although there are 8 known genotypes, specifically genotypes Genotype A and Genotype B of Giardia intestinalis have been previously found in both monkeys and humans. The genotypes are characterized by differences in the sequencing of the beta-giardin, glutamate dehydrogenase, and triose phosphate isomerase genes (Sprong et al. 2009). Genotype A is marked by the WB and DH isolates whereas Genotype B is marked by the GS isolate (Adam et al. 2013). I hypothesize that the prevalence of Genotypes A and B will be positively correlated with proximity of howler monkeys to village communities. The measurement of genotypes A and B in the samples will be found in higher prevalences within rural and village communities compared to isolated groups due to a higher chance of human howler monkey interactions in those areas. Village howler monkey groups will have the highest prevalences of Genotypes A and B as a result of higher human-howler interactions. Since isolated communities have zero interaction with humans, there is an unlikely chance that those monkeys will have Genotypes A and B. This project is important because it targets two issues: conservation of biodiversity and global health. Giardia has a large network of hosts, including endangered species such as the black and gold howler monkeys. Developing nations are particularly susceptible to threats of infectious diseases as they usually include high levels of biodiversity and strong ecological factors for disease transmission. At the same time, these countries are industrializing and increasing their GDPs at the social and environmental cost of introducing anthropogenic factors of transmission. By understanding the prevalence and diversity of Giardia in this

3 system, it is possible to assess how this parasite is affecting conservation of the howler monkeys and human and primate health. Background Giardia cysts are environmentally resistant and are tolerant to chlorine disinfection as a strong outer shell protects the organism. Data show that there is an increase in cysts, and therefore, giardiasis, in the late summer (Molina 2011). Giardia goes through a two-stage life cycle: the cyst stage and the trophozoite stage. Once the infectious cysts are passed through feces and ingested by an organism, acids and salts in the stomach catalyze the release of trophozoites, which then attach to the intestinal walls (Monis 2003). There are currently 6 different species recognized in scientific literature: Giardia muris (rodents), Giardia ardeae (birds), Giardia psittaci (birds), Giardia microti (muskrats and voles), Giardia agilis (amphibians), and Giardia intestinalis (mammals); G. intestinalis is also known as G. duodenalis and G. lamblia (Monis 1999). Within the species G. intestinalis, eight genotypes or assemblages have been discovered thus far. Genotypes A (AI and AII) and B can infect both humans and animals, with the greatest risk of infection occurring in the AI subgroup (Thompson 2004). Some genotypes have specific host preferences while others have a broader range (Appelbee et. al 2005, Minivielle and Molina 2008). The black and gold howler monkeys, Alouatta caraya, are hosts to a variety of parasites and are considered to be sentinels of ecosystem health (Kowalewski and Gillespie 2009, Kowalewski et al. 2011). A high prevalence of Giardia in the howler monkeys across all types of habitats provides a strong indication that the monkeys are reservoirs of the parasite (Kowalewski et al. 2011). The non-human primates either carry Giardia as a natural component of their parasite communities, or they are being exposed to the waterborne parasite through interactions with humans, livestock, or companion animals. Howler monkeys are model organisms due to their ability to survive in fragmented forests that force them to increase their terrestrial travel, allowing more contact with other species (Kowalewski et al. 2011). They also drink water from streams, rivers, and lagoons, especially during dry seasons and when water holes in trees remain empty (Kowalewski et al. 2011), increasing their potential to contract and transmit Giardia. Previous studies of Giardia in Argentina have demonstrated a varied prevalence from 6-36% in humans, depending on local levels of sanitation and personal hygiene (Molina 2011). Human outbreaks of Giardia normally occur through

4 unfiltered surface and groundwater systems that have been affected by surface runoff or sewage discharges (Jakubowski 1984). These outbreaks seem to be most prevalent in developing countries where human population and industries are growing at the expense of clean water sanitation (human to human transmission) and where humans are encroaching on wildlife habitats (animal to human transmission). The intersections between human, wildlife, livestock, and pet cycles of Giardia are yet to be determined regarding the frequency and source of transmission (Thompson 2000). Research Methods Molecular analysis will be used to identify the genotypic variability of Giardia in black and gold howler monkeys. I will extract DNA from the samples and perform Polymerase Chain Reaction (PCR) and gel electrophoresis to ascertain which samples are positive for Giardia. I will then purify the samples and send them to Genewiz for genotype sequencing. Professional expertise on this protocol will be taken from previous successful protocols, such as Read et al and Salzer et al I will also ask Dr. Johanna Salzer from CDC about the specific molecular techniques for analyzing Giardia so that I can conduct my project as accurately as possible. The faculty members on my thesis committee Dr. Julie Clennon and Dr. Karen Levy as well as Dr. Thomas Gillespie will serve expert advisors on this project. Samples will undergo DNA extraction and molecular analysis to determine the genotypes of the howler monkey individuals. I will extract DNA from the RNAlater-preserved fecal samples using the FastDNA Spin Kit for Soil from MP Biomedicals LLC following the manufacturer s protocol. Next, I will amplify the glutamate dehydrogenase (gdh) gene using a semi-nested Polymerase Chain Reaction (PCR) method as described in Read et al This gene is a known amplification target for Giardia species and genotype identification (Caccio et al. 2005). The semi-nested PCR method is most effective in sequencing the different genotypes and sub-assemblages (Read et al 2004). A 432 base pair fragment of DNA will be amplified using the gdh external forward primer and the gdh internal reverse primer in the first reaction and the gdh internal forward primer and the gdh internal reverse primer in the secondary reaction (David et al. 2014). Each reaction will have a volume of 25uL; the gdh external forward primer and the gdh internal reverse primer will be used in the first PCR reaction with 1uL of DNA. Next, 1uL of the products from the first PCR reaction will be added to the gdh internal forward primer and the gdh internal reverse

5 primer in a secondary PCR mixture (Read et al. 2014). In all reactions, a positive control and a negative control will be included to confirm positive samples. The positive control is already available in the Gillespie Lab. PCR will be done with a thermal cycler in two cycles, as delineated in Read et al ul of PCR products will be run through 1% agarose gel electrophoresis that is stained with ethidium bromide. Band visualization by ultraviolet light will determine which samples are positive for Giardia. The Giardia-positive PCR products will be purified and submitted for sequencing at Genewiz LLC in both directions of DNA. During sequencing, reference isolates will be used to distinguish the assemblages for each positive sample. Reference isolates for genotypes (Read et al. 2004) Reference isolates Genotype A Bac1; p1c10; Bah 134; Bah45c5 Genotype B Bah 12; Bah 15c1; Bah34c8 Primers for PCR (Lasek-Nesselquist et al. 2010) Target Primer Sequence GDH Initial F1 CACGCTYCCVATGGGYGGCGGCAAG GDH Initial R1 ACCTCCTCRGMYGTCCACTGGAG GDH Nested F2 GTCTACTTCCTBGAGGAGATG GDH Nested R3 CTCGTTCTGRGTRGCGCARGGCATGATGC *instead of R1, can use R2 *instead of R3, can use R2 R2 = CTCAMGGARTTCTGRGACATCTC Genotype-specific primers (van Keulen et al. 2002) Genotype Primer Sequence A GGTGGATCCTGCCGGAGCG B GGTGGATCCTGCCGGAATC *the bolded dntps are genotype-specific for genotypes A and B Project Timeline

6 Project Activity Sept. Oct. Nov. Dec. Jan. Feb. Samples arrive DNA extraction of all samples PCR and Gel electrophoresis Sequencing Project Goals This project will create a baseline assessment of the prevalence of Giardia intestinalis in black and gold howler monkeys in Corrientes, Argentina and will also serve as a pilot for my master s thesis for the Environmental Science 4+1 BS/MS program, which will evaluate the distribution of Giardia in Corrientes, Argentina among humans, black and gold howler monkeys, and companion animals. Studying the interactions among humans, howler monkeys and domesticated animals, such as pets and livestock, can give insight on the health of the individuals and the health of the system as a whole. This project will be completed in four parts: Summer 2016, Fall 2016, Summer 2017, and Fall I have already collected howler monkey samples in Corrientes, Argentina as SIRE funds have allowed me to be successful in my first field season; I would like to use Fall 2016 Lester funding to do molecular analysis on the 130 samples from the summer collection to establish the distribution of Giardia genotypes in howler monkeys. Since my budget is over the Lester award limit, a previous Lester award and the Gillespie Lab will fund the rest of the money required for the project. In my second field season of this project in Summer 2017, I hope to pinpoint the source of Giardia and sample humans and domestic animals as well as re-sample Giardia-positive groups of howler monkeys. I also hope to assess risk factors for contracting the parasite in the system of Corrientes. If genotypes found in howler monkeys are also found in humans or other animals within a singular geographical location, then there is a strong potential that Giardia intestinalis at that site is zoonotic. Summer 2017 and Fall 2017 will be devoted to creating a larger and more diverse sample set and evaluating the samples to determine if and how this system

7 encourages zoonosis of Giardia. The final product of the project will be a master s thesis written during the Spring of However, I would like to submit a preliminary paper to undergraduate journals based on my initial season of data collection and molecular analysis in Spring During the course of this project, I will be able to utilize my prior knowledge gained from working on microscopy and molecular analysis in the Gillespie labs; however, I will also be able to develop new skills, such as tracking animals and sample collection. I have the opportunity to link the fieldwork done during the summer to my future coursework in disease ecology and spatial analysis. I want to apply remote sensing techniques to the data collected to understand possible environmental factors that relate to the patterns of infection (i.e. movement of water or close contact between humans and animals due to habitat loss). Since Giardiasis is a common waterborne disease that affects humans and animals equally, I would like to determine the source direct or indirect of the parasite and its interactions within the system. I hope my findings will contribute to a greater understanding of the effect of Giardia in Corrientes. References Adam, R.D., Dahlstrom, E.W., Martens, C.A., Bruno, D.P., Barbian, K.D., Ricklefs, S.M., Hernandez, M.M., Narla, N.P., Patel, R.B., Porcella, S.F., Nash, T.E Genome sequencing of Giardia lamblia genotypes A2 and B isolates (DH and GS) and comparative analysis with the genomes of genotypes A1 and E (WB and Pig). Genome Biol Evol 5: Appelbee A. J., Thompson, R.C.A., Olson M.E Giardia and Cryptosporidium in mammalian wildlife-current status and future needs. Trends in Parasitology 21: Caccio, S.M. Thompson, R.C.A., McLauchlin, J., Smith, H.V Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21: David, E.B., Patti, M., Coradi, S.T., Oliveira-Sequeira, T.C.G., Ribolla, P.E.M., Guimaraes, S Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Revista do Instituto de Medicina Tropical de Sao Paulo 56:49-54.

8 Erlandsen, S.L., Meyer, E.A Giardia and Giardiasis: Biology, Pathogenesis, and Epidemiology. Springer Science & Business Media. Feng, Y., and L. Xiao Zoonotic Potential and Molecular Epidemiology of Giardia Species and Giardiasis. Clinical Microbiology Reviews 24: Hunter, P.R., Thompson, R.C.A The zoonotic transmission of Giardia and Cryptosporidium. International Journal for Parasitology. 35: Jakubowski, W Detection of Giardia Cysts in Drinking Water. Giardia and Giardiasis Kowalewski, M., Salzer, J.S., Deutsch, J.C., Raño, M., Kuhlenschmidy, M.S., Gillespie, T.R Black and Gold Howler Monkeys (Alouatta caraya) as Sentinels of Ecosystem Health: Patterns of Zoonotic Protozoa Infection Relative to Degree of Human-Primate Contact. American Journal of Primatology 73: Lasek-Nesselquist, E., Welch, D.M., Sogin, M.L The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. International Journal for Parasitology 40: Minvielle, M. C., and Molina, N.B First Genotyping of Giardia lamblia from Human and Animal Feces in Argentina, South America. Mem. Inst. Oswaldo Cruz Memórias Do Instituto Oswaldo Cruz 103: Molina, N High Prevalences of Infection with Giardia intestinalis Genotype B among Children in Urban and Rural Areas of Argentina. Annals of Tropical Medicine & Parasitology 105: Monis, P. T., and R. H. Andrews Molecular Systematics of the Parasitic Protozoan Giardia intestinalis. Molecular Biology and Evolution 16: Monis, P.T, and R.C.A Thompson Cryptosporidium and Giardia- Zoonoses: Fact or Fiction? Infection, Genetics and Evolution 3:

9 Read, C.M., Monis, P.T., Thompson, R.C.A Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR- RFLP. Infection, Genetics and Evolution 4: Sahagun, J., Clavel, A., Goñi, P., Seral, C., Llorente, M.T., Castillo, F.J., Capilla, S., Arias, A., Gomez-Lus, R Correlation between the presence of symptoms and the Giardia duodenalis genotype. European Journal of Clinical Microbiology and Infectious Diseases. 27: Salzer, J. S., Rwego, I.B., Goldberg, T.L., Kuhlenschmidt, M.S., Gillespie, T.R Giardia sp. and Cryptosporidium sp. Infections in Primates in Fragmented and Undisturbed Forest in Western Uganda. Journal of Parasitology. 93: Sprong, H., Caccio, S.M., van der Giessen, J.W.B Identification of Zoonotic Genotypes of Giardia duodenalis. PLoS Neglected Tropical Diseases 3:558. Thompson, R. C. Andrew Giardiasis as a Re-emerging Infectious Disease and Its Zoonotic Potential. International Journal for Parasitology 30: Thompson, R.C. A The Zoonotic Significance and Molecular Epidemiology of Giardia and Giardiasis. Veterinary Parasitology 126: van Keulen, H., Macechko, P.T., Wade, S., Schaaf, S., Wallis, P.M., Erlandsen, S.L Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis. Veterinary Parasitology 108: