The Effect of Chemicals on the Viability of Schistosoma mansoni and Schistosoma haematobium. By Anne Frelick A thesis submitted to the USF Honors College in conformity with the requirements for the degree of Honors University of South Florida Tampa, FL May 2012 Thesis Director: Jason Rohr, Ph.D. Assistant Professor, Department of Integrative Biology College of Arts and Sciences 1
Acknowledgments First and foremost, I would like to express my sincere and utmost gratitude to my Thesis Director, Dr. Jason Rohr. Dr. Rohr always made time for guidance and to assist me with my experiments. He was extremely helpful, supportive and created a great learning environment. I would like to give a special thanks to Neal Halstead, PhD candidate, who directed my research and guided me through the processes. Thank you, Anne Frelick 2
Table of Contents Abstract 4 I. Introduction 5 A. Background 5 I. Schistosomiasis: Important Considerations 5 II. Historical Background 6 III. Basic Life Cycle 7 IV. Species of Worldwide Significance 11 B. Objectives and Hypotheses 12 II. Methods 13 III. Results 15 IV. Discussion 18 V. References 20 3
Abstract Schistosomiasis is a parasitic disease that is most commonly found in developing countries in Africa, Asia, and South America. Schistosomiasis is caused by several different species of trematodes, which are a parasitic worm that use snails as the primary host. These infected snails are often found in freshwater areas like lakes, rivers, and ponds, which are also used as a primary source of water for many people in developing countries. Once the parasite has infected the secondary host, the human, it can damage internal organs, impair growth and cognitive development in children, and cause bladder cancer in adults. Herein, we investigate the effects of the insecticide, Chlorpyrifos, the herbicide, Atrazine, and inorganic fertilizers on two species of human trematodes, Schistosoma mansoni and Schistosome haematobium. Specifically, the hatching and survival rates of the miracidial stages of the species will be observed in addition to the ability to infect snails. Collectively, our results demonstrate that the combination of the chemicals can prevent hatching, kill the miricidia, or lower the ability to infect, relative to the absence of the chemicals. 4
I. Introduction A. Background I. Schistosomiasis: Important Considerations The parasitic helminths of the Schistosomatidae family are estimated to infect 200 to 300 million people worldwide thus representing a considerable portion of the burden of illness on humans. With an increase in global human population came an increase in the difference in living conditions of the poorest and richest nations. This vast difference in standard of living led to the United Nations adopting Millennium Development Goals in order to solve these disparities in wealth and health (15). One of the goals was to increase agricultural production with industrial and chemical means. Agrochemicals have had a number of direct and indirect effects on aquatic communities including the viability of Schistosoma eggs and their miracidial survival. It is important to note that macroparasites, such as schistosomes, do not multiply within the definitive host (1-4). Increases in the population of the pathogen occur only in the snail intermediate host while adult worms in mammals are unable to expand their numbers without acquisition of new infections from the environment. Another important consideration is the host-parasite relationship. The number of parasites in the host correlates to the severity of the disease. There are other factors to be considered in the infection of the host, such as the genetics of the parasite or host, nutrition of the host, and the role of chemicals in the environment. It is clear that the infection and disease in schistosomiasis is a multi-factorial process. 5
II. Historical Background Schistosomiasis is a helminth disease of human beings and animals in many subtropical and tropical regions of the world. The infection is sometimes called snail fever due to the freshwater snails that serve as intermediate hosts in the life cycle of the parasite. It has been recorded that schistosomes have plagued humans for at least four millennia. The Papyrus of Kuhun hieroglyphics are the oldest known medical text of any kind and dates back to 1800 BCE. The translation reveals what looks like references to hematuria, which is a clinical sign of urinary schistosomiasis (5). Schistosome eggs have been found in Egyptian mummies from 3000 BCE and Chinese mummies from 2100 BCE (6). The modern study of schistosomiasis began in the nineteenth century with the descriptions of the Katayama syndrome during the early stages of infection in Japan and descriptions of adult worms found in Egypt. After sixty years of debate, it was proven that aquatic snails played a role in the life cycle and that two distinct species occurred in Egypt, Schistosoma mansoni and S. haematobium, along with a single species, S. japonicum, in Japan (7). Once the life cycle was clear, it was possible to create effective management programs. These management programs have had several setbacks such as cost and national perceptions of the importance of the disease. Aquatic snail habitats continue to increase with the construction agricultural irrigation ditches, drinking water reservoirs, and reservoirs formed behind hydroelectric dams. There are five species of human-infecting schistosomes. S. japonicum, S. mansoni, S. haematobium, S. intercalatum, and S. mekongi all give rise to serious disease 6
within humans. The latter two species have more restricted distributions thus more focus is placed on S. japonicum, S. mansoni, and S. haematobium. III. Basic Life Cycles Figure 1. Life cycle of schistosomes (8). Schistosomes are digenetic trematodes of the family Schistosomatidae. The adults are dioecious with separate sexes. A dorsoventrally flattened male clasps a cylindrical female in a ventral groove. The gynaecophoric canal, otherwise known as the schist, is created by the infolding of the male s lateral margins, so that the pair assumes a nematoid, wormlike form that is best suited to life within the vascular system. 7
Both females and males have oral and ventral suckers but it is the male that has sufficient strength to hold firmly onto the blood vessel walls (9). All schistosomes essentially follow the same complex life cycle. Juvenile flukes develop rapidly in the nutrient-rich environment of the liver s hepatic sinusoids. As they reach maturity, the worms pair up and leave the liver. The paired worms are carried by the male against the bloodflow of the hepatic portal vein to either the proximal branches of the hepatic portal vein around the intestine or to the veins around the bladder. Eggs are then laid within these veins and must travel to enter the lumen of the bladder or intestine and from that point they are voided in the urine or feces. The eggs must reach freshwater in order to hatch and release the miracidium (9). If the miracidia are successful in penetrating a compatible freshwater snail, they continue development and begin the process of asexual multiplication within the intermediate host. Once penetration has occurred, the miracidium changes into a primary sporocyst then multiple secondary sporocysts develop over a period of one to two weeks. Once developed and released, the secondary sporocysts travel to the nutrient-rich environment of the snail s digestive glands. Asexual multiplication takes place again with the secondary sporocysts creating infective offspring called cercariae (9). The cercariae exit the snail and continue the life cycle if they successfully penetrate the healthy skin of a definitive host, usually a human. The parasite secretes enzymes that break down the skin's protein to enable penetration of the cercarial head through the skin. The stable temperatures of the warm-blooded definitive hosts allows for stable development times. Within the skin, the head of the cerariae transform into an endoparasitic larva, the schistosomula, and enters the vascular system. The newly 8
transformed schistosomulae may remain in the skin for 2 days before finding a postcapillary venule; from here the schistosomulae travel to the lungs where they undergo further developments necessary for movement to the liver. Eight to ten days after penetration of the skin, the parasites travel to the liver sinusoids. From this point, there is variation in timelines amongst the different species. A comparison of all five schistosoma species is listed in Table 1. 9
Table 1.Comparison of Schistosoma species infecting human beings (9). Schistosoma japonicum migrates more quickly than S. mansoni, and usually reaches the liver within 8 days of penetration. Juvenile S. mansoni and S. japonicum worms develop an oral sucker in the liver and feed on red blood cells. Worm pairs of S. 10
mansoni and S. japonicum travel to the rectal or mesenteric veins. S. haematobium schistosomulae migrate from the liver to the perivesical venous plexus of the bladder, ureters, and kidneys through the hemorrhoidal plexus. The parasites reach maturity at six to eight weeks, at which time they start producing eggs. Adult S. mansoni pairs residing in the mesenteric vessels can produce up to 300 eggs per day. S. japonicum can produce up to 3000 eggs per day. Many of the eggs pass through the walls of the blood vessels through the intestinal wall, to be passed out of the body in feces. S. haematobium eggs pass through the ureteral or bladder wall and into the urine. Mature eggs are capable of infecting the digestive tract. Up to half the eggs released by the worm pairs become trapped in the mesenteric veins, or will be washed back into the liver, where they will become lodged. They can live in the body from an average of four years and up to twenty years (9). IV. Species of Worldwide Significance This research is focused on the two species of schistosomes that cause the most destruction worldwide. S. mansoni is the species that is the most widespread of the schistosomes infecting humans. It is the cause of intestinal schistosomiasis found in over 83 million people and is present in 54 countries (10). It is predominant in Africa, South America, the Caribbean, and the Middle East regions. The hepatic (intestinal) form of schistosomiasis is the most damaging to the human body. Granulomas are formed around the lodged eggs in the tissue, which give rise to fibrosis of the liver. The number and location of eggs lodged in the tissue influences the symptoms and progression of the disease. Fibrosis of organs is most often irreversible and can lead to organ failure (14). 11
Schistosoma haematobium is the cause of urinary schistosomiasis and can be found in Africa, the adjacent islands in the Atlantic and Indian Oceans, and Southwest Asia. The dispersal of eggs from the body is accompanied by blood loss that stains the urine red. This hematuria is a characteristic symptom of the infection (11). The majority of infections are asymptomatic. However, acute schistosomiasis (Katayama's fever) may occur weeks after the initial infection, particularly by S. mansoni. Symptoms consist of fever, cough, abdominal pain, diarrhea, hepatosplenomegaly, and eosinophilia. Granulomatous lesions around lodged eggs in the spinal cord from S. mansoni and S. haematobium infections can result in transverse myelitis with flaccid paraplegia (11). Continuing infection may bring about granulomatous reactions and fibrosis in the affected organs, which may result in colonic polyposis with bloody diarrhea (S. mansoni); portal hypertension with hematemesis and splenomegaly (S. mansoni); cystitis and ureteritis (S. haematobium) with hematuria, which can progress to bladder cancer; pulmonary hypertension (S. mansoni); glomerulonephritis; and central nervous system lesions (14). B. Objectives and Hypotheses The objective of this experiment is to observe the effects of the insecticide, Chlorpyrifos, the herbicide, Atrazine, and inorganic fertilizer (nitrate and phosphate) on S. mansoni and S. haematobium egg viability and the hatching and survival rates of the miracidial stages. The two different species were incubated in the livers of mice and the intestines of hamsters. The eggs were collected from the mammals organs and placed in saline to prevent hatching. In the lab, the trematode eggs were exposed to the chemicals 12
alone and in combination, in 24 well culture plates at their estimated environmental concentrations calculated using US EPA software. I then quantified how many miracidia hatched from the eggs and how long the miracidia survived. It is hypothesized that egg viability and miracidial survival will respond to the chemical mixtures in a manner consistent with each chemical in isolation. II. Methods Egg collection process Mature S. mansoni eggs were harvested from the livers of Swiss-Webster mice while mature S. haematobium eggs were harvested from the bladders of Siberian Golden hamsters. The tissues were then homogenized in a 1.4% sodium chloride solution and eggs were passed through a series of sieves and collected on a U.S.A. Standard Sieve of 45 microns. The eggs were then maintained in a 1.4% saline solution until trials were begun. Before the trials, a subsample of mature eggs was placed in fresh artificial spring water to determine initial successful hatching rates. Experimental design The insecticide, Chlorpyrifos, the herbicide, Atrazine, and fertilizer consisting of nitrate and phosphate were researched and the estimated environmental concentrations (EEC) was calculated using the US EPA GENEEC-v.2.0 software. In total, twelve treatments were applied beginning with each individual chemical at 1x and 2x the EEC for a total of six treatments. Three treatments consisted of pair-wise combinations (1x EEC) of each chemical. One treatment consisted of a three-chemical combination at 2/3x 13
EEC of each chemical. Lastly, there were individual treatments for a water control and solvent (acetone) control. All chemicals were mixed from a 10x EEC stock solution. For administrating the treatments, a 24 well tissue culture plate was used. Each well contained a final volume of three milliliters. All wells received 1900 microliters of artificial spring water, 500 microliters of eggs, and 600 microliters of the chemical treatment. All 1x treatments received 300 microliters of 10x EEC stock and 300 microliters of 10x EEC solvent stock. All of the 2x EEC treatments received 600 microliters of 10x EEC stock. The pair-wise mixtures received 300 microliters of each chemical of 10x EEC stock. The three-chemical combination received 200 microliters of each stock chemical. The solvent control received 600 microliters of 10x EEC solvent. The water control received 600 microliters of artificial spring water. Egg Viability Experiment Each chemical was mixed in the wells. Three 24 well tissue culture plates were used. These 24 well plates equaled six replicates of each treatment. After the chemicals were added to the wells, 500 microliters of mature eggs in 1.4% saline were added. Based on the initial hatching rates, a target of ten miracidia was established. After one hour, the number of miracidia swimming in each well was counted to determine the hatching success rate. Once counted, Lugol s Iodine was added to the wells in order to stain and preserve the eggs. Miracidia Survival Experiment 1900 microliters of artificial spring water and 500 microliters of eggs were added 14
to each well of the plate. After one hour, the number of swimming miracidia was counted. Chemical treatments were then added to each well. At that point, the number of swimming miracidia was counted every hour for eight hours. Once counted, Lugol s Iodine was added to the wells in order to stain and preserve the eggs. Statistical Analyses We tested for the main effect of the eleven treatments, blocked by the 24-well plate, and weighted the analyses by the number of eggs placed in each well. If there were any main effects of treatment, we conducted Fisher s Least Significant Difference Posthoc tests to determine which treatments were different from one another. Statistical analyses were conducted with Statistica 9.1 (Statsoft, Tulsa, OK). III. Results There was often a significant effect of block (24-well plate), but there was no effect of treatments on Schistosoma mansoni miracidial survival (Fig. 1) or egg viability (Fig. 2) or on S. haematobium miracidial survival (Fig. 3) or egg viability (Fig. 4; all p>0.05). Legend for Figures 1-4: Treatment Name H2O Treatment Components Artificial spring water S A1x 10x EEC solvent (acetone) 1x EEC Atrazine 15
A2x F1x F2x C1x C2x AF AC CF ACF 2x EEC Atrazine 1x EEC Fertilizer 2x EEC Fertilizer 1x EEC Chlorpyrifos 2x EEC Chlorpyrifos 1x EEC Atrazine + 1x EEC Fertilizer 1x EEC Atrazine + 1x EEC Chlorpyrifos 1x EEC Chlorpyrifos + 1x EEC Fertilizer 2/3x EEC Atrazine + 2/3x EEC Chlorpyrifos + 2/3x EEC Fertilizer Figure 1. Mean (± 1 SE) effects of treatments on the miracidial survival of Schistosoma mansoni. 16
Figure 2. Mean (± 1 SE) effects of treatments on the egg viability of Schistosoma mansoni Figure 3. Mean (± 1 SE) effects of treatments on the miracidial survival of Schistosoma haematobium 17
Figure 4. Mean (± 1 SE) effects of treatments on the egg viability of Schistosoma haematobium IV. Discussion Through our research, we conclude that atrazine, chlorpyrifos, and inorganic fertilizers do not affect miracidial hatching or survival. Previous experiments conducted by other investigators have shown that Atrazine, Chlorpyrifos, and inorganic fertilizers increased the overall number of snails and infected snails. These results suggest that the chemicals are not having a major impact on the schistosoma eggs or miracidia. If the chemicals were having an impact then the number of infected snails would be expected to be less. Our research reinforces these previous findings. While it seems counterintuitive that such chemicals would actually increase the number of snails, the mechanisms in which this occurs sufficiently explain this phenomenon. The insecticides altered the top-down regulation of aquatic food webs by reducing the number of the consumers of the snails. The inorganic fertilizers, containing 18
nitrogen and phosphorus, increased the primary productivity thus increasing the number of infected snails by increasing the amount of periphyton, snail food. The herbicide, Atrazine, caused a decrease in phytoplankton, which led to an increase in light penetration and an increase in periphyton abundance. The increase in periphyton, the food for snails, led to an increase in snail abundance. This research was concerned with just three agrochemicals however there are thousands of chemicals being used by the agricultural community today. In isolation and combination, these chemicals have a wide variety of effects on the aquatic community. Consequently, we recommend further research on the effects of other agrochemicals on human pathogens and firewater communities. This research will lead to a more comprehensive understanding of the effects of agrochemicals on disease and freshwater ecosystems. Future experiments should attempt to identify agrochemicals that might maximize agricultural productivity but do not increase human disease risk. VI. References 1. Anderson RM and May RM (1985). Adv. Parasitol. 24: 1. 2. Anderson RM and May RM (1991). Infectious Diseases of Humans: Dynamics and Control (Oxford University Press, New York), 538 pp. 3. Mahmoud AAF (1989). Science 246: 1015. 4. Fulford AJC, Butterworth AE, Ouma JH and Sturrock RF (1995). Parasitology 10: 307. 19
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