The Chinese liver fluke Clonorchis sinensis: an environmental investigation into a

foodborne parasite

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By

Gary Lee Klase

Graduate Program in Public Health

The Ohio State University

2013

Master's Examination Committee:

Dr. Jiyoung Lee, Advisor

Dr. Song Liang

Dr. J. Mac Crawford

Copyright by

Gary Lee Klase

2013

Abstract

The Chinese liver fluke Clonorchis sinensis is a foodborne parasitic trematode transmitted through the consumption of raw or undercooked fish, and is responsible for a substantial burden of morbidity and mortality in China and other Asian countries. The present study includes two investigations into environmental factors related to clonorchiasis transmission in China. In the first study, a field investigation was carried out in Guangdong province, China to investigate the relationship between water quality factors and the abundance and infection status of the snail intermediate of C. sinensis in rural Chinese fishponds. No host snails were found during the study. From the microbial source tracking analysis it was found that a large proportion of the water samples from fishponds showed evidence of human fecal contamination (84%), pig fecal contamination

(40%), and antibiotic resistant bacteria (80%), raising concerns about potential public health risks related to these fishponds. In the second study, the results of a clonorchiasis prevalence study conducted by the Jiangmen City Center for Disease Control in

Guangdong province were combined with satellite imagery and geographic information systems (GIS) techniques to investigate the relationship between the proportion of water in the nearby landscape and the risk of clonorchiasis infection. It was found that proximity to water in the landscape, as measured by the proportion of water in a 4 km radius around each village, was a strong risk factor for human infection of liver flukes.

Using logistic regression, it was found that doubling the proportion of water around a ii village was associated with an odds ratio of 4.6 of increasing the liver fluke infection (p <

0.001).

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Dedication

This thesis is dedicated to the ideals of wise use and ecological engineering:

Abundance through harmony

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Acknowledgments

Many people were instrumental in supporting the work of this thesis.

I would like to thank Dr. Song Liang for initiating and supporting my research in China,

and Dr. Nancai Zheng and Bonian Liang at the Jiangmen City CDC for their substantial support of this research.

I would like to thank Dr. Jiyoung Lee and her lab members, Seungjun Lee, Feng Zhang, and Dr. Kuo Tseng, for their support in the laboratory.

I would like to thank Dr. John Mac Crawford for the much-needed reminders that nothing is ever perfect, and as such, I should not worry so much when things aren’t perfect.

I would like to thank the Department of Chemistry and Biochemistry at Ohio State for entrusting me with the education of 150 students in the laboratory over the course of two years, which has been one of the most rewarding aspects of my academic career.

And most of all, I would like to thank my family. I have achieved what I have achieved because of their support. v

Vita

1999...... Hoover High School, North Canton, Ohio

2007...... B.A. English, Otterbein College

2009...... A.S. Environment and Natural Resources,

The Ohio State University

2011...... B.S. Environmental Science,

The Ohio State University

2012 to present ...... Graduate Teaching Associate, Department

of Chemistry, The Ohio State University

Fields of Study

Major Field: Public Health

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1: Introduction ...... 1

1.1 Clonorchis sinensis ...... 2

1.2 Epidemiology and global burden ...... 3

1.3 Ecology...... 5

1.4 Control Strategies ...... 7

1.4.1 Treating human and reservoir hosts ...... 7

1.4.2 Health education ...... 8

1.4.3 Snail control ...... 9

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1.4.4 Treating feces ...... 10

1.4.5 Hazard Analysis and Critical Control Points ...... 11

1.4.6 Control Summary ...... 12

1.5 Ecological approach to parasite control ...... 14

Chapter 2: Environmental investigation of water quality and health risks in an endemic region of Clonorchis sinensis in Guandgong province, China ...... 17

2.1 Introduction ...... 17

2.2 Methods ...... 19

2.2.1 Study sites ...... 19

2.2.2 Snail sampling ...... 21

2.2.3 Water sampling ...... 22

2.2.4 Physiochemical water quality parameters ...... 22

2.2.5 Fecal indicators ...... 22

2.2.6 Water filtration ...... 23

2.2.7 DNA extraction...... 24

2.2.8 Microbial source tracking and antibiotic resistance ...... 25

2.2.9 Quantitative polymerase chain reaction (qPCR) ...... 25

2.2.10 Chlorophyll-a measurement ...... 26

2.2.11 Nutrient measurement ...... 26

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2.2.12 Statistical analysis...... 27

2.3 Results ...... 28

2.3.1 Snail sampling ...... 28

2.3.2 Physiochemical parameters ...... 28

2.3.3 Fecal indicators ...... 30

2.3.4 Microbial source tracking (human, pig), and antibiotic resistance ...... 32

2.3.5 Eutrophic status ...... 34

2.3.6 Statistical analyses ...... 34

2.4 Discussion ...... 37

2.4.1 Fecal contamination ...... 37

2.4.2 Microbial source tracking and antibiotic resistance ...... 38

2.4.3 Eutrophic status ...... 39

2.4.4 Physiochemical parameters ...... 41

2.4.5 Snail sampling ...... 42

2.5 Conclusion ...... 42

Chapter 3: Epidemiology and spatial analysis of the liver fluke Clonorchis sinensis in

Guangdong province, China ...... 44

3.1 Introduction ...... 44

3.2 Materials and Methods ...... 46

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3.2.1 Epidemiological survey ...... 46

3.2.2 Cluster analysis ...... 46

3.2.3 Remote sensing and Normalized Differential Water Index ...... 47

3.2.4 Statistical analysis...... 50

3.3 Results ...... 50

3.3.1 Clonorchis sinensis infection ...... 50

3.3.2 Cluster analysis ...... 53

3.3.3 Remote sensing and NDWI ...... 53

3.3.4 Logistic regression model ...... 56

3.4 Discussion ...... 59

Chapter 4: Conclusions ...... 62

4.1 Summary ...... 62

4.2 Future research ...... 62

References ...... 65

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List of Tables

Table 1: A summary of the physiochemical measurements ...... 29

Table 2: Microbial source tracking and antibiotic resistance results ...... 33

Table 3: Correlations between dissolved oxygen and both temperature and pH ...... 35

Table 4: Correlations between chlorophyll-a, conductivity, and secchi depth ...... 36

Table 5: Correlation between coliforms and E. coli ...... 36

Table 6: Survey summary by gender ...... 50

Table 7: Survey summary by age...... 51

Table 8: Survey summary by village ...... 52

Table 9: The regression output ...... 58

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List of Figures

Figure 1: The location of the study sites in Guangedong province, China ...... 20

Figure 2: The location of the study sites in reference to Jiangmen city ...... 20

Figure 3: E. coli concentrations and relevant guidelines ...... 31

Figure 4: Chlorophyll-a concentrations by pond ...... 34

Figure 5: A sample scene, showing: original satellite image, NDWI-index image, binary water map ...... 49

Figure 6: A sample scene used to subjectively validate the NDWI procedure ...... 55

Figure 7: A plot of prevalence vs. percent water cover in 4 km radius by village ...... 56

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Chapter 1: Introduction

In the current study, several health risks associated with rural integrated aquaculture operations in southern China are investigated, with a primary focus on the ecology of the Chinese liver fluke Clonorchis sinensis. Integrated aquaculture is an agricultural management practice in which resource efficiency is maximized by utilizing the wastes from one process as inputs for other processes (Prein, 2002). For example, crop residues and human or animal excrement are often added to fish ponds as food or fertilizer, recycling nutrients among multiple production processes and maximizing the gain for the farmer.

Rural integrated aquaculture has substantial benefits and drawbacks. These operations produce high quality protein, economic benefits, and potentially do so in a sustainable manner due to the efficient use of natural resources (Prein, 2002). However, there are public health risks associated with integrated aquaculture, particularly when excrement is used as a fertilizer. Both fishborne zoonotic trematodes (FZTs) and antibiotic resistant bacteria have been identified as potential health risks of integrated aquaculture in Asia (Su, 2011; Boerlage, 2013).

In this study, we investigated the Chinese liver fluke Clonorchis sinensis in rural fishponds in the Jiangmen city region of Guangdong province, China. During this investigation, we also found evidence of antibiotic resistant bacteria in a high proportion of the fishponds studied. Our goal was to better understand the ecology of the Chinese 1 liver fluke and the role played by aquaculture ponds in the transmission of the disease to humans in this endemic region. Ponds were randomly sampled and no distinction was made between integrated and non-integrated aquaculture, but we note that the health risks identified are related to the high levels of fecal contamination detected in the fishponds and suggest that integrated aquaculture ponds in particular, while important for their role in resource-efficient food production, must be managed with great care to minimize the risk to the associated human population.

1.1 Clonorchis sinensis

Liver flukes of the family Opisthorchidae (class Trematoda), particularly the species Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis, pose a major public health burden in Southeast Asia. Humans are infected through consumption of freshwater aquatic products (primarily fish) contaminated by the pathogens. It is estimated that 35 to 45 million individuals are infected with liver flukes—with another

600 million at risk (Sripa, 2008; Lim, 2011)—and chronic infections can lead to a range of liver and bile duct conditions, including cholangiocarcinoma, a malignant and generally fatal cancer (Lim, 2011). Due to the strong association of liver fluke infections and cholangiocarcinoma, the International Agency for Research on Cancer (IARC) has recently classified C. sinensis and O. viverrini as Group 1 known human carcinogens

(Hong & Fang, 2012).

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1.2 Epidemiology and global burden

Clonorchiasis, the infection caused by C. sinensis, poses a great burden of

morbidity to the population of Southeast Asia. C. sinensis is known to infect humans in

China, Korea, Vietnam, Taiwan, and Russia (Hong & Fang, 2012). Within these

countries, the distribution of Clonorchiasis is known to be very heterogeneous. For

example, a survey of Chinese infection rates between 2001 and 2004 reported an

estimated national infection prevalence of 0.58%. Another study of 27 provinces in

China reported an overall infection prevalence of 2.4%, with prevalence of infection

ranging from no detected human infections in eight provinces, less than 4% prevalence in

16 provinces, 4 to 10% in two provinces (including 9.8% in Guangxi), and 16.4% in

Guangdong province (Hong & Fang 2012). Within Guangdong province, Lun et al

(2005) report infection rates as high as 85% in some communities, with infection rates being particularly high near streams connected to the Pearl and Han rivers.

Showing a similar heterogeneity, Hong & Fang (2012) note a survey reported by

Kim et al. (2004), revealing a nationwide prevalence of 2.9% in Korea, with some endemic regions showing infection rates above 10%. Qian et al. (2012) report infection rates ranging from 0.2% to 40.1% for communities in northern Vietnam, although there are some concerns given regarding potential misclassification of disease for the surveys reviewed. Surveys reported by Figurnov et al. (2002) and Rim (2005), respectively, report a similar heterogeneity of infection in Russia and Taiwan. Hong & Fang (2012) note that infections tend to cluster in river basins, with substantially lower rates occurring in upland regions.

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Hong & Fang (2012) put the number of infected globally at 15 to 20 million individuals, with another 200 million at risk. The bulk of these infections occur in China, with an estimated 12.5 million cases of Clonorchiasis, with another 1.3 million cases in

Korea, 1 million or more in Russia and Vietnam, and an unknown number of cases in

Taiwan. A review by Lun et al. (2005) puts the global estimate higher, at approximately

35 million cases, with 15 million in China.

There is evidence to indicate that the prevalence of clonorchiasis is rising in

China (Lun et al., 2005; Qian et al., 2012), possibly having doubled between the early

1990s and 2004. The authors suggest this is due to an increase in the consumption of raw freshwater fish, which is considered a delicacy in some regions. In Korea, a substantial decrease in infection prevalence was reported between 1971 and 1997 (from 4.6% to

1.4%), followed by an increase (to 2.4%) by 2004.

Clonorchiasis is a serious medical concern, although this may not be apparent at first glance—even to the infected individuals. In fact, a majority of the infected may show mild or no symptoms to indicate their disease (Hong & Fang, 2012; Lun et al.,

2005). However, C. sinensis is a long-lived parasite; infections can last decades (Rim,

2005; Lun et al., 2005), and as the disease progresses serious symptoms and complications occur.

After a human ingests food contaminated with the liver fluke, the juvenile flukes migrate toward the bile ducts, to which they attach themselves (Lun et al., 2005). The flukes can also migrate to the pancreatic ducts, and occasionally the gall bladder, duodenum, and stomach. Common symptoms of infections include abdominal

4 discomfort, diarrhea, or malaise. As infections intensify, symptoms can progress to fever, weight loss, fatigue, or abdominal distension. Chronic cases may progress to include hypertension, jaundice, liver damage, and a range of other conditions, and ultimately, death may result. In addition, infection may result in cholangiocarcinoma, a rare but lethal form of cancer, with an estimated 5,000 cases occurring annually as a result of Clonorchiasis (Qian et al, 2012). Unfortunately, the lack of serious symptoms among the majority of the infected individuals may lead to an underestimation of the severity of the disease, both among the infected and among national and international policymakers (Sripa, 2008).

1.3 Ecology

Aquatic ecology lies at the heart of C. sinensis transmission, and as such it is critically important to recognize and integrate into any effective control program. Like other foodborne trematodes, C. sinensis has a complex life cycle including definitive hosts (in this case, humans and other fish-eating mammals), as well as first and second intermediate hosts (WHO, 2010).

The C. sinensis life cycle begins as eggs are passed from the definitive

(mammalian) host via the feces. The egg must be washed into a freshwater environment, where it remains dormant until it is consumed by an aquatic snail. C. sinensis is highly selective of its first intermediate host, and the egg will only hatch and develop if the consuming snail is of the appropriate species (Qian et al., 2012), including about eight species of aquatic snails in China from the order Mesogastropoda, genera Alocinma,

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Parafossarulus, Bithynia, Semisulcospira, Melanoides, and Assiminea, with the major

hosts considered to be A. longicornis, P. striatulus, and B. fuchsianus (Lun et al, 2005).

If the egg is consumed by the appropriate snail, the C. sinensis miracidium

hatches and parasitizes the host snail, eventually developing into sporocysts, then redia,

then free-swimming cercaria, which will burrow out of the snail host and swim in search

of a fish host. While C. sinensis is highly selective of its first intermediate host, this is not true of the second intermediate host; Qian et al. (2012) note that there are at least 132

species of fish across 46 genera and 11 families, as well as three species of shrimp, that

can serve as the second intermediate host of C. sinensis.

If the free-swimming cercaria is able to locate a fish host, it will burrow itself into

the fish’s flesh and encyst itself, where it will lie dormant until consumed by a primary

host, such as a human. The C. sinensis cyst allows the fluke to pass through the stomach

unharmed to the small intestine, where the parasite will begin to migrate toward the liver,

where it will feed and reproduce, restarting the cycle of transmission and infection (Lun

et al., 2005).

The ecology of Clonorchiasis has major consequences in the transmission and

control of the disease. As noted previously, infection tends to cluster in river basins, and

an increase of aquaculture fish production is suspected as substantially contributing to an

increase in the prevalence of infection in China and other Asian countries (Qian et al.,

2012). Also, while the low selectivity of the parasite toward its fish hosts, coupled with

the economic and nutritional importance of fish in endemic areas, makes fish a poor

target for liver fluke control, the parasite’s high specificity toward it’s snail hosts may

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provide an excellent opportunity for controlling the transmission of the disease by

controlling the snail hosts.

1.4 Control Strategies

The transmission of clonorchiasis presents a complex problem. There are

biological, ecological, social, and cultural dimensions pertaining to the spread of disease

that should be addressed in a holistic, integrated control program. Researchers have been

working to address these issues and developed a number of intervention strategies,

however these have traditionally neglected the ecological nature of the disease, instead

focusing primarily on the human link.

Describing a control program of the closely-related disease, opisthorchiasis, in

Thailand, Sithithaworn et al. (2012) list three primary intervention strategies: the

treatment of human and animal reservoir hosts, improved sanitation, and health

promotion education.

1.4.1 Treating human and reservoir hosts

Clonorchiasis and opisthorchiasis can be effectively treated in mammalian hosts

through the use of the anti-helminthic drug praziquantel; by treating human and other

reservoir hosts, the excretion of C. sinensis eggs can be stopped, theoretically breaking

the cycle of infection. However, C. sinensis can infect a wide range of mammalian hosts, including pets such as cats and dogs, livestock such as pigs and cows, and wildlife such as rabbits and rats, among others (Guoqing et al, 2001; Lun et al, 2005). Additionally,

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the prevalence of infection in animal hosts can be quite high; Lun et al. (2005) reported

cat and dog infection rates between 50 and 100% in six of nine Chinese provinces

surveyed. Rat infection rates, while generally low, were measured at 10 and 14% in two

of these provinces. While host treatment strategies can lead to a reduction in

transmission, due to the extent of infection in animal hosts, a total elimination of

reservoir transmission is unlikely. Additionally, the treatment of reservoir hosts can be

very cost-prohibitive (Lun et al., 2012).

A similar shortcoming exists with improving sanitation infrastructure and practices in endemic regions: the practice, while beneficial, may not sufficiently interrupt the life cycle of the parasite, as long as animal reservoir hosts exist as a source of continual contamination.

1.4.2 Health education

The third intervention strategies noted above—health education—exists as the

most effective intervention strategy of all, at least in potential. Human infection by C.

sinensis occurs through the ingestion of raw or undercooked contaminated fish products,

or through the cross-contamination by contaminated fish (Hong & Fang, 2012).

Theoretically, the spread of new disease in humans could be sharply reduced by halting

the consumption of raw fish and through the uptake of hygienic food preparation

methods. However, eating raw fish is deeply rooted in culture in some endemic areas,

and is viewed as a cultural delicacy (Lun et al, 2005), and centuries of tradition have

proven difficult to overcome (Rim, 2005). On the one hand, there is evidence that

8 campaigns directing individuals to properly cook fish have shown some success in preventing the disease (Rim, 1986), however despite decades of intervention efforts there are indications that infection prevalence has been increasing in China (Lun et al, 2005).

While these education campaigns may continue to be an important part of an overall control strategy, unfortunately this intervention has proven insufficient and must be integrated into a more comprehensive strategy.

Additional control strategies are discussed by Lun et al. (2005). In addition to the above methods, the authors suggest the control of host snail populations, the treatment or sterilization of feces, and the implementation of food production safety regulations such as Hazard Analysis and Critical Control Points (HACCP) principles.

1.4.3 Snail control

The control of host snail populations holds the potential to be a very effective method of clonorchiasis control; however it has a mixed reputation in the literature. Lun et al. (2005) recommend it as a potential control strategy; however, Sithithaworn et al.

(2012) suggest this may not be an ineffective strategy, noting the widespread distribution of host snails in the environment and the ecologically-detrimental effects of molluscicides in aquatic environments. Additionally, there is evidence that an incomplete reduction of host snail populations may have little or no effect on C. sinensis transmission rates, due to the fact that high levels of fish and human infection can be maintained by a relatively small number of infected snails (Sithithaworn et al., 2012), although this point remains unclear. In a report by the WHO (1997), concerning Asian

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liver flukes, it is briefly noted that “snail control measures are not cost-effective,” in part

due to the high cost of the molluscicide niclosamide. The advantage of snail control

measures is that, if proven successful, they could interrupt the disease at a critical

juncture: unlike interrupting the disease in humans, which leaves reservoir hosts to carry

on transmission, if the host snails are controlled then there is no (known) second path by

which transmission could occur. Transmission would be controlled for as long as the

snails are controlled.

Addditionally, the critiques of the snail control approach often assume chemical

control methods; alternative methods potentially exist, such as biologic or environmental

control. It should also be noted that if critical focal points of transmission can be

identified in the landscape, the snail control measures could be targeted to a relatively

small area. For example, if infection of fish were found to occur primarily in aquaculture

hatcheries prior to the fish being sold to regional ponds, then chemical or ecological

control at the hatchery might prove very effective without the cost and ecological damage of a wide-spread control program—however, such ideal focal points may not exist.

1.4.4 Treating feces

Another possible control method noted by Lun et al. (2012) is the treatment or

sterilization of feces. This could achieve a measure of control by inactivating the

pathogens present in the feces of humans and livestock, preventing the infection of snails

in the pond environment. One such method, small-scale anaerobic digestion, has gained attention over the last decade and shown promising results for a wide range of pathogens,

10 although in the case of C. sinensis this method would likely suffer the same drawbacks as improved sanitation, that is, the spread of the parasites via reservoir hosts.

Small-scale anaerobic digestors, or biodigesters, are a relatively inexpensive treatment technology which makes use of anaerobic conditions and bacteria to decompose organic materials including human and animal feces, vegetation, and slaughter residues, converting them into biogas fuel (consisting primarily of methane) and a solid residue suitable as an agricultural soil amendment (Brown, 2006). An additional benefit to this process is the reduction of a wide range of pathogens. While it is not known if biodigesters would be effective in inactivating C. sinensis eggs in feces, one study by Mentz et al. (2004) did show that anaerobic digestion was capable of inactivating the eggs of the sheep liver fluke helminth, Fasciola hepatica. These results demonstrate that biodigesters may have the potential to eliminate the eggs of the helminth

C. sinensis from the feces of infected humans and animals, although currently research into this avenue is lacking.

1.4.5 Hazard Analysis and Critical Control Points

The final control strategy suggested by Lun et al. (2012) is a strategy already employed in the U.S. known as HACCP. HACCP, or Hazard Analysis and Critical

Control Points, is defined by the FDA as “a management system in which food safety is addressed through the analysis and control of biological, chemical, and physical hazards from raw material production, procurement and handling, to manufacturing, distribution and consumption of the finished product” (FDA, 2012). HACCP is an integrated control

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strategy in which all stages of food production, from source to consumer, undergo a

hazard analysis to identify procedures and processes which can prevent or eliminate a

health hazard. Currently in the U.S. HACCP procedures are required for the meat,

seafood, and fruit juice industries, as well as for school lunch preparation.

Garrett et al. (1997) briefly discuss a study conducted in Vietnam where aquaculture was conducted in two adjacent fishponds, the control pond managed by traditional methods and the experimental pond under HACCP procedures. Water supply, fish fry, and fish feed were identified as critical control points. The results showed that

HACCP procedures were able to eliminate C. sinensis from silver carp in the experimental pond, while fish infection prevalence was 45% in the control pond. The authors also note that similar experiments showed that HACCP successfully controlled the closely-related parasite Opisthorchis viverrini in aquaculture ponds in Thailand and

Laos. Clearly, HACCP shows promise in controlling liver fluke infections in aquaculture ponds. One potential barrier to implementation is that HACCP is a very structured process, including intensive process analysis, monitoring, record-keeping, and

verification, which would require the training and education of a large number of rural

fishpond managers and workers to be effective in controlling clonorchiasis.

1.4.6 Control Summary

The control of Clonorchis sinensis remains a developing field of study. Ideally,

infection prevention would be as simple as the elimination of raw and undercooked fish

from the human diet in endemic regions, however this goal has so far proven

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unattainable. Cultural motivations continue to drive individuals to eat raw fish, out of

preference or as a sign of affluence, so developing strategies to protect food fish from

infection remains a major focus of clonorchiasis control.

Major control efforts, such as those discussed above, have focused around the

human link in the chain of infection. The treatment of infected individuals is clinically

feasible, which also aids in reducing transmission of C. sinensis eggs back to the aquatic

ecosystem. However, barring the alteration of eating habits and/or protecting fish from

infection, the reinfection of treated individuals remains a problem. Similarly,

transmission of liver fluke eggs can also be reduced by implementing improved sanitation

infrastructure in endemic regions, or possibly by treating feces by using anaerobic

digesters or another treatment method. Unfortunately, these efforts do little to prevent

the flow of eggs from reservoir hosts, and even achieving 100% improved sanitation is

unlikely to be sufficient in controlling clonorchiasis, although this could reduce

prevalence and is a worthwhile goal. While some or all of these control measures may

prove critical to a holistic, integrated strategy of liver fluke control, these human-focused interventions have thus far been insufficient in protecting human health.

For this reason, there is a pressing need to broaden the scope of liver fluke control to encompass the entire life-cycle of the parasite. In limited published reports, Hazard

Analysis and Critical Control Point (HACCP) methods have proven successful in protecting aquaculture fish from liver fluke infection by analyzing and monitoring those critical production processes where infection may be promoted or reduced.

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Snail control, like health education, theoretically offers the complete control of

liver fluke transmission by interrupting the life-cycle of the parasite by removing access to a necessary intermediate host. However, also like health education measures, snail control efforts have faced substantial barriers that have prevented them from being successful. The expense of molluscicides and their unintended ecological damage, plus the presence of widespread snail populations, have made pesticide-based snail control

measures infeasible. However, alternative ecological intervention strategies may exist

which may make snail control a much more attractive candidate for control in the future.

From our perspective, there exists a strong rationale for developing ecological-

based liver fluke control methods. Just as HACCP’s success stems from its holistic,

source-to-consumer ideology, we believe that an effective, long-term strategy of liver fluke control must begin with a fundamental understanding of the ecology of the parasite, and expand beyond human-focused interventions to look at the big picture of the disease.

While foundational ecological studies have already taken place, our understanding of the environmental factors that affect (and are affected by) liver flukes is still relatively superficial.

1.5 Ecological approach to parasite control

In our attempts to develop a sustainable approach to liver fluke control, it’s worth considering the experience of pest control in agriculture. Over the past several decades, agriculture has shifted away from the old paradigm of a primary reliance on pesticides to a more integrated approach to pest management, which takes into consideration the

14 ecology of the pests of interest including their interaction with their environment, potential predators, and the crops that managers are looking to protect (Gurr et al., 2004).

In this time frame there has been a rising interest in the field of ecological engineering, which has been described as “the design of human society with its natural environment for the benefit of both” (Mitsch & Jorgensen, 1989). Ecological engineering in pursues sustainable ecosystems that maximize the beneficial output of agriculture while maintaining ecosystem resilience to disruption and minimizing the harm caused to humans by the ecosystem and harm caused to the ecosystem by humans. Additionally, maintaining systems with a minimum of external inputs such as energy or nutrients is another goal of ecological engineering.

In agriculture, the basic tenants of ecological engineering include designing and adapting agriculture to the ecosystems of the region, using biological elements to optimize the system (such as using predator species to control pests), and minimizing induced changes to natural systems and minimizing the use of non-renewable resources

(Gurr et al., 2004). This benefits producers and community members by maximizing resource efficiency and maintaining stable, sustainable agricultural operations.

Additionally, reduced reliance on pesticides helps to curtail the development of pesticide resistance in pest species (Weiss et al., 2009). The integrated aquaculture ponds of southern China are themselves a form of ecological engineering, in which nutrients are recycled between several trophic layers within the farm, reducing the need for external inputs and reducing the waste generated by the farm.

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In the discipline of public health, there has also been a growing interest in the

relationship between ecosystems and humans, particularly in the relatively new fields of

Ecohealth and One Health. These fields recognize that human health is intimately tied to

the health and integrity of ecosystems and animals, and seek to broaden our

understanding of the specific relationships between these factors (Zinsstag, 2013).

The control of liver flukes in Chinese fishponds lies directly at the intersection of these emerging paradigms of ecological engineering, Ecohealth, and One Health, and we believe that substantial progress can be made by pursuing a better understanding of liver fluke ecology. The basic ecology—the life-cycle of the parasites and intermediate

hosts—is well-documented, but we know little about how C. sinensis and its hosts

interact with their environment: how they move in the landscape, how they interact with

other species, or how they are affected by anthropogenic manipulation of the

environment.

By investigating the relationship between the liver fluke and the environment, it is

our goal to elucidate ecological and landscape factors that influence human infection.

While this preliminary study is unlikely to directly affect liver fluke control efforts, it

may serve as a foundation for future investigation into liver fluke ecology. Further, an

ecological engineering approach to controlling liver flukes in fishponds may have

additional public health benefits, such as a reduction in the prophylactic use of antibiotics

in livestock and fish, which may serve to reduce the occurrence of antibiotic resistant

bacteria in the environment and in the food supply.

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Chapter 2: Environmental investigation of water quality and health risks in an endemic region of Clonorchis sinensis in Guandgong province, China

2.1 Introduction

Aquaculture is a major economic activity in China, directly employing about 4.3 million rural workers (FAO, 2005). Integrated aquaculture operations—those that utilize animal waste or plant residue as a fertilizer for fishponds—can be a resource-efficient method of producing food for consumption or sale (Prein, 2002). However, the use of human or animal excrement as a pond fertilizer carries certain risks to consumers and workers that should be understood and addressed. Fecal-oral pathogens, antibiotic resistant bacteria, and fishborne zoonotic trematodes (FZTs) have been identified as potential health risks of waste-fed aquaculture in Asia (Do et al. 2007; Su et al., 2011;

Boerlage et al., 2013).

We have investigated water quality and health risks associated with rural aquaculture operations in the Jiangmen city region, Guangdong province, China. 25 fishponds across 10 villages were sampled between June and August of 2012 in collaboration with the Jiangmen City Center for Disease Control (CDC). We attempted to locate the host snails of the FZT Clonorchis sinensis, a liver parasite that is endemic in the region, and develop estimates of their abundance in each pond. The degree of fecal contamination in each pond was measured in terms of Eschericia coli concentrations, and microbial source tracking methods were used to identify ponds which were contaminated

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with human feces and/or pig feces. Antibiotic resistance within fishponds was also

investigated. Concentrations of chlorophyll-a was measured in order to characterize the

eutrophic status of the ponds with an interest in understanding the possible relationship

between eutrophication and the intermediate host snails of the FZT C. sinensis, and basic

physiochemical parameters were also measured.

Human and animal fecal contamination of fishponds is a public health concern

due to the potential for the transmission of pathogens via the fecal-oral route. The World

Health Organization (WHO) (2006) notes many human pathogens that may be present in feces and wastewater, including bacteria (such as Salmonella spp., Campylobacter jejuni,

Vibrio cholerae, and Shigella spp.), helminth eggs (including hookworms, Schistosoma

mansoni, and C. sinensis), protozoa (including Cryptosporidium parvum and Giardia

intestinalis), and viruses (including rotavirus and other enteric viruses). The risks to

consumers of fish raised in pathogen-contaminated water are not clear, however studies

have shown that workers and community members in contact with waste-fed aquaculture

water are at increased risk of diarrheal diseases (Do et al., 2007) and skin diseases (van

der Hoek et al., 2005). These risks may be increased if antibiotic resistant bacteria are

present in the water or fish: while the antibiotic resistant bacteria may not be pathogenic

themselves, non-pathogenic antibiotic resistant bacteria can transfer antibiotic resistant

genes to pathogens (Wang et al., 2006; Li & Wang, 2010), reducing the effectiveness of

antibiotics in treating those pathogens (WHO, 2013).

The eutrophic status of these fishponds was studied primarily for the potential of

eutrophic conditions to affect the ecology of the intermediate host snails of FZTs such as

18

C. sinensis, which is endemic in the study region. While the snails themselves were not located during the course of this study, the possible public health implications of fishpond eutrophication will be considered. The available concentrations of the major plant nutrients nitrogen and phosphorus were also measured to better characterize the eutrophic conditions of the ponds.

2.2 Methods

2.2.1 Study sites

Twenty five fishponds across 10 villages were selected for investigation in the

Jiangmen city region, Guangdong province, China, between June and August, 2012. We attempted to keep fishpond selection as random as possible, but accessibility and the ponds’ managers’ willingness to collaborate were also factors for selection (Figures 1 and

2).

19

Figure 1: The location of the study sites in Guangdong province, China

Figure 2: The location of the study sites in reference to Jiangmen city 20

2.2.2 Snail sampling

To develop an estimate of the densities of the host intermediate snail species in the study ponds, we used an approach suggested by clonorchiasis researchers with the

Guangdong Province CDC (Guangdong Province CDC, unpublished data). Under this method, two researchers searched the rocks and soil of the pond, near the shore, for a period of twenty minutes. We were informed by our collaborators at the Guangdong

Province CDC that in this region of China, the host snails of C. sinensis are

Parafossarulus striatulus, Alocinma longicornis, and Bithynia fuchsianus, and these three species of snails were the targets of our search. Snails matching the description of the three hosts species were to be collected in plastic jars labeled with the location and date, stored on ice, and stored at 4°C in the lab until they could be identified by a trained investigator and assessed for infection status. Further, three large pieces of palm leaf from a local tree, each with a surface area of approximately 1 square meter, were placed in the water, secured with rope, weighted down, and left for one week. After this time, the vegetation was recovered and snails were again collected and stored.

It was our intention that this pattern would be followed as consistently as possible, so that snail counts would reflect snail densities in the pond and be yield results that could be used for comparison between ponds. However, due to extreme difficulty in finding the host snails and insufficient results, in practice the method was altered as the study progressed, and different variations were attempted at different locations.

21

2.2.3 Water sampling

For microbial assessment, approximately 200 mL of pond water was collected from a depth of approximately 0.5 meter using a sterile Whirl-Pak collection bag (Nasco,

Fort Atkinson, Wisconsin). Another 400 mL sample for nutrient and chlorophyll analysis was collected from a depth of 1 meter in a hard plastic bottle. Both containers were stored on ice, then placed in storage at 4°C when they were returned to the lab.

2.2.4 Physiochemical water quality parameters

Standard water quality indicators were assessed at each pond using a direct- reading sonde (YSI inc., Yellow Springs, Ohio). These parameters included temperature, dissolved oxygen, and pH. Having been previously calibrated in the lab using known standard solutions, the probe was lowered into the water to a depth of approximately 1 meter, and the readings were recorded on paper as well as in the device itself. Turbidity was analyzed at each site through the use of a secchi disk. While standing on a dock or boat, an investigator would lower the secchi disk into the water until it disappeared from view, and then would pull the disk back up until it was visible once again. The depth of reappearance was measured and recorded.

2.2.5 Fecal indicators

The fecal indicator bacteria, coliforms and Eschericia coli, were counted using commercial Coliplate test kits (Bluewater Biosciences, Mississuagua, Ontario, Canada), due to their inherent transportability and ease of use. These kits use a 96-well, most

22

probable number technique in which each well of the kit is filled with sample water

(approximately 1 mL per well) and incubated at 35°C for 24 hours. After incubation,

wells testing positive for coliforms turned blue, and wells testing positive for E. coli

turned blue and also fluoresced under UV light. After incubation, all colored and

fluorescent wells were counted and recorded for each kit, and the most probable number

of bacteria colony forming units (CFU) per 100 mL of sample were read from a most

probable number chart following the manufacturer’s instructions.

Due to the high concentration of fecal indicator bacteria in the water, samples

were diluted with sterile phosphate buffer saline (PBS) solution for use with the Coliplate

test kits, to reduce the sample concentrations to below the maximum limit of detection of

the tests. Aseptic techniques were used throughout the procedure to prevent contamination.

2.2.6 Water filtration

For each site, three water filtrations were conducted at the lab: chlorophyll samples were filtered in duplicate, and a single microbial source tracking sample was filtered. Chlorophyll samples were filtered and prepared following Bergman and Peters

(1980). For each algae sample, a 47 mm Whatman (Kent, UK) GFF glass filter was placed in a clean, acid-washed glass filter funnel and washed by first filtering 200 mL of deionized water, which was then discarded. Then 100 mL of sample was placed in the funnel and filtered. The filtrate was taken for nutrient analysis, and the filter was placed in a plastic centrifuge tube along with 10 mL of 95% ethanol. The glass filter was

23 crushed for 15 minutes using a clean glass stirring rod, the centrifuge tube was capped and wrapped in foil to prevent light from degrading the chlorophyll, and allowed to extract into the ethanol for 24 hours at 4°C.

To filter the microbial source tracking samples, more care had to be taken to maintain the sterility of the filtration equipment. Disposable filter cups were used in the funnel assembly, and a 0.45 µm pore size cellulose fiber membrane filter (Millipore,

Billerica, Massachusetts) was used to filter up to 100 mL of sample, depending on the filterability of the water. After filtration, the filter was placed in a sterile, 2-mL micro centrifuge tube labeled with the site, date, and volume information, and stored at -20°C until further processing.

2.2.7 DNA extraction

Following Lee & Lee (2012), the membrane filters were placed in a 15 mL sterile tub along with 2 mL of sterile phosphate buffered saline solution, and the tube was vigorously vortexed, then mildly sonicated to increase the efficiency of cell extraction.

After sonication, the sample was centrifuged at 10,000 g for 15 minutes at 4°C and the supernatent liquid was removed, leaving the cell pellet behind.

The cell pellet was resuspended in 1 mL of ASL buffer and the DNA was extracted using a QIAmp DNA Stool Mini Kit (Qiagen, Venlo, Netherlands) under the manufacturer’s instructions. Then the DNA was concentrated by evaporating the final elution of 200 µL, then resuspended with 20 µL of elution buffer. The final

24

concentration of DNA was determined using a NanoDrop (Thermo Fisher Scientific,

Waltham, Massachusetts) system and checked for purity via gel electrophoresis.

2.2.8 Microbial source tracking and antibiotic resistance

The methods of Lee et al. (2012) were followed for the DNA extraction and microbial source tracking. DNA extraction was performed using the commercial QIAmp

DNA Stool Kit (Qiagen, Venlo, Netherlands). For microbial source tracking analyses, two human-specific assays (gyrB (Lee & Lee, 2010) and HF183 (Haugland et al., 2010)), a pig-specific assay (PF163 (Dick et al., 2005)) were performed. In addition, an antibiotic resistance assay (tetQ (Nikolich et al., 1994)) was performed.

2.2.9 Quantitative polymerase chain reaction (qPCR)

A 48-well StepOne Real Time System (Applied Biosystems, Foster City,

California) was used to perform quantitative PCR with the gyrB, HF183, tetQ, and PF163 gene-based primers and probes. The amplification reactions were carried out in optical microplates with 30 µL of reagents: the 1x TaqMan universal PCR master mix (PCR buffer, deoxynucleoside triphosphates, AmpliTaq Gold polymerase, internal reference signal 6-carboxy-x-rhodamine [ROX], Amp Erase uracil N-glycosylase [UNG], MgCl2

(Applied Biosystems, Foster City, California)), 500 nM of each primer, and 250 nM of

TaqMan Minor Groove Binding probe labeled with 6-coboxy fluorescein. For each reaction, a negative control was produced by reacting all of the PCR reagents without any template DNA in the mix. Thermal cycling consisted of an initial cycle (50 °C for 2

25 minutes and 95 °C for 10 minutes), followed by 40 cycles of denaturation (95 °C for 15 seconds each cycle) and annealing and extension (60 °C for 1 minute each cycle).

2.2.10 Chlorophyll-a measurement

Chlorophyll samples were analyzed according to the methods given in Bergman and Peters (1980). To analyze the extracted chlorophyll samples, they were removed from cold storage and centrifuged at 4,000 rpm for 20 minutes. Approximately 3 mL was decanted into a centrifuge tube, and absorbance readings were taken at 649, 665, and 750 nm. The same readings were also taken using the ethanol solvent alone as a blank.

Chlorophyll a was determined using the equation:

(13.7(A665 − A750) − 5.76(A649 − A750))v Chl a (ug/L) = V * l

Where: v = the volume of ethanol used in the extraction in mL

V = the volume of the sample filtered in L l = the path length of the cuvette

2.2.11 Nutrient measurement

The filtered sample from the algae analysis was stored at 4°C until it could be analyzed for nitrogen and phosphorus content. Both nutrients were analyzed colorimetrically using NitraVer 6, NitriVer 3, and PhosphVer 3 (Hach, Loveland,

Colorado). 26

To analyze nitrate and nitrite, 10 mL of filtered sample was added to a 25 mL

flask. The contents of one NitraVer 6 reagent packet was also added, and the flask was

swirled continuously for 3 minutes, and allowed to settle for 2 minutes. This step of the

process was used to reduce the nitrite in the sample to nitrate. Then 5 mL of sample was

decanted into a second flask, and one NitriVer 3 packet was added. The flask wash

swirled for one minute and allowed to stand for 10 minutes. The NitraVer 6 reagents

turned pink in the presence of nitrate; the absorbance of the treated sample was measured

at 507 nm using a Phenix (Shanghai, China) UV1901PC spectrophotometer and recorded,

as was the absorbance of an untreated sample used as an absorbance blank.

To measure phosphorus in the sample, 10 mL of filtered sample was added to a 25

mL flask, along with the contents of one PhosVer 3 reagent packet. The flask was

swirled to mix, allowed to develop for 2 minutes, then the treated sample and an

untreated sample were measured for absorbance at 890 nm.

To convert the absorbance readings for nitrogen and phosphate into

concentrations, first a standard curve was developed by creating known solutions over a

range of concentrations, which were assessed using the aforementioned process. These

concentration and absorbance values were plotted and a trend line produced, which was

used to calculate concentration from absorbance data.

2.2.12 Statistical analysis

To analyze the relationship between water quality parameter and host snail densities, we intended to use linear regression using SPSS version 20 (IBM, Armonk,

27

New York). Correlations between the various water quality parameters were assessed using Spearman’s correlations. Fecal contamination as shown by concentrations of E. coli were evaluated in comparison to the World Health Organization (WHO) guidelines for waste-fed aquaculture and the United States Environmental Protection Agency (US

EPA) standard for recreational water bodies. The proportion of ponds testing positive for each of the four genetic markers was also assessed, and the concentration of gene copies per 100 mL of pond water were calculated and plotted. For microbial source tracking results, ABI 48-well StepOne Real-Time System data were analyzed using StepOne software v2.0 (Applied Biosystems). Subsequent threshold cycle (Ct) values were exported to Excel 2010 (Microsoft, Redmond, Washington).

2.3 Results

2.3.1 Snail sampling

We were unable to locate the host snails of the liver fluke during the course of our investigation, likely due to our inexperience with the search protocol.

2.3.2 Physiochemical parameters

A summary of the results of the water quality survey is given in Table 1.

28

Table 1: Most of the physical and chemical parameters were within acceptable ranges for fish production, except for concentrations of chlorophyll-a and the nutrients nitrogen and phosphorus, which were high. The chlorophyll-a concentrations indicate excessive eutrophication in the fishponds.

Std. Physiochemical N Minimum Maximum Mean Deviation Temp (ºC) 25 29.88 34.22 31.83 1.04 pH 25 6.99 8.94 8.04 0.52 Cond (S/cm) 25 0.1 1.15 0.38 0.29

O2 (mg/L) 25 0.63 16.16 8.29 3.99 Secchi depth (cm) 25 15.5 67.6 33.08 15.96 Chlorophyll-a (ug/L) 25 16.79 657.05 240.25 153.24 Nutrients

NO3-N (mg/L) 25 0.06 22.95 3.94 6.1

PO4-P (mg/L) 25 0.4 88.9 11.57 19.15

Our measurements showed that the available nitrogen (as nitrate and nitrite) and

phosphorous (as phosphate) were high in the ponds. Nitrate and nitrite nitrogen ranged

from 0.06 /L to 22.95 mg/L with a mean of 3.94 mg/L, and phosphate phosphorous

ranged from 0.4 to 88.9 mg/L with a mean of 11.57 mg/L (Table 1). Guidelines

published by the Western Regional Aquaculture Center (Conte, 1993) suggest that in

freshwater aquaculture ponds, nitrate-N levels should be maintained below 0.67 mg/L,

nitrite-N below 0.06 mg/L, and phosphate-P below 3.0 mg/L. Our measurements could

not differentiate between nitrate and nitrite, but conservatively assuming that the entire

fraction was nitrate, this guideline was exceeded in 48% of the ponds sampled, while the

phosphate guideline was exceeded in 64% of the ponds.

29

In the study ponds, pH ranged from 6.99 to 8.94, with a mean of 8.04, within guidelines for fish production suggested by the WHO (Enderlein et al., 1997). Dissolved oxygen ranged from 0.63 to 16.16 mg/L, with a mean of 8.29 mg/L. Most (84%) of the ponds met or exceeded the WHO recommendation of at least 5 mg/L dissolved oxygen for warm water fish production. Secchi depth in the ponds ranged from 15.5 to 67.5 cm, with a mean of 33.08 cm, which demonstrate low levels of clarity of the pond water and suggest eutrophic conditions (Carlson, 1977). Water temperature ranged from 29.88ºC to

34.22ºC, with a mean of 31.83ºC.

2.3.3 Fecal indicators

The levels of fecal indicator bacteria (coliforms and E. coli), as measured using the Coliplate test kits, were very high. Concentrations of E. coli, the indicator with available guidelines, ranged from 1.2x102 to 9.3x104 CFU / 100 mL of pond water, with a mean concentration of 1.5x104 CFU / 100 mL.

30

100000

WHO aquaculture consumer guideline 10000

WHO aquaculture worker guideline 1000 coli (CFU / 100 mL) (CFU / coli E. US EPA freshwater recreational criteria

100 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Pond #

Figure 3: E. coli concentrations and 3 relevant guidelines. The E. coli levels in the fishponds were very high, indicating high levels of fecal contamination.

The U.S. EPA has a recreational water single-day maximum criteria for E. coli of

235 CFU/100 mL in freshwater (EPA, 1986). Beaches and lakes found in excess of this concentration may be closed to public use until the concentrations fall below this level, due to the increase risk of disease transmission through the fecal-oral route. Fifteen of the 16 ponds (94%) in our study exceeded this E. coli single-day maximum criteria.

The WHO uses a less-conservative guideline for waste-fed aquaculture such as these Chinese fishponds, suggesting that ponds should maintain E. coli levels below

10,000 CFU / 100 mL to protect consumers and 1,000 CFU / 100 mL to protect pond workers and community members (WHO, 2006). In our analysis, the ponds sampled exceeded these two guidelines in 5 of 16 (31%) and 12 of 16 (75%) of cases, respectively

(Figure 3).

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2.3.4 Microbial source tracking (human, pig), and antibiotic resistance

The microbial source tracking analysis results showed that the majority of the ponds were positive for human fecal contamination (56% positive for HF183, 84% positive for gyrase B). Tetracycline antibiotic resistance was observed in 80% of the water samples. Ten of the 24 ponds (40%) were shown to be contaminated with pig feces

(Table 2).

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Table 2: Microbial source tracking and antibiotic resistance results; most ponds were contaminated with human feces and AR genes, almost half contaminated with pig feces.

Gene copies per 100mL Pond HF183 GyrB Pig TetQ 1 0 0 0 0 2 0 8.4E+03 0 2.0E+06 3 6.3E+03 6.2E+03 0 4.6E+05 4 8.7E+03 3.7E+03 0 1.1E+07 5 0 0 0 0 6 0 1.2E+04 0 4.4E+06 7 8.2E+03 5.1E+03 5.8E+03 3.2E+06 8 4.2E+03 4.1E+03 4.5E+03 2.1E+07 9 3.0E+03 1.5E+03 5.1E+04 1.9E+07 10 0 1.7E+04 1.1E+03 6.0E+04 11 2.1E+03 1.5E+03 9.5E+03 4.0E+07 12 0 3.0E+03 0 1.1E+04 13 1.2E+04 6.3E+03 0 0 14 8.4E+05 4.0E+05 9.9E+02 8.1E+07 15 3.8E+03 2.8E+03 4.6E+04 2.3E+07 16 0 0 0 0 17 6.3E+03 3.0E+03 0 7.8E+06 18 0 6.6E+03 0 1.3E+07 19 7.9E+03 5.5E+03 0 5.4E+05 20 8.0E+03 3.9E+03 1.2E+04 6.6E+06 21 0 3.9E+03 0 8.2E+03 22 2.4E+03 6.5E+03 4.4E+03 5.8E+07 23 2.4E+03 4.7E+03 3.8E+03 9.0E+06 24 0 7.0E+03 0 2.6E+06 25 0 0 0 0 Percent positive 56% 84% 40% 40% 80%

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2.3.5 Eutrophic status

Very high concentrations of chlorophyll-a were measured in the fishponds. Ponds may be considered “highly eutrophic” when chlorophyll-a conentrations exceed 30 µg/L

(US EPA, 2003), a level which was exceeded by 24 of the 25 ponds sampled (Figure 4).

Figure 4: Chlorophyll-a concentrations were very high, indicating excessive eutrophication in the fishponds.

700

600

500

400

300 a concentration (ug / L) (ug a concentration - 200

100 Chlorophyll 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Pond

2.3.6 Statistical analyses

Relationships between the physiochemical water quality variables were assessed using Spearman’s correlation. Four pairs of measurements were found to be significantly correlated with each other.

Table 3 shows the correlations between dissolved oxygen (in mg/L) and both pH and temperature. Temperature increases as a result of sunlight being absorbed in the 34 water, and as light and temperature increase, photosynthesis occurs at an increased rate within the pond. As the rate of photosynthesis increases, primary producers absorb CO2

(much of which exists in water as carbonic acid) from the water, increasing the pH of the water. During photosynthesis, primary producers also release O2, increasing the concentration of dissolved oxygen in the pond.

Table 3: Positive correlations between dissolved oxygen and both temperature and pH.

O2 pH Correlation Coefficient 1.000 .692**

O2 Sig. (2-tailed) . .000 N 25 25 Correlation Coefficient .692** 1.000 Spearman's rho pH Sig. (2-tailed) .000 . N 25 25 Correlation Coefficient .571** .200 Temp Sig. (2-tailed) .003 .337 N 25 25

Significant correlations were also found between specific conductivity and both chlorophyll-a and secchi depth (Table 4).

35

Table 4: A positive correlation between conductivity and chlorophyll-a, and a negative correlation between conductivity and secchi depth.

Chl-a cond Correlation Coefficient 1.000 .497* Chl-a Sig. (2-tailed) . .011 N 25 25 Correlation Coefficient .497* 1.000 Spearman's rho cond Sig. (2-tailed) .011 . N 25 25 Correlation Coefficient -.202 -.615** Secchi Sig. (2-tailed) .334 .001 N 25 25

The final significant correlation, which should be expected, is between coliforms

and E. coli, both indicators of fecal contamination (Table 5). To perform this analysis,

the censored values were treated as uncensored (i.e., greater than 26664 was treated as

26664).

Table 5: A positive correlation between coliforms and E. coli.

coliforms Correlation Coefficient 1.000 coliforms Sig. (2-tailed) . N 16 Spearman's rho Correlation Coefficient .694** E. coli Sig. (2-tailed) .003 N 16

36

2.4 Discussion

2.4.1 Fecal contamination

The high levels of fecal contamination in these fishponds suggest that those workers and community members who come in contact with the fishpond water, and consumers who eat fish produced in the pond, are at an elevated risk of infectious disease. The WHO guidelines for waste-fed aquaculture suggest that E. coli

concentrations should remain below 10,000 CFU / 100 mL to protect consumers, and

below 1000 CFU / 100 mL to protect workers. 31% of the ponds sampled exceeded the

consumer-protection guideline, and 75% of the ponds sampled exceeded the worker-

health guideline, by as much as a factor of 10. In the United States, the U.S. EPA criteria for recreational waters (such as freshwater beaches) dictate that E. coli concentrations should remain below 235 CFU / 100 mL from a single day measurement, a concentration which was exceeded by 94% of the ponds in this study.

Fecal contamination at these levels puts workers and fish consumers at elevated risk of a wide range of fecal-transmitted infectious diseases. A wide range of pathogens may be present in contaminated water, including bacteria such as Salmonella spp. and

Campylobacter jejuni, protozoa such as Cryptosporidium parvum and Giardia

intestinalis, as well as enteric viruses and helminth eggs (WHO, 2006). While the WHO

report notes that the specific risks associated with consuming fish from waste-fed aquaculture are unclear and often related to food handling and hygiene, there is evidence of increased risk to workers and community members coming into contact with water from waste-fed aquaculture ponds, such as enteric diseases (Do et al, 2007) and skin

37

diseases (van der Hoek, 2005) such as contact dermatitis. Fecal contamination of

fishponds may also serve to transport the eggs of C. sinensis to the aquatic environment,

allowing the liver fluke the opportunity to continue its life cycle and produce a new

generation of infective organisms, elevating the risk of infection and reinfection among

fish consumers.

2.4.2 Microbial source tracking and antibiotic resistance

The results of our microbial source tracking analysis confirm that the majority of

the ponds studied (84%) tested positive for contamination with human waste, further

demonstrating the potential pathway for the completion of the liver fluke life cycle, as

well as other fecal-transmitted diseases.

The antibiotic resistance analysis also demonstrated a high proportion of ponds

(80%) contaminated with bacteria resistant to tetracycline antibiotics. A high prevalence of antibiotic resistant bacteria has previously been documented in seafood in Guangzhou, the capital city of Guangdong province (Ye, 2013). Antibiotic resistance is a major public health concern; resistant pathogens are less susceptible to medical treatment and increase the duration of illness and the risk of death among infected patients (WHO,

2013). While there is no specific indication that the resistant bacteria found in this study are pathogenic, previous studies have shown antibiotic resistant pathogens in foods imported from China (Kiessling et al., 2003; Zhao et al., 2003), and in vitro studies have shown that non-pathogenic, antibiotic resistant bacteria isolated from food may transfer their antibiotic resistance traits to human pathogens (Wang et al., 2006; Li & Wang,

38

2010), suggesting that antibiotic resistant bacteria in the food supply may play an

important role in the rise of antibiotic resistant pathogens in humans. As with other

infectious disease risks, the risks associated with antibiotic resistant bacteria may be

heightened for aquaculture workers and community members who come in direct contact

with the pond water.

Two different sources of the antibiotic resistant bacteria detected in the ponds are

likely. Especially in developing countries such as China, antibiotics are used in

aquaculture as a preventive measure to reduce the occurrence of costly disease outbreaks,

a practice that has been linked to the emergence of antibiotic resistant bacteria (Cabello,

2006). Antibiotics are also used in animal husbandry for similar purposes, and antibiotic

resistant bacteria may be present in the animal feces used to fertilize these fishponds. A

study in Guangdong province found a high prevalence of antibiotic resistant bacteria in

the feces of ducks and pigs raised on integrated fish farms (Su et al., 2011). Ultimately, the public health impact of the regular use of antibiotics in food production is unknown, it has been demonstrated that the potential exists for the emergence and transfer of antibiotic resistant bacteria to humans through to the prophylactic use of antibiotics in aquaculture and animal husbandry. Further investigation, including other types of antibiotic resistance other than tetracycline, are warranted.

2.4.3 Eutrophic status

As we expected prior to our investigation, the fishponds we studied were extremely eutrophic, as indicated by the high levels of chlorophyll-a detected in the

39

ponds. While concrete guidelines regarding chlorophyll-a concentrations are difficult to find, a useful criteria for comparison is that in the state of Connecticut, waterbodies with a chlorophyll-a concentration of greater than 30 µg/L in midsummer may be considered

“highly eutrophic” (EPA, 2003), and at this point the ecology of the pond or lake is heavily impacted by eutrophication. Only one pond in our study was below this level (17

µg/L), the mean concentration of the ponds was 8 times this level (240 µg/L), and the maximum concentration measured was 22 times this level (660 µg/L).

In the case of the fishponds we investigated, we found that pond managers often raised pigs next to the fishponds, and it was not uncommon for them to wash the pig feces directly into the ponds in order to fertilize algal growth, in order to feed the pond fish. Additionally, these ponds were also often contiguous with crop agriculture, and runoff containing plant fertilizers can also drive the eutrophication process (Johnson et al., 2007).

It is important to note that eutrophication, itself, is not necessarily good or bad, but rather is context-dependent (Sawyer, 1966). In the case of fishponds, eutrophication is often desirable for the increased ability of a pond to produce fish. However, there is also theoretical potential for an increased pathogen risk. For example, Johnson et al.

(2007) have demonstrated that excessive pond nutrients can raise the transmission risk of a trematode parasite of amphibians, Ribeiroia ondatrae, by supporting increased populations of the parasite’s snail intermediate hosts. Daldorph and Thomas (1991) suggest that an increased eutrophication status in may increase populations of some species of host snails of schistosomiasis, while decreasing populations of other host snails

40 of the same disease. In Guangdong province, the Chinese liver fluke C. sinensis is an endemic FZT responsible for a substantial burden of morbidity in the population (Zheng et al., forthcoming). The effect of eutrophication on the ecology of the host intermediate snails of C. sinensis is unknown, however there is enough evidence to suggest that anthropogenic eutrophication in fishponds may be supporting increased populations of host snails, and may be one factor in the high prevalence of infection in the region.

If it was found that high levels of eutrophication support an increased risk of FZT transmission in the region, this might lead to a conflict with the economic interest of the pond managers, who intentionally maintain this hyper-eutrophic state to produce more economic gain. Implementing any eutrophication control measures would necessitate finding some way of balancing the economic concerns of pond managers with public health concerns, or developing alternative management methods that maximize the economic output of the ponds while maintaining low levels of pathogens.

2.4.4 Physiochemical parameters

Most of these parameters were within normal ranges, except for the nitrogen and phosphorus. There were high available concentrations of these nutrients, which may place stress on the fish and certainly support pond eutrophication, but present little public health risk.

41

2.4.5 Snail sampling

Our inability to find the host snails of C. sinensis during the course of this study is likely due to our inexperience with the search methods or seasonal effects. In later searches, our collaborators at the Jiangmen City CDC have been successful in locating the host snails in the region in the winter of 2012, and we hope to repeat this portion of our study at a later date.

2.5 Conclusion

In this investigation we documented high concentrations of fecal contamination in aquaculture ponds in the Jiangmen city region of Guangdong province, China, and microbial source tracking methods confirm that, in addition to pig waste produced during animal production, many of the ponds are also contaminated with human fecal matter.

Many of these ponds also tested positive for antibiotic resistance genes. These findings together suggest elevated health risks to workers and community members who come into contact with the water from these fishponds, as well as a potential risk to consumers of fish from these ponds. These ponds also showed a high degree of eutrophication, which may potentially affect the risk of FZT infection—and particularly the endemic

FZT C. sinensis—of pond fish by affecting the abundance of the intermediate host snails, although the directionality of this effect is currently unknown. Freshwater aquaculture continues to play an important role in providing protein and economic support to millions of rural Chinese, however it is important to understand the potential health risks

42 associated with pond management practices such as using untreated human and animal waste to fertilize ponds.

43

Chapter 3: Epidemiology and spatial analysis of the liver fluke Clonorchis sinensis in Guangdong province, China

3.1 Introduction

The Asian liver fluke Clonorchis sinensis is a pathogen of major public health importance, and one whose transmission is heavily mediated by the landscape (Wang,

2011). C. sinensis is estimated to infect 35 million individuals, including 15 million in

China (Lun et al., 2005). This infection, clonorchiasis, is often nearly symptomless, but it is also highly chronic and without treatment the infection can develop serious complication, including cholangiocarcinoma, a highly lethal form of liver cancer (Lim,

2011).

C. sinensis had a life-cycle that is rooted in aquatic ecology (Lun et al., 2005).

The liver fluke has a multi-host life cycle, with eggs hatching and developing inside of freshwater snails, burrowing out of the snail and into freshwater fish, and ultimately being ingested into a mammalian host such as humans, but also potentially livestock, rodents, or domestic animals (Lun et al., 2005). Major control efforts of the past three decades have focused on the human component of this life-cycle, relying on chemical treatment, education programs, and improved sanitation methods to prevent humans from completing the fish to human and human to snail links in the infection cycle

(Sithithawarn, 2012), however these efforts have so far shown themselves insufficient in controlling the spread of disease (Rim, 2005).

44

There has been some movement among researchers towards developing and

integrating a better understanding of the ecology of the parasite into infection control

programs. Discussing the closely-related disease opisthorchiasis, Wang et al. (2011) call for a “landscape approach” to liver fluke control, noting that better understanding the environmental factors—particularly water—related to the spread of infection can help us understand and intervene in the patterns and processes that promote the chain of infection.

This disease is spread via the consumption of raw or undercooked freshwater fish, and it has often been observed that infections tend to cluster near rivers (Lun et al., 2005;

Figurnov et al., 2002). However, to our knowledge this relationship has not been

quantified. The primary purpose of this study was a preliminary investigation into

quantifying the relationship between proximity to water in the landscape and prevalence

of infection of local residents by combining the results of a recent survey of clonorchiasis

infection (Zheng, forthcoming) with landscape information obtained using geospatial and

remote-sensing techniques. For this, we have utilized spatial scanning statistics, satellite

images, and geographic information systems (GIS) methods to investigate a particular

cluster of infection located in the Pearl River delta in the Jiangmen city region of

southern China.

45

3.2 Materials and Methods

3.2.1 Epidemiological survey

From May to December 2011, the Jiangmen City Center for Disease Control conducted a clonorchiasis prevalence survey of 13,243 residents of the Jiangmen region

(Zheng, forthcoming). The region includes four cities (Taishan, Kaiping, Enping, and

Heshan) and three districts (Jianghai, Xinhui, and Pengjiang). From each city and district, five or six towns were randomly selected, and between approximately one to five hundred residents from each town were tested for infection with Clonorchis sinensis using the Kato-Katz fecal smear method, which also quantifies the intensity of infection among cases (Hong, 2003). To perform the Kato-Katz test, three specimen slides were smeared with 41.7 mg of feces and liver fluke eggs were detected under microscope by trained technicians. To estimate the number of eggs per gram of stool, the average number of eggs per slide was multiplied by 24 (Katz, 1972). Additionally, age and gender information were also collected for each study participant.

3.2.2 Cluster analysis

After initially mapping the survey data, it appeared that clonorchiasis infections were clustered in the eastern region of the study area, coinciding with the Pearl River delta region. To assess the statistical significance of these observed clusters, a spatial scan approach was followed similar to that used by Odoi et al. (2004) in their study of giardiasis clusters in Canada. The presence and location of infection clusters were identified using the SaTScan software program (Kulldorf, 2009), using a Bernoulli spatial

46 scan statistic described by Kulldorf (1997). In this process, a circular window of variable radius is moved across the map, and the observed number of cases inside of the window is compared to the number of cases expected under the null hypothesis of no disease clusters. (SaTScanTM is a trademark of Martin Kulldorff. The SaTScanTM software was developed under the joint auspices of (i) Martin Kulldorff, (ii) the National Cancer

Institute, and (iii) Farzad Mostashari of the New York City Department of Health and

Mental Hygiene.)

3.2.3 Remote sensing and Normalized Differential Water Index

The cluster analysis was able to determine whether or not the disease prevalence was unevenly distributed in the study region, but unable to evaluate any reason for the distribution. Due to the visually observed association between the river network and clonorchiasis prevalence, and the association between river valleys and clonorchiasis clusters reported by previous authors (Figurnov et al.2002; Rim, 2005), we chose to further evaluate the possible association between water and infection by creating a normalized difference water index (NDWI) for the study region. The NDWI is a method that utilizes satellite images of a region to identify areas of open water, particularly rivers and fishponds in our study region (Rogers and Kearney, 2004).

The first step in creating our NDWI was to obtain remotely-sensed images of the study region, specifically images from the United States Geological Survey (USGS)

Global Land Survey 2010 (GLS2010). These were downloaded from the EarthExplorer website (http://earthexplorer.usgs.gov/). The GLS2010 images are created by the USGS

47

using images from the Landsat 7 Enhanced Thematic Mapper Plus (ETM+) sensor and

the Landsat 5 Thematic Mapper (TM) sensor, using images primarily from 2009 and

2010 and selected to maximize image quality and minimize cloud cover (US Geological

Survey, 2013) The data in these images are separated into discrete bands of electromagnetic wavelength; the relevant bands for this study were the red spectrum

(Landsat band 3) and the shortwave infrared spectrum (Landsat band 5).

After acquiring the images, they were loaded into the ArcGIS software package

(Esri, Redlands, California) to compute the NDWI for each image. This was done

following the approach by Rogers and Kearney (2004), by dividing the difference of the

bands 3 and 5 by the sum of bands 3 and 5, using the raster calculator function in ArcGIS

to perform the operation on each pixel in the image:

(band3 − band5) NDWI = (band3+ band5)

Using this method, pixels with a positive NDWI value are identified as water.

With the NDWI image created, it was validated empirically by overlapping the

NDWI image with a visible-spectrum image of the same scene, and verifying that water

bodies were included in the NDWI, and that non-water features were excluded (Rogers

and Kearney, 2004) (Figure 6). Finally, to simplify the mathematical evaluation of water

features, the NDWI image was reprocessed so that pixels with a value greater than 0 were

given a value of 1, and all other pixels were given a value of 0.

48

Figure 5: A sample scene, showing: original satellite image (left) NDWI-index image (center) binary water map (right)

With the NDWI prepared, the coordinates of the study villages were imported into

ArcGIS, and for each village the percent of water coverage in both a 2 and 4 km radius by creating buffers with those dimensions and calculating the mean value of the binary

NDWI (where for each pixel 1 represents water, and 0 represents non-water) within each buffer. These percent water coverage variables were produced for each study village, and these values were assigned to the individuals from each village.

49

3.2.4 Statistical analysis

Using the results of the 2011 Jiangmen survey combined with the water coverage data produced in ArcGIS, we performed a logistic regression analysis using SPSS version

20, comparing the binary dependent variable of disease presence to the independent variables of age, gender, and percent water coverage in either a 2 or 4 km radius.

3.3 Results

3.3.1 Clonorchis sinensis infection

Of the 13,243 individuals tested during the 2011 survey, a total of 1350

individuals tested positive for infection with C. sinensis, yielding an overall prevalence of

10.2% in the region. Gender information was missing for 184 subjects, age was missing

for 448 subjects, and village was missing for 1 subject. Village prevalence ranged from

0% to 36.1% (Table 6).

Of the 6252 males in the survey, 820 tested positive for liver fluke infection,

giving an infection prevalence of 13.1%. The prevalence proportion among females was

nearly half that of males, with 498 positive out of 6707, for a prevalence of 7.4%.

Table 6: Infection prevalence among men was approximately twice that among women.

Gender Males Females Missing Subjects 6252 6707 184 Cases 820 498 32 Prevalence 13.10% 7.40% 17.40%

50

There were also substantial differences in infection by age, with a prevalence

proportion of less than 4% for those under 20, and greater than 10% for those over 30.

Infection seemed to peak for subjects from age 61 to 70 (prevalence of 15.2%), then

declining somewhat in later decades (Table 7).

Table 7: Infection prevalence increased with age until 70 years, and then decreased.

Age Prevalence Subjects 0 to 10 2.1% 1524 11 to 20 3.7% 1006 21 to 30 8.7% 1557 31 to 40 10.0% 2488 41 to 50 13.6% 2601 51 to 60 13.5% 2015 61 to 70 15.2% 1020 71 to 80 12.9% 435 > 80 11.6% 147 Age missing 9.1% 450 Total 10.2% 13243

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Table 8: A summary of the infection survey data by village.

District Village Subjects Cases Prevalence (%) Water (2km) Water (4km) Pengjiang Hetang 557 201 36.1% 22.10% 24.60% Duruan 290 34 11.7% 1.10% 1.50% Tangxia 431 129 29.9% 6.70% 9.00% Huanshi 150 20 13.3% 3.50% 2.90% Chaolian 83 18 21.7% 30.40% 32.40% Sub-total 1511 402 26.7% Jianghai Jiangnan 365 22 6.0% 3.30% 2.10% Jiaotou 248 13 5.2% 12.70% 11.80% Waihai 219 19 8.7% 12.90% 25.50% Lile 421 38 9.0% 2.20% 18.10% Jiaobei 192 29 15.1% 3.60% 3.80% Sub-total 2956 523 8.4% Xinhui Daao 401 125 31.2% 49.30% 45.70% Yamen 401 62 15.5% 32.90% 23.80% Siqian 401 119 29.7% 10.40% 16.80% Shadui 405 101 25.0% 25.50% 25.90% Sanjiang 402 48 11.9% 13.70% 15.90% Sub-total 4966 978 22.7% Taishan Douhu 301 3 1.0% 0.50% 7.50% Guanghai 408 0 0.0% 0.70% 6.40% Dajiang 470 14 3.0% 2.90% 4.30% Haiyan 424 0 0.0% 5.50% 4.30% Sijiu 445 3 0.7% 2.80% 2.30% Sub-total 2048 20 1.0% Kaiping Urban areas 536 18 3.4% 20.70% 16.80% Jinji 297 0 0.0% 0.90% 1.30% Longsheng 307 0 0.0% 3.00% 4.60% Magang 301 0 0.0% 2.00% 1.70% Changsha 331 6 1.8% 0.10% 1.50% Shagang 300 42 14.0% 6.70% 19.50% Sub-total 2072 66 3.2% Heshan Shaping 795 100 12.6% 3.40% 6.50% Zhishan 301 39 13.0% 10.70% 7.10% Zhaiwu 291 20 6.9% 3.00% 2.80% Hecheng 291 2 0.7% 1.90% 1.40% Gulao 301 33 11.0% 47.40% 35.90% Sub-total 1979 194 9.8% Continued

52

Table 8 continued Enping Langdi 328 10 3.1% 2.50% 4.60% Niujiang 348 13 3.7% 1.60% 3.30% Liangxi 328 13 4.0% 1.80% 1.50% Dongcheng 345 21 6.0% 3.70% 4.80% Encheng 508 25 4.9% 3.50% 3.50% Dahuai 357 10 2.8% 1.60% 1.00% Sub-total 2214 92 4.2% Total 13243 1350 10.2%

3.3.2 Cluster analysis

Visually interpreting the data, it appears that clusters of high infection prevalence exist in the east of the study region, coinciding with the Pearl River delta (Table 8).

Formal cluster analysis using Bernoulli analysis via the SaTScan software similarly identified a statistically significant cluster of infection (p<0.001) centered on the village of Daao in Xinhui at geographic coordinates 22.481274 N, 113.217762 E, and extending in a radius of 43.42 kilometers. This cluster included the districts of Pengjian, Jianghai, and Xinhui, as well as two villages in Heshan. This cluster included 5697 (43%) of the study subjects. Under the null hypothesis of even disease distribution, 579 cases were expected, however 1095 were observed within this radius, with an estimated risk ratio of

5.71 within the radius of the cluster.

3.3.3 Remote sensing and NDWI

After processing the Landsat images provided by the United States Geological

Survey (USGS) and analyzing them in ArcGIS (Esri, Redlands, California), it was determined that within a 2 km radius of each village, the surface water coverage varied

53 from 0.1% to 49.3%, and within a 4 km radius, water coverage varied from 1.0% to

45.7%, with higher proportions of water in the east of the study region, toward the cluster previously identified and coinciding with the Pearl River delta. This water consisted of two primary features: the rivers and tributaries themselves, and the fishponds that have been heavily developed near the rivers. Subsequent subjective validation indicated that, while imperfect, the NDWI did a satisfactory job of identifying water in the landscape.

54

Figure 6: A sample scene used to subjectively validate the NDWI procedure. Most, but not all, of the fishponds are identified as water by the NDWI procedure, and all land features appear to be correctly identified as land.

55

40 R² = 0.4204 35

30

25

20

15

Infection (%) prevalence Infection 10

5

0 0 5 10 15 20 25 30 35 40 45 50 Water cover in 4 km (%)

Figure 7: A plot of prevalence vs. percent water cover in 4 km radius by village. The data showed a general positive association.

3.3.4 Logistic regression model

A logistic regression model was fitted, initially using age and percent water coverage as continuous predictors, and gender as a categorical predictor. Two models were fitted this way, using the two different water coverage variables produced in

ArcGIS; it was found that water cover in the 4 km radius was more predictive of infection, and that model was used subsequently and the 2 km data discarded. While all terms were found to be highly significant, further analysis showed that the continuous predictors of age and percent water failed the assumption of linearity with the log-odds of

56 the dependent variable (infection), which was tested using the Box-Tidwell approach as described by Hosmer and Lemeshow (1989).

To correct this, the 4 km water term was transformed by taking the natural log of the variable, which was then found to be both significant (p<0.001) and linear in the log- odds of the outcome. However, because the prevalence of infection first increased, then decreased with age, it could not achieve linearity using a simple transformation as was done with water cover. It is possible that it could have been acceptably transformed using a quadratic term, however, it was deemed more reasonable to categorize the age variable into decades of age and compare each group to the group with the lowest infection prevalence (0 to 10 year olds).

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Table 9: The regression output. The odds of infection were positively associated with water cover in 4km radius around the village and with being male. Infection increased with age until age 70, declining after.

Variables in the Equation B S.E. Wald df Sig. OR 95% C.I.for OR Lower Upper ln_water in 4 km 0.79 0.033 562.75 1 <0.001 2.20 2.06 2.35 age 209.7 8 <0.001 11 to 20 years 0.506 0.257 3.874 1 0.049 1.66 1.00 2.75 21 to 30 years 1.493 0.216 47.897 1 <0.001 4.45 2.92 6.79 31 to 40 years 1.703 0.206 68.378 1 <0.001 5.49 3.67 8.22 41 to 50 years 2.043 0.203 101.67 1 <0.001 7.71 5.18 11.47 51 to 60 years 2.106 0.205 105.99 1 <0.001 8.22 5.50 12.27 61 to 70 years 2.15 0.212 102.82 1 <0.001 8.59 5.67 13.01 71 to 80 years 2.069 0.242 73.314 1 <0.001 7.92 4.93 12.71 > 80 years 1.872 0.309 36.741 1 <0.001 6.50 3.55 11.91 gender (males) 0.721 0.063 131.02 1 <0.001 2.06 1.82 2.33 - Constant 6.053 0.217 775.29 1 <0.001 0.00

In the final model, all covariates were highly significant (p<0.001), and the transformed continuous predictor (natural log of water cover) was linear in the log-odds.

As has been found in previous studies (Yu, 2003; Kim, 2002), men have a higher prevalence of infection over women, with an estimated odds ratio of 2.056. Water cover within a 4 km radius of each village was also positively associated with higher prevalence proportions (Table 9). For each unit increase in the natural log of percent water cover in

4 km, there was a 2.2 unit increase in the log-odds of infection. Put in simpler terms, a village with twice as much water in the surrounding area as a reference village had an odds ratio of 4.604. Regarding age, compared to the reference group (0 to 10 years), each 58 other group had an increased odds of infection, ranging from an OR of 1.659 in the 11 to

20 year group, to greater than 8 from 51 to 70 years, and declining back to 6.500 for those age 80+.

3.4 Discussion

The results of this analysis are consistent with the findings of previous studies, that infection prevalence is higher among males, and that infection prevalence increases with age until peaking in late middle age, then decreasing with age. It has been reported that the prevalence of clonorchiasis infection in males is twice that in females, not only in southern China (Yu, 2003), but also in Korea (Kim, 2002). Yu (2003) suggests that this trend may be due to social differences between the genders, such as men more frequently eating at restaurants and engaging in social activities.

Both authors also found similar trends of infection by age. In our current study, we found the highest prevalence (13 to 16%) between the ages of 41 and 70. Yu (2003) noted the highest infection prevalence among individuals between 30 and 69, and Kim

(2002) reported peaks between 30 and 49. Although the proportions of infected individuals reported by Yu were much higher (around 50%) than those reported here, the age distribution was very similar.

The results of the cluster analysis demonstrates the non-homogeneous nature of infection prevalence (infection cluster with radius of 44 km centered on village of Daao, p<0.001), and NDWI analysis confirms what we expected, that water in the landscape is significantly related to clonorchiasis infection (Odds of infection increase with the natural

59

log of percent water cover in 4 km radius: β = 0.79, p < 0.001). In his review on

clonorchiasis, Rim (2005) reports that several major clonorchiasis hot spots—including

in Korea, Russia, and Vietnam—occur along rivers. Lun (2005) specifically indicates

that the Pearl River in Guangdong is linked to liver fluke infection. Given that this

disease is transmitted by consumption of freshwater fish, this connection to water is self- evident, however, none of these studies have attempted to quantify this link.

With the present study, we have identified a cluster of disease coincident with the distributaries of the Pearl River delta with a clonorchiasis risk ratio of 5.71. Using a combination of remote sensing, GIS software, and logistic regression, we have also been able to concurrently account for the effects of age, gender, and water on infection.

However, these results are themselves only preliminary, a first step toward a landscape investigation into clonorchiasis.

For example, while our spatial scan analysis was able to detect and outline the

Pearl River cluster, the accuracy of the cluster boundaries is limited by the lack of study villages to the north or east of the detected cluster. With the current information, is it unknown if the cluster extends through the rest of the delta network. Similarly, with the

NDWI analysis we have been able to demonstrate statistically the connection between proximity to water in the landscape and a high prevalence of infection, however at its current level our analysis our analysis is unable to disentangle the relative contributions of the river network vs. the extensive aquaculture farming in the landscape. We suspect that both factors are contributing substantially to the burden of disease in the region; further, there is a high potential for a synergistic effect when we consider that the extreme

60 hydraulic interconnectedness of the fish farm / river delta system provides an excellent migration network for both the liver flukes and their snail hosts while also linking them directly to the human food supply. These hypotheses require a deeper level of investigation than we have provided here, however, the methods outlined in this study may provide an excellent tool to future investigations into clonorchiasis and other landscape-mediated infectious diseases.

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Chapter 4: Conclusions

4.1 Summary

This study characterized several qualities of rural aquaculture ponds in

Guangdong province, China, including extreme levels of eutrophication and a substantial prevalence of human fecal contamination and contamination with antibiotic resistant bacteria. This contamination suggests that workers and community members in contact with the contaminated water, and consumers who eat fish produced in the contaminated ponds, may be at an increased risk of infectious disease. While these ponds are important to the economy and food supply of rural China, it is important to consider the implications of pond management practices in terms of public health.

Our epidemiology and spatial analysis study was successful in quantifying the association between a measure of water in the landscape and human infection with the liver fluke, confirming the positive relationship between proximity to water and infection status, while also confirming the relationships between age, gender, and infection status that have been reported in other studies.

4.2 Future research

During the course of the water quality investigation, we searched for the host snails of Clonorchis sinensis in the aquaculture ponds we sampled, however we were unable to locate any of the host snails in the summer of 2012. While the snails were not 62

found during this study, our collaborators have since managed to locate the host snails in

the region during the winter of 2012, and the study methods used here may be refined and

used again in another attempt to investigate the relationship between eutrophication in

fishponds and the host snails of the Chinese liver fluke. We have enough evidence to

suggest that pond management is probably affecting the risk of liver fluke transmission in

the region, however the true effect of eutrophication on liver flukes in unknown and

deserves further investigation.

In addition to the snail component, the fish component of the C. sinensis life- cycle may also be investigated. It could be very informative to map the transfer of fish from hatcheries to ponds to markets to consumers, and to test a sample of fish for infection at each junction. It would be possible to see where infection is occurring and how far it is being transferred, which would have useful implications in terms of targeting control efforts.

Specific management practices may also be investigated for their impact on water quality indicators and specific health risks, and pilot interventions may be applied to contaminated systems. For example, feces may be treated, such as by using anaerobic biodigesters, to reduce the pathogen load of the fecal matter before it is applied to the fishponds.

Additional studies may also be performed with the DNA. For example, these

DNA extracts may be tested for other forms of antibiotic resistance, or for specific human pathogens to better characterize the health risks to consumers, workers, and community members. The sources of antibiotic resistance may also be further investigated.

63

Antibiotic resistant bacteria may be transferred to the ponds in the feces of animals (such as pigs) treated with antibiotics, in the feces of humans, or antibiotic resistance may be selected for within the ponds due to the use of antibiotics in fish feed. Working with our collaborators at the Jiangmen City and Guangdong Province CDCs, the incidence of antibiotic resistant infections in the region may be investigated, as well as the potential link between these infections and the fishponds.

At this stage our landscape analysis has only been performed for one region, using the results of one epidemiology study, but the methods we outlined may easily be used in conjunction with the results of other liver fluke studies, both past and present, to characterize this relationship across a wide variety of regions. The method outlined could also be further developed to account for a wider range of landscape factors, such as differentiating between rivers and ponds in the analysis, as well as considering the effects of proximity to vegetation and urban areas in relation to infection status. The model can also be expanded to include additional demographic factors as they become available, such as village socioeconomic status.

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