• unlimited access with print and download
    $ 37 00
  • read full document, no print or download, expires after 72 hours
    $ 4 99
More info
Unlimited access including download and printing, plus availability for reading and annotating in your in your Udini library.
  • Access to this article in your Udini library for 72 hours from purchase.
  • The article will not be available for download or print.
  • Upgrade to the full version of this document at a reduced price.
  • Your trial access payment is credited when purchasing the full version.
Buy
Continue searching

The transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment

Dissertation
Author: Eric Jorge Nelson
Abstract:
Understanding the transmission of cholera has importance for public health officials attempting to provide sanitation in a resource-scarce environment, and for the vaccinologist attempting to improve vaccine efficacy. Vibrio cholerae is the etiologic agent of the diarrheal disease cholera. V. cholerae is a facultative pathogen that resides in the environment, and on occasion, finds its way into the human host where the actions of cholera toxin cause devastating dehydration and mortality rates that reach 40%. With simple rehydration therapy, mortality rates drop below 1%. Three critical factors affect, or are likely to affect, transmission: (i) the culturability of V. cholerae in the aquatic environment, (ii) the increased infectivity of in vivo derived V. cholerae, and (iii) lytic vibriophage that prey on V. cholerae. The first goal of this thesis was to quantify these factors upon passage from the human host into the aquatic environment. The second goal was to assess the relevance of any one factor in relation to the other factors. The data reveal a model for transmission that pertains to events inside and outside the human host. Inside the host, the model suggests that V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, culturable counts of V. cholerae drop, and other microorganisms bloom. Outside, in the pond water, the model suggests that a loss of culturable cells (for reasons independent of phage) and a rise of lytic phage block transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment. Future research on rice-water stools that harbor both low titers (included in this work) and high titers (not included) of phage will provide further understanding of the impact of lytic phage on transmission. The model proposed herein is supported by epidemiological findings that suggest if an index cholera case passes lytic phage in his/her stool (assayed by darkfield microscopy as a proxy for lytic phage) household contacts are at a decreased risk of being infected with V. cholerae. These findings should provide public health officials with a renewed sense of urgency and an opportunity for sanitary interventions. In terms of vaccine development, transcriptional analysis traced the transformation of V. cholerae as the bacteria passage from patients into the aquatic environment. The nature of the final transcriptome in pond water was a function of the source from which the cells were derived. This finding is important to the vaccinologist because producing a vaccine with 'environmental' antigens from in vitro derived bacteria may not yield the same 'environmental' antigens from patient derived bacteria. Therefore, a vaccine that has antigens relevant to those expressed by V. cholerae in the natural environment may be more difficult to produce than originally considered. Diarrheal disease continues to be the second most common cause of death among children under 5 years of age globally--it is the leading cause of morbidity. I hope these public health and vaccine-oriented findings find relevance to the poverty stricken households of Bangladesh in the near future.

TABLE OF CONTENTS

8

TABLE OF CONTENTS Chapter I: Introduction.....................................................................................................15 SECTION I: CLINICAL ASPECTS OF CHOLERA.................................................16 Taxonomy and history of Vibrio cholerae................................................................16 Epidemiology............................................................................................................17 Pathophysiology of cholera.......................................................................................19 Clinical presentation.................................................................................................22 Diagnosis...................................................................................................................23 Vaccinology..............................................................................................................24 SECTION II: VIRULENCE FACTORS AND THE ToxR REGULON....................27 Overview...................................................................................................................27 Toxin co-regulated pilus...........................................................................................28 Cholera Toxin...........................................................................................................28 ToxR Regulon...........................................................................................................29 Animal models..........................................................................................................32 Methods to measure the in vivo expression of virulence factors..............................34 Summary...................................................................................................................38 SECTION III: ADAPTATION of Vibrio cholerae TO THE ENVIRONMENT........39 Overview...................................................................................................................39 Adapting to low osmolarity......................................................................................40 Nutrient limitation in fresh water ecosystems..........................................................42 Phosphate limitation..................................................................................................43 Fixed nitrogen limitation...........................................................................................45 Summary...................................................................................................................47 SECTION IV: Regulating the switch from patients into the environment..................48 Overview...................................................................................................................48 Quorum sensing in V. cholerae.................................................................................49 Additional sensory inputs into the quorum-sensing cascade....................................50 Global regulation by the secondary messenger c-di-GMP.......................................52 RpoS and the stress response....................................................................................55 Regulation of RpoS...................................................................................................57 Stringent response by RelA and SpoT......................................................................59 Nucleoid-associated proteins HN-S, Fis, and IHF....................................................62 Summary...................................................................................................................65 SECTION V: TRANSMISSION OF V. cholerae.......................................................66 Overview...................................................................................................................66 Defining the viable but non-culturable state.............................................................66 Infectivity of VBNC bacteria....................................................................................72 V. cholerae in the natural aquatic environment........................................................76 Infectivity of V. cholerae..........................................................................................84 Lytic phage of V. cholerae........................................................................................88 Summary...................................................................................................................99 Chapter II: Complexity of rice-water stool from patients with.......................................101 ABSTRACT................................................................................................................102 INTRODUCTION......................................................................................................103

9

RESULTS...................................................................................................................106 One-half of the culture positive patients shed DF- stool........................................106 DF- stools have a 1,000-fold lower V. cholerae viable count, but a similar direct count, to DF+ stools................................................................................................108 Darkfield status is in part dependent on the presence of lytic V. cholerae phage..109 There are four distinct populations of V. cholerae O1 in rice-water stool.............112 Non-V. cholerae bacteria are increased substantially in DF- culture positive samples. .................................................................................................................................115 Household contacts of a DF+ index case are at an increased risk of infection with V. cholerae...................................................................................................................116 Household contacts of a DF- index case are at an increased risk of diarrhea of unidentified etiology...............................................................................................116 DISCUSSION.............................................................................................................119 Chapter III: The transmission of Vibrio cholerae is antagonized by..............................124 The transmission of Vibrio cholerae is antagonized by..................................................124 ABSTRACT................................................................................................................125 INTRODUCTION......................................................................................................126 RESULTS...................................................................................................................131 Sample collection from cholera patients at the ICDDR,B......................................131 Chemical analysis of pond water............................................................................131 Exponential drop of culturability in a pond microcosm.........................................133 Lytic phage from patients bloom in the pond microcosm......................................136 Hyperinfectivity is maintained for at least 5 h of dialysis in pond water...............139 Culturable V. cholerae are the major contributors to infection..............................140 Coinfection of lytic phage and V. cholerae alters the burden of infection.............145 Global transcriptional analysis of ABNC V. cholerae in the aquatic environment.150 Regulon specific analysis of ABNC V. cholerae in the aquatic environment........160 DISCUSSION.............................................................................................................165 CHAPTER IV: Thesis Summary and Future Directions...............................................171 EXPERIMENTAL PROCEDURES...............................................................................187 Bacterial strains and growth conditions......................................................................188 Analyses of darkfield microscopy data from index cases and follow-up of household contacts (2001-2005)..................................................................................................188 Collection of stools from patients with suspected cholera at the ICDDR,B (2006 and 2007)...........................................................................................................................191 Darkfield microscopy and bacteriophage assays........................................................192 Fluorescence microscopy............................................................................................192 Pond microcosm system.............................................................................................194 Preparation of stool and in vitro derived V. cholerae for incubation in the pond microcosm...................................................................................................................195 Infection experiments in the infant mouse model.......................................................196 Analysis of Cell Surface Polysaccharides...................................................................199 Microarray experiments..............................................................................................200 Validation of microarray by quantitative qRT-PCR...................................................202 REFERENCES...............................................................................................................203 APPENDIX.....................................................................................................................226

10

LIST OF FIGURES

11

LIST OF FIGURES Figure 1. Ion exchange across the colonic epithelium.....................................................21 Figure 2. Virulence gene regulation in V. cholerae.........................................................30 Figure 3. Four quorum-sensing systems in V. cholerae at high and low cell densities...51 Figure 4. Model for the link between quorum sensing and c-di-GMP............................54 Figure 5. Control of stringent response............................................................................61 Figure 6. Cholera outbreak at burial feast and treatment with phage therapy.................90 Figure 7. Seasonality of vibriophage in water and cholera cases (Calcutta) in 1930......94 Figure 8. V. cholerae culture results and darkfield status (DF) for rice-water stools collected from 2001-2005 at the ICDDR,B............................................................107 Figure 9. Direct counts of V. cholerae and non-V. cholerae from rice-water stools.....110 Figure 10. Populations of V. cholerae and non-V. cholerae found in rice-water stool.114 Figure 11. Change in concentration of biologically relevant elements upon passage from patients to the pond environment............................................................................132 Figure 12. V. cholerae and phage counts during a 24 h incubation in pond water........135 Figure 13. Phage positive patient derived V. cholerae have irregular shape at 24 h ….138 Figure 14. ID 50 experiments of V. cholerae incubated in pond water............................142 Figure 15. Hyperinfectivity is not induced in vitro.........................................................144 Figure 16. Coinfection of infant mice with V. cholerae and lytic vibriophage.............149 Figure 17. Transcriptional profiles of patient derived and in vitro derived V. cholerae incubated in a pond microcosm..............................................................................153 Figure 18. Transcriptional profiles of patient derived V. cholerae with and without phage incubated in a pond microcosm..............................................................................156 Figure 19. Change in gene expression at 0, 5, and 24 h for each individual sample.....158 Figure 20. Quantification of genes induced or repressed over 24 h in pond water.......159 Figure 21. Model for the impact lytic vibriophage have on the microbial community within a patient infected with V. cholerae..............................................................177 Figure 22. Relationship between culturability, phage, and infectivity in pond water...180

12

LIST OF TABLES

13

LIST OF TABLES Table 1. Dependence of dark field status on presence of Vibriophage.........................111 Table 2. Vibriocidal antibody titer increase in household contacts grouped by DF status of the household index case....................................................................................117 Table 3. Incidence of diarrhea of unidentified etiology (DUE) among household contacts grouped by the DF status of the household index case...........................................118 Table 4. Median qRT-PCR values per biological comparison (N=3) a ..........................161 Table 5. Bacterial strains and primers used in this study................................................189 Table 6. – Appendix Node 1 for patient (phage +) and in vitro microarray...................227 Table 7. – Appendix Node 2A for patient (phage +) and in vitro microarray...............230 Table 8. – Appendix Node 2B for patient (phage +) and in vitro microarray...............234 Table 9. – Appendix Node 3 for patient (phage +) and in vitro microarray..................237 Table 10. – Appendix Node 4 for patient (phage +) and in vitro microarray.................243 Table 11. – Appendix Node 1 from phage (+/-) patient microarray...............................246 Table 12. – Appendix Node 2 from phage (+/-) patient microarray...............................251 Table 13. – Appendix Microarray data in Excel format.................................................253

14

The transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment

15

Chapter I: Introduction (Sections I-V)

16

SECTION I: CLINICAL ASPECTS OF CHOLERA

Taxonomy and history of Vibrio cholerae The history of cholera can, in many ways, be told through its taxonomy. The Gram negative bacterium Vibrio cholerae is the etiological agent of the secretory diarrheal disease cholera [1,2]. V. cholerae taxonomy is complicated because of the great diversity of strains within the species classification. Strains of V. cholerae are classified in a confusing, semi-hierarchical manner by their biotype, serogroup, and serotype. There are more than 200 serogroups of V. cholerae, but only two of these, serogroups O1 and O139, have been associated with epidemic disease, even though the others may cause illness in individual patients [3,4]. When a strain of V cholerae is isolated on selective media, the first test the technician carries out is the bacterial agglutination test with O1 and O139 antisera. If the strain agglutinates with either of these sera, the strain is then known to be a V. cholerae O1 (or O139). If it does not agglutinate, it is known as a non- O1, non-O139 V. cholerae. If the strain agglutinates with O1 antiserum, the strain is further tested to determine its Inaba or Ogawa serotype (rarely, there are strains that agglutinate with both Ogawa and Inaba antisera, and these strains are called serotype Hikojima). Unlike strains of the O1 serogroup, strains of the O139 serogroup are not further subdivided [5].

The biotype is another way to specifically classify and subdivide the O1 V. cholerae serogroup [5]. The biotype depends on the biological properties of the bacterium, beyond

17

the nature of the LPS. The two biotypes of V. cholerae are Classical and El Tor. The 5 th

and 6 th pandemics of cholera were caused by the classical biotype, but the 7 th (current) pandemic is caused by El Tor. Strains from earlier pandemics are not available so it is not confirmed which biotypes were responsible. In addition to the serotype and biotype, V. cholerae strains vary in their capacity to produce cholera toxin [3]. Strains that do not produce the toxin are not associated with epidemics even though they may be an O1 or O139 serogroup. For strains of the serogroup O139, the strains are only subdivided as being a producer (toxigenic) or non-producer of cholera toxin. Serogroup O139 may have evolved from strains of O1 El Tor, as they share many properties with El Tor strains [6,7]. The one major difference is that O139 have an O-antigen capsule and LPS with a different O-antigen composition, which causes a different agglutination reaction with antisera [8].

Epidemiology V. cholerae has been endemic in South Asia, the Middle East, and Africa from the time of recorded history. As mentioned above, there have been 7 major pandemics. It is believed that V. cholerae originated in what are modern-day Bangladesh and the state of West Bengal in India [5]. The first recorded pandemic of cholera began in 1817 and reached Europe by the 1830s; there are recorded comments about cholera that date back even to Greek times. It was not until the 5 th pandemic, during the year of 1849 (and republished in 1855), that John Snow published the groundbreaking article On the mode of communication of cholera [9]. In this epidemiological masterpiece, Snow provided evidence that cholera was spread by contaminated water in London, and at the same time

18

disproved the miasma theory. At the same time that Snow was working, Filippo Pacini discovered the cholera bacillus during an outbreak in Florence in 1854 [1]. Robert Koch later discovered the causative agent of cholera in Kolkata in 1883, and for decades, he was incorrectly honored as the discoverer of the etiologic agent [2]. The seventh pandemic was caused by the El Tor biotype, which was first isolated in 1905 in El Tor, Egypt from Indonesian pilgrims traveling to Mecca [5]. In the 1960s, El Tor spread from Indonesia, throughout Asia, to the eastern Mediterranean, Africa, Europe and North and South America. Today, the El Tor biotype has replaced the Classical biotype as the dominant cause of cholera [10]. Currently, O1 and O139 co-exist on the Indian subcontinent. In the spring of 2002 in Dhaka, Bangladesh, O139 cholera cases exceeded the number of cases of El Tor cases and therefore it is postulated that 0139 might be the cause of an 8 th pandemic of cholera [11]. However, more recently, O139 cases have begun to fall.

During cholera outbreaks in non-endemic areas, children and adults both contract the disease. However, because adults are more mobile and have more of an exposure risk outside the home, they are typically infected more often than children. In endemic areas the situation is reversed. Because most adults in an endemic area have been previously exposed to cholera, the children have higher rates of infection [12,13,14]; note that the absolute number of cases however is higher in the adult population because there are more adults than children in the population. For reasons that are not clear, stains of O139 have tended to have equal or higher rates in adults than children [15].

19

The attack rate (percentage of cases per susceptible population per year) is approximately 0.2% in endemic areas [15]. However, attack rates (AR) can be much higher. In an endemic area with very poor sanitary conditions, the attack rate is generally 0.6%. The attack rate can be even higher in rural communities with 5000 people or less (2%) and in refugee camps with a high-risk population due to a significant percentage of malnourished people (5-8%) [16].

Pathophysiology of cholera After ingestion, V. cholerae passes through the acidic environment of the stomach and enters the small intestine where the bacteria colonize. The bacteria are sensitive to acid; thus, persons with little acid in their stomach are more susceptible [17]. For persons with normal stomach acid production, the numbers of bacteria ingested must be high enough so that some bacteria can survive to reach the small intestine (acid tolerance and infectious dose are discussed in greater detail in section V). V. cholerae is propelled by a single polar flagellum. Once V. cholerae reaches the small intestine, it uses filamentous surface structures called toxin co-regulated pili (TCP) to colonize the intestinal mucosal surface. V. cholerae does this without inducing a gross inflammatory response [18]. However, inflammatory indicators of innate immunity are detected in sera [19]. This intimate relationship between the bacterium and the mucosa allows cholera toxin to be efficiently delivered to the mucosal cells to cause secretory diarrhea (TCP and cholera toxin are discussed in more detail in Section II). For now, it is sufficient to state that the devastating loss of fluid is caused by the actions of the A subunit of cholera toxin.

20

The result is a net outpouring of fluid into the lumen of the small intestine [20]. The fluid loss is too much for the colon to reabsorb [21,22], especially given that the toxin may also inhibit water absorption from the colon [23]. This secretion leads to what is referred to as acute secretory diarrhea. Secretory diarrhea is characterized by the active secretion of fluid and solutes. Osmotic diarrhea, on the other hand, is the passive shift of water to the lumen of the intestine when the lumen harbors a non-absorbable osmolyte [24,25].

Water is absorbed from the lumen of the colon in an attempt to aid the renal system by retaining fluid when the body faces dehydration [21,22]. Unlike the more proximal regions of the gastrointestinal tract, the epithelial tight junctions in the colon do not permit the passive diffusion of Na + (Figure 1). However, K + can diffuse across the tight junctions. During states of hypovolemia, aldosterone stimulates the absorption of Na + in an active exchange for K + . Cl - also exchanges with HCO 3 - across the surface facing the lumen, and the Cl - anion continues across the baso-lateral surface to follow Na + into the bloodstream. At the same time, K + slips through tight junctions into the lumen [24,25]. The net result is that some water is absorbed, and K + and HCO 3 - are lost in the lumen. Because of the dramatic nature of cholera and the finding that cholera toxin may actually disrupt the Na + /K + pumps, the colon fails to adequately retain water, and large amounts of K + and HCO 3 - are lost in the rice-water stool. The stool ultimately becomes isotonic with blood. The electrolyte losses result in potassium depletion and metabolic acidosis, and may present with ileus and rapid respiratory rates, respectively. For these reasons, the International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) developed Cholera Saline for IV transfusion that replaces the required K + and HCO 3 - .

21

Figure 1. Ion exchange across the colonic epithelium. ‘Aldo’ represents aldosterone which is produced by the adrenal glands in response to low blood pressure via the renin- angiotensin system. Figure is adapted from First Principles of Gastroenterology by A.B.R Thomson and E. A. Shaffer [26].

22

Even though the patient experiences K + depletion, usually the serum K + concentration is normal when the patient first presents for treatment. However with correction of the metabolic acidosis, the serum K + can fall quickly if the rehydration solution does not replace K + . This is due to the movement of K + into the cells when the metabolic acidosis is corrected [24,25].

Clinical presentation The most common presentation of cholera is a very acute onset of profuse watery diarrhea and vomiting without abdominal pain or cramping. However, spasmodic abdominal pain might occur. Muscle cramps in the extremities can cause severe pain; it is thought that these cramps may be due to calcium abnormalities. Typically the onset of diarrhea is in the middle of the night or the very early hours of the morning. It is unclear if the onset of disease is a function of the time of incubation and infection and/ or a function of the host physiology. Dehydration can occur rapidly -- severe dehydration can occur after approximately 4-6 hours of purging. As a result, many patients may present to health centers with severe dehydration in the early morning (within 8 hours after symptoms start). In endemic areas, a short duration of profuse watery diarrhea (“rice- water stool”) with signs of dehydration, especially severe dehydration, must be assumed to be cholera.

Vomiting is common and can also be a significant source of fluid loss and dehydration, especially if excessive vomiting prevents the patient from taking enough oral rehydration solution (ORS). It is not clear if the vomiting reduces the systemic acidosis. In terms of

23

the rice-water stool, patients with severe cholera can purge more than 200 mL/ kg/ day of diarrhea; some patients receiving fluid replacement therapy can purge a volume in excess of their body weight during the course of disease [20]. This stool typically looks like rice-water and is relatively homogeneous and light colored. Signs of severe dehydration include having a low-volume or absent radial pulse, sunken eyes, extremely reduced skin turgor, and anuria. Patients may be restless and thirsty with moderate dehydration, but when they progress to severe dehydration they become lethargic or may lose consciousness and they may be unable to drink. They might also have Kussmaul breathing due to metabolic acidosis from the loss of basic diarrheal fluid; Kussmaul breathing is a deep rapid respiration typical of acidosis. Even in overwhelming outbreaks in refugee camps, fluid replacement therapy is completely effective. When used properly, intravenous or oral fluid replacement therapy drops case fatality rates from 20- 40% to below 1 % [27]. For patients that are capable of drinking, oral rehydration therapy is completely adequate. For patients that are unable to drink because of hypotensive shock, IV fluid replacement with cholera saline is also completely adequate. However, for severely dehydrated patients, antibiotics are also provided to shorten the course of disease.

Diagnosis Diagnosis of cholera should include a laboratory based diagnosis in addition to a clinical diagnosis. In the past, ‘laboratory confirmation’ has been limited to growth on selective media. Microscopy has played a mixed role for diagnosis. Benenson et al. introduced darkfield microscopy as a tool for the rapid identification of V. cholerae in rice-water

24

stools in 1964 [28]. This technique involves examining fresh human stool under darkfield microscopy (400x magnification) for vibriod-shaped cells (0.75 - 2.0 µM) with distinct darting motility, and designating these samples darkfield positive (DF+). The results are confirmed by using antibodies specific for V. cholerae LPS to neutralize motility of the observed organisms [28,29,30]. Without enriching for V. cholerae, the sensitivity for diagnosis by darkfield microscopy ranges from 0.50 to 0.80, with a specificity greater than 0.92 [28]. Chapter 2 provides an explanation for why the sensitivity is so low but the specificity is so high. Currently, darkfield microscopy is used in laboratory research but not in standard clinical diagnosis.

A breakthrough in diagnosis occurred over the past few years. A rapid dipstick is now available for detecting V. cholerae directly from rice-water stool [31,32,33]. This dipstick is available from Span Diagnostics and the test does not need to be performed by a specialist. This dipstick can be used on a representative sample of specimens to confirm V. cholerae as the cause of an outbreak and the test results are available in only 5 minutes. At the minimum, a representative positives sample must still be sent to a reference laboratory for confirmation by culture and antibiotic sensitivities. The dipstick is incredibly useful for rapid diagnosis of cholera, but the gold standard for diagnosis is still growth on selective media for V. cholerae.

Vaccinology The types, formulations, and recommendations for cholera vaccinations are changing rapidly [34,35]. Recently, oral vaccines have been developed that are proving beneficial,

25

and the WHO is now recommending the use of vaccines in certain high-risk situations, especially among refugees [36]. Dukoral is an inactivated (dead) vaccine that is given as two doses, two weeks apart along with a buffer; Dukoral is a mixture of the classical serogroup (both Inaba and Ogawa serotypes) and the El Tor serogroup (only the Inaba serotype) plus purified cholera toxin B subunit [37]. Another inactivated vaccine is made in Vietnam and other Asian countries and is similar to Dukoral except that it does not require a buffer and is less expensive [38,39,40,41]. The implementation of the Vietnam vaccine is centered in Asia and the scope is not intended to have global impact. The Vietnam vaccine is a derivative of Dukoral. A recent step to reduce the cost of production of the Vietnam vaccine has been to engineer the vaccine strain to produce an immunogenic version of the cholera toxin B subunit such that it is trapped in the cells. Therefore, isolating the bacteria will by default purify over expressed B subunit. This eliminates the standalone HPLC purification of B subunit that is required in the purification of Dukoral [42]. This is a significant advancement and may may increase the fieldability of Dukoral-like cholera vaccines.

The other major cholera vaccines include a live-attenuated vaccine (Orochol), which requires only a single dose; this vaccine has been removed from the market [34]. A second live-attenuated vaccine, called Choleraguard, is under development but it is not yet licensed and it is unclear if development will continue [43,44]. Lastly, S. Schild, E. Nelson, and A. Camilli (Tufts University School of Medicine, Boston, MA) have developed an acellular vaccine that is based on outer membrane vesicles naturally

26

produced in culture by V. cholerae; this vaccine has not undergone any human trials. To date, Dukoral is the main vaccine considered for use in high-risk populations.

Recommendations from the World Health Organization (WHO) are still evolving because of ongoing clinical research [36]. “Since 1999, the WHO recommends the use of killed oral WC/rBS (Dukoral) vaccine as a tool to prevent cholera in populations at risk of a cholera epidemic. Such high-risk populations may include, but are not limited to, refugees and urban slum residents” [36]. In 2002, the WHO recommended that demonstration projects with oral cholera vaccines be performed in populations living in endemic settings [36]. The WHO is in the process of developing guidelines to clearly define when either of these vaccines should be used in refugee situations or in endemic areas. The stance at the ICDDR,B is that a cholera vaccine should be used in endemic areas when the rate of cholera is > 1/1000 annually; however, the currently licensed vaccines are still too expensive to manufacture and require complex logistics (cold chain and systems for distributing and administering the vaccine). As newer vaccines or improvements in formulations become available, it is likely that vaccinations will be used widely for both refugees at high risk and for endemic areas.

Full document contains 254 pages
Abstract: Understanding the transmission of cholera has importance for public health officials attempting to provide sanitation in a resource-scarce environment, and for the vaccinologist attempting to improve vaccine efficacy. Vibrio cholerae is the etiologic agent of the diarrheal disease cholera. V. cholerae is a facultative pathogen that resides in the environment, and on occasion, finds its way into the human host where the actions of cholera toxin cause devastating dehydration and mortality rates that reach 40%. With simple rehydration therapy, mortality rates drop below 1%. Three critical factors affect, or are likely to affect, transmission: (i) the culturability of V. cholerae in the aquatic environment, (ii) the increased infectivity of in vivo derived V. cholerae, and (iii) lytic vibriophage that prey on V. cholerae. The first goal of this thesis was to quantify these factors upon passage from the human host into the aquatic environment. The second goal was to assess the relevance of any one factor in relation to the other factors. The data reveal a model for transmission that pertains to events inside and outside the human host. Inside the host, the model suggests that V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, culturable counts of V. cholerae drop, and other microorganisms bloom. Outside, in the pond water, the model suggests that a loss of culturable cells (for reasons independent of phage) and a rise of lytic phage block transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment. Future research on rice-water stools that harbor both low titers (included in this work) and high titers (not included) of phage will provide further understanding of the impact of lytic phage on transmission. The model proposed herein is supported by epidemiological findings that suggest if an index cholera case passes lytic phage in his/her stool (assayed by darkfield microscopy as a proxy for lytic phage) household contacts are at a decreased risk of being infected with V. cholerae. These findings should provide public health officials with a renewed sense of urgency and an opportunity for sanitary interventions. In terms of vaccine development, transcriptional analysis traced the transformation of V. cholerae as the bacteria passage from patients into the aquatic environment. The nature of the final transcriptome in pond water was a function of the source from which the cells were derived. This finding is important to the vaccinologist because producing a vaccine with 'environmental' antigens from in vitro derived bacteria may not yield the same 'environmental' antigens from patient derived bacteria. Therefore, a vaccine that has antigens relevant to those expressed by V. cholerae in the natural environment may be more difficult to produce than originally considered. Diarrheal disease continues to be the second most common cause of death among children under 5 years of age globally--it is the leading cause of morbidity. I hope these public health and vaccine-oriented findings find relevance to the poverty stricken households of Bangladesh in the near future.