INVISIBLE MICROBES AND VIROLOGY

12 May

Long  before  scientists could  define the nature  of specific viruses, viral diseases—smallpox   and   rabies—had   provided   the   most   significant and  dramatic   examples  of  the  potential   of  preventive  inoculations. Because  the  meaning  of  the  Latin  word  virus has  undergone   many changes in two millennia of usage, the modern reader is likely to be con- fused upon finding the term in ancient texts. The first and most general meaning of virus was slime, presumably  unpleasant, but not necessarily dangerous. However, Latin authors  increasingly used the term with the implication  of poison  or  venom,  something  menacing  to  health,  or  a mysterious,  unknown  infectious  agent.  Thus,  both  the  Roman  writer Celsus (ca. 14-37) and Louis Pasteur  could speak of the virus of rabies.

Medieval scholars generally used virus as a synonym for poison. In

medical treatises of the sixteenth and seventeenth  centuries, translators usually replaced the Latin term virus with the English word venom. Seventeenth-century  writers  referred   to  a  virus  pestiferum  or  virus pestilens in discussing  infectious  diseases.  Eighteenth-century  medical writers  applied  the  term  virus  to  the  contagion   that  transmitted an infectious  disease, as in Edward  Jenner’s  discussion  of the  ‘‘cow-pox

virus’’ in the pustular  lymph that  transmitted the disease. For  medical writers  in  the  early  nineteenth   century,  virus  stood  for  the  obscure causative  principles  of infectious  diseases. The  vagueness  of the  term made it particularly attractive.

After  the  establishment   of  germ  theory  in  the  late  nineteenth century, virus was used in the general sense of ‘‘an agent with infectious properties.’’ When submicroscopic filterable infectious agents were discovered in the 1890s, the term virus was applied to these mysterious entities.  Even if the causative  agent  for  an  infectious  disease had  not been identified, Pasteur  argued that  ‘‘Every virus is a microbe.’’ Specu- lating  about  the  still unknown  causes  of  various  infectious  diseases, Koch   suggested  that   pathogenic   organisms   different   from  bacteria might be discovered. The exceptional agents that were known at the time were  larger  than   bacteria,   most  notably   the  protozoan that   causes malaria,  but  there  was no theoretical  reason  to rule out  the existence of smaller parasites. Among microbiologists, however, respect for Koch’s postulates   might  have  inhibited   virology,  as  well  as  protozoology, because  it was virtually  impossible  to culture  such entities in artificial media. Koch himself did not let bacteriological  dogma inhibit his work on tropical  medicine even where the microbes could not be cultured  in the laboratory.

By the  end  of  the  nineteenth  century,  the  techniques  of  micro- biology  were sufficiently  advanced  for  scientists  to  state,  with  a high degree of confidence that  certain  diseases were caused by specific bac- teria  or  protozoa. However,  the  infectious  agents  of  some  diseases refused  to  be isolated  by conventional techniques.  Eventually,  exotic, but visible, pathogens  (rickettsias,  chlamydias,  mycoplasmas,  and bru- cellas) joined the classical fungi, bacteria,  and protozoa. Because some of the exotic pathogens  had  complicated  life cycles and  were difficult to  culture  in vitro,  it seemed  possible  that  members  of these  groups might be the undiscovered  agents of various  infectious diseases.

Therefore, in the early twentieth century, the term virus was gener-

ally  restricted   to  the  class  of  ‘‘filterable-invisible’’  microbes.   Such microbes  were defined  operationally in terms  of their  ability  to  pass through  filters that trapped  bacteria and their ability to remain invisible to the light microscope.  The criterion  of filterability  was the outcome of work conducted by Pasteur’s associate Charles Chamberland (1851–1908), who discovered that a porous porcelain vase could be used to separate visible microorganisms  from their culture medium. This technique  could  be  used  in  the  laboratory  to  prepare   bacteria-free liquids and in the home to prepare  pure drinking  water. Chamberland was also  instrumental in the  development  of  the  autoclave,  a  device for   sterilizing   materials   by  means   of  steam   heat   under   pressure. However,   technique-based  criteria   provided   little   insight   into   the genetic  and  biochemical  nature  of viruses.  As scientists  closed  in on

the  invisible-filterable-viruses,   they  discovered  that  their  operational criteria  were not  necessarily linked.  The failure of infectious  agents to grow in vitro was not  a satisfactory  criterion  either, because scientists could not exclude the possibility that exotic microbes might need special media and growth conditions.  A more radical explanation for the failure to identify the causative  agents of some apparently infectious  diseases was that  some microbes might be obligate parasites  of living organisms that  could not be cultured  in vitro on any cell-free culture medium.

Although  for the sake of human  health, it would have been better if viruses had totally  destroyed  all tobacco  plants,  progress  in virology owes a great deal to this pernicious product  of the New World, because it was the tobacco  mosaic virus (TMV) that  established  Adolf Eduard Mayer,  Martinus Beijerinck, and Dimitri  Ivanovski  as the founders  of virology. The study of plant virology can be traced to 1886 when Adolf Eduard   Mayer  (1843–1942) discovered  that   tobacco   mosaic  disease (TMD) could be transmitted to healthy plants by inoculating  them with extracts  of sap from the leaves of diseased plants.  Unable  to culture  a tobacco  mosaic disease microbe  on artificial  media, Mayer  filtered the sap and  demonstrated that  the filtrate  was still infectious.  Mayer  was certain  that  his microbe  must  be a very unusual  bacterium.  In  1892, Dimitri  Iosifovitch  Ivanovski  (1864–1920) demonstrated that  the infec- tious  agent  for  tobacco  mosaic  disease could  pass  through  the  finest filters  available,  but  all  attempts   to  isolate  or  culture  the  ‘‘tobacco microbe’’ were failures.

Apparently unaware of Ivanovski’s work, Martinus Willem Beijer- inck  (1851–1931) also  reported  that  a  filterable  agent  caused  TMD. Thinking  about  the  way  in  which  a  small  quantity   of  filtered  plant sap transmitted the disease to a large series of plants,  Beijerinck con- cluded  that  TMD  must  be caused  by a contagium vivum fluidum that could pass through  filters and reproduce  within the living plant  tissues. On the basis of reports  in the botanical  literature,  Beijerinck thought soluble germs could cause many other  plant  diseases.

Similar observations were made by Friedrich  Loeffler (1862–1915)

and Paul Frosch  (1860–1928) in their studies of foot-and-mouth disease (FMD), the first example of a filterable virus disease of animals. Attempts to culture bacteria from lesions in the mouths and udders of sick animals were unsuccessful.  Even  after  passage  through  a  Chamberland  filter, however,  the  apparently bacteria-free  fluid  from  FMD  lesions  could transmit  the disease to cattle  and  pigs. Filtered  fluids from  these ani- mals could  transmit  the  disease to  other  experimental  animals.  Their experiments and calculations  suggested that only a living agent, capable of reproducing  itself could  continue  to cause the disease after  passage through  a series of animals.  Loeffler  and  Frosch  suggested  that  other infectious diseases, such as smallpox, cowpox, and cattle plague, might be caused  by similar  filterable  microbes.  Nevertheless,  they continued

to  think  of the infectious  agent  as a very small and  unusual  microbe rather  than  a fundamentally different  entity.

Scientists later demonstrated that  FMD  is a highly infectious, air-

borne  viral  disease  that  attacks  cloven-hoofed  livestock  animals  like cows, sheep, goats, and pigs. The FMD  virus is a member of the picor- navirus family, which includes many important human  pathogens, such as poliovirus, hepatitis A virus, and rhinovirus.  Picornaviruses  are char- acterized by a small RNA  genome. FMD  is generally not regarded as a threat  to humans  who consume meat or pasteurized  milk from affected animals,  but  people in close contact  with infected animals  can acquire the disease. In the 1830s scientists apparently infected themselves with FMD  by inoculation and by drinking  milk from infected cows. Proven cases of FMD  in humans  have occurred  in several countries  in Europe, Africa,  and  South  America.  Nevertheless,  human  cases appear  to  be extremely rare, even when large numbers  of farm animals are affected.

Foot-and-mouth disease was introduced  into the Americas in 1870. The disease was soon reported  in parts of the United  States, Argentina, Chile, Uruguay,  Brazil, Bolivia, Paraguay, and Peru. Known  outbreaks of FMD  occurred in the United States from the 1870s to the 1920s, from New England  to California. In the 1950s, the disease was reported  in Venezuela,  Colombia,  Ecuador,  and  Canada. A Pan-American Foot- and-Mouth Disease Center was established in 1951. Using information gathered  from  participating countries,  the Center  developed  plans  for FMD   eradication.  By  2000,  Chile,  Uruguay,   Argentina,   Paraguay, and parts of Brazil were declared free of FMD. Although  many authori- ties assumed  that  FMD  had  been  virtually  eradicated  from  Western Europe,  a major  epidemic in 2001, the first since 1967, costs millions of dollars and resulted in the destruction of more than 1 million animals in the United  Kingdom  alone.  Vaccination  is used in countries  where the disease is still endemic, but because vaccinated  animals test positive for  FMD  antibodies,  countries  where vaccination  is practiced  cannot call  their  livestock  ‘‘disease-free’’ and  they  cannot   export  to  other nations.   British  scientists  think  meat  from  animals  with  FMD   was illegally brought  into England  and fed to pigs.

In  1915,  Frederick   William  Twort  (1877–1950) discovered  that

even bacteria  could  fall victim to  diseases caused  by invisible viruses. As Jonathan Swift (1667–1745) had  suggested  in a satirical  poem  on the microscope, naturalists might use the instrument  to prove that  fleas were preyed  on  by smaller  fleas that  were,  in turn,  attacked  by still smaller fleas. While trying  to grow viruses in artificial  medium,  Twort noted  that  colonies  of  certain  bacteria   growing  on  agar  sometimes became  glassy and  transparent. If  pure  colonies  of  this  micrococcus were touched by a tiny portion  of material from the glassy colonies, they too  became transparent. Like the infectious  agent of many mysterious plant and animal diseases, these so-called Twort particles were filterable.

World  War I interrupted Twort’s work on this problem  and his paper had  little  immediate   impact   on  microbiology.   Twort   later   became obsessed with speculative work on the possibility that  bacteria  evolved from viruses that  had developed from even more primitive forms.

While working  on the dysentery  bacillus at the Pasteur  Institute, Fe´lix  d’He´relle  (1873–1949) also  discovered  the  existence of bacterial viruses. In 1917, he published his observations on ‘‘An invisible microbe that  is antagonistic to the dysentery bacillus.’’ He never acknowledged that  Twort,  who published  two years earlier, had  discovered the same phenomenon. Because the invisible microbe could not grow on labora- tory  media  or  heat-killed  bacilli,  but  grew  well  in  a  suspension  of washed bacteria  in a simple salt solution,  d’He´relle concluded  that  the anti-dysentery microbe  was an obligate bacteriophage,  that  is, an eater of  bacteria.  Bacterial  viruses  were sometimes  called  Twort–d’He´relle particles.  The invisible microbe  was found  in stool samples of patients recovering from bacillary dysentery. When an active filtrate  was added to a culture of Shiga bacilli, bacterial  growth soon ceased and bacterial death and lysis (dissolution) followed. A trace of the lysate produced the same  effect  on  a  fresh  Shiga  culture.  More  than  50  such  transfers gave the  same  results,  indicating  that  a  living agent  was responsible for bacterial  lysis.

Speculating on the general implications  of the phenomenon he had discovered, d’He´relle predicted  that  bacteriophages would be found for other pathogenic  bacteria.  Although  the natural parasitism  of the invis- ible microbe seemed species specific, d’He´relle believed that  laboratory manipulations could transform bacteriophages into ‘‘microbes of immu- nity’’ with activity against  human  pathogens. d’He´relle suggested that phages  were involved  in  natural recovery  and  the  end  of  epidemics. American  novelist  Sinclair  Lewis  (1885–1951), in  collaboration  with medical writer and microbiologist  Paul de Kruif (1890–1971), explored this idea in Arrowsmith (1925). Although  experimental  tests of ‘‘phage therapy’’   were  generally   abandoned  when  antibiotics   appeared   as

‘‘miracle drugs,’’ the method has been used in the former Soviet Union,

and  certain  traditional Indian  cures  may  employ  naturally  occurring bacteriophages. In 1896, for example, a Western scientist reported  that water  from  the  Ganges  River  in  India,  traditionally  known  for  its curative properties,  was lethal to the cholera  vibrio.

The hope that bacteriophages could be trained in the laboratory to serve as weapons in the war on bacteria was not realized in the twentieth century,  but researchers  continue  to explore the possibility that  viruses might  be  recruited   to  attack   drug-resistant  bacteria.   An  estimated

90,000 Americans  died in 2000 of hospital-acquired infections  caused by antibiotic-resistant bacteria.  Some scientists think  that  phages  that prey on the tubercle bacillus might provide useful insights into the microbe’s pathogenicity, as well as new methods  of diagnosis and drug

screening. Studies of the genomes of phages that attack tubercle bacteria suggest  genetic  exchanges  between  phages  and  their  bacterial  hosts. Many  researchers  are skeptical about  this approach, primarily  because of possible adverse effects caused by introducing a self-replicating virus into  a  patient’s  bloodstream. Some  drug  companies  have,  however, explored  the  use  of  genetic  engineering  to  control  potentially  useful

‘‘therapeutic phages’’ that could be given orally or as topical treatments.

In order to find a virus that could kill a specific bacterial pathogen, researchers  had  to  use a mixture  of viruses.  Critics  warn  that  phage preparations might  be  contaminated with  unknown   strains.  Even  if highly purified preparations are used, dangerous  strains of viruses might arise through  recombination or mutation. Moreover,  replicating viruses might also acquire  and express genes for toxins or learn how to attack the cells of the patient  instead of the bacterial  target.  Some researchers hope  that  genetic  engineering  can  be  used  to  produce  very  specific viruses and, therefore, reduce the risks. Others argue that naturally occurring  viruses—already  engineered  by  Mother   Nature—are likely to be superior to, as well as less costly to produce, than modified viruses. One  approach is to  use phages  to  kill Salmonella  and  Listeria,  often associated  with food poisoning,  during  food preparation.

In  a practical,  rather  than  philosophical  sense, many  arguments

about  the  nature  of viruses  faded  from  the  picture  as researchers  in the 1930s and 1940s examined  them with new biochemical  techniques. By the 1940s, biochemists  were discovering  just how complicated  bio- logical macromolecules  could be. Advances  in biochemistry  supported the concept of the virus as a complex entity on the borderline  between cells, genes, and  molecules.  Viruses  could,  therefore,  be described  as particles  composed  of a protein  overcoat  and  an inner core of nucleic acid that is capable of entering a host cell and taking over its metabolic apparatus. As  to  just  what  viruses  are  and  where  they  fit  into  the scheme of things among plants and animals, microbes and macromole- cules, living and nonliving, French microbiologist  Andre´ Lwoff’s (1902–

1994) paraphrase of a famous  line by Gertrude Stein seems the most

appropriate  answer:  ‘‘Viruses should  be  considered  viruses  because viruses are viruses.’’

Stories  about  the  Human   Genome  Project  are  commonly  pub- lished in the popular  press and newspapers. In contrast, the sequencing of microbial genomes generates little publicity. Microbial genomics may have  many  practical  applications  for  better  vaccines,  safer  fermented foods  and  beverages,  biodefenses,   cleaner  environment,  and   better health. Although  the complete genomes of some one hundred  microbes were sequenced by 2003, scientists note that  we really know very little about  the microbial  world.

Research on diseases attributed to slow viruses, viroids, and prions

suggests that other invisible, mysterious, and perhaps menacing creatures

may  well exist  in  the  submicroscopic   world.  Unlike  viruses,  viroids appear  to be pathogens  consisting of small, single-stranded RNA  mole- cules without  a protein  coat.  Between 1971, when Theodor  O. Diener (1921–) discovered that the infectious agent responsible for potato  spin- dle tuber  disease was a novel pathogen  consisting of naked  RNA,  and

2001, about  30 viroid species and hundreds  of variants  had been stud- ied. Viroid  diseases affect  many  plants,  from  avocadoes  to  coconuts, but viroids may also be involved in tumor  formation and other diseases of animals.  Despite the excitement generated  by studies of viroids and other small RNAs,  many questions  remain about  how viroids replicate, move from cell to cell, and cause disease. Viroids have been called evo- lutionary  fossils and relics of pre-cellular evolution,  but their discovery has  stimulated  research  into  interaction  between  foreign  RNA  mole- cules and  human  diseases.

Viroids have been called ‘‘naked intruders,’’ but because they con-

tain nucleic acid, they still seemed to fit into the fundamental framework, or  Central  Dogma,  of molecular  biology,  that  is, the  flow of genetic information from nucleic acids to proteins.  Prions,  the most bizarre of all the infectious  agents discovered in the twentieth  century,  challenge the Central Dogma, as well as the idea that ‘‘viruses are viruses,’’ at least in the case of disorders that were originally attributed to ‘‘slow viruses.’’ In 1982, Stanley B. Prusiner (1942–) coined the term prion, which stood for ‘‘proteinaceous infectious particle.’’ The diseases attributed to prions are known as transmissible spongiform encephalopathies (TSE), that is, degenerative  diseases of the central  nervous  system. Prion  diseases of animals include scrapie in sheep and goats, transmissible mink encepha- lopathy,  chronic wasting disease of mule deer and elk, feline spongiform encephalopathy, and  bovine  spongiform  encephalopathy (BSE,  com- monly known as mad cow disease). Human  diseases attributed to prions include Creutzfeldt–Jakob Disease (CJD) and a new variant (vCJD) that appears  to  be related  to  BSE,  Fatal  Familial  Insomnia,  Gerstmann– Sra¨ u¨ ssler–Scheinker syndrome, and kuru.

The  idea  that  some  neurological  degenerative  diseases  might  be

caused by a novel infectious agent was suggested by Carleton Gajdusek’s (1923–) studies of kuru, a disease found only among the Fore people of New Guinea.  Based on field studies, Gajdusek  came to the conclusion that  kuru  was transmitted by mourning  rituals  during  which  women and children handled and ate the brains of deceased relatives. After cannibalism was outlawed, the incidence of the disease decreased. Using brain tissue from victims of kuru, Gajdusek  and his associates were able to transmit  the disease to chimpanzees. Symptoms did not appear,  how- ever, until about two years after inoculation. Laboratory experiments by Gajdusek  and  others  suggested that  kuru,  scrapie,  and  CJD  might be caused by similar infectious agents. Gajdusek, who was awarded a Nobel

Prize in 1976, thought that  the infectious  agent must be an unconven- tional ‘‘slow virus.’’

Many  aspects  of  the  history  of  kuru  seem  relevant  to  the  still

unfolding  story  of BSE and  vCJD.  Scrapie,  an old Scottish  name  for a  disease  of  sheep  and  goats,  has  been  known  since  the  eighteenth century,  but until the 1980s when BSE first appeared  in England,  there was no evidence of transmission to cows or humans.  Scientists believe the  BSE  epidemic  began  when  a  nutritional supplement   containing the rendered  remains  of sheep and  cows was added  to cattle  feed. By essentially  transforming  domesticated  herbivores   into  carnivores,   or even cannibals  like the Fore  victims of kuru,  the new dietary  regimen presumably  created  an  unprecedented opportunity  for  scrapie  agents to  infect  cattle.  As the  mad  cow epidemic  reached  its peak  in 1992, millions  of  cattle  were  destroyed,   but  by  then  contaminated  meat products had probably entered the food chain. The World Health Organization  warned   in  2003  that   many  countries,   particularly  in Eastern   Europe   and   Southeast   Asia,   were  at   risk   for   mad   cow disease,  even though  the  worst  appeared  to  be over  in Britain.  Mad cow  disease  appeared   in  areas  in  Europe,   Southeast   Asia,  Canada, North  Africa, and the United  States that  used contaminated feed.

Not  all prion  infections  can  be transmitted from  one  species to

another,  but the new variant  of CJD, designated vCJD, has been attrib- uted to the consumption of beef from animals  with BSE. Much  about the transmission of prion diseases remains obscure, as demonstrated by the  relatively  small  number  of  human  cases  that  occurred  in  Great Britain,  compared  to the millions of people who must have eaten con- taminated  meat.  The  panic  caused  by  mad  cow  disease  has  raised awareness of all the prion  diseases. Perhaps,  the emergence of new dis- eases, such as BSE and  vCJD,  might  be related  to the ways in which human  beings have affected the environment, especially through  global exchanges of previously isolated plants,  animals, and infectious agents.

In  1972, after  one of his patients  died of CJD,  Stanley  Prusiner

began studying the literature that linked CJD to kuru and scrapie. Creutzfeldt–Jakob  Disease  appears   to  strike   sporadically,   affecting about  one in one million people  over the age of 60 years throughout the  world.  When  Prusiner  isolated  the  scrapie  agent  from  the  brains of diseased hamsters,  he was surprised  to find that  it apparently con- sisted of a specific protein.  All previously known infectious agents, even the smallest viruses, contained  genetic material  in the form  of nucleic acids, either DNA  or RNA.  Prusiner’s ‘‘protein only hypothesis’’ was initially regarded  as heresy, but within a few years, genes that  encoded prion  proteins  were found  in all animals tested, including humans.

Despite continuing  skepticism and controversy,  by the early 1990s

many  scientists  had  accepted  Prusiner’s  prion   hypothesis.   In  1997, Prusiner  was awarded  the Nobel  Prize in Physiology  or Medicine  for

discovering  prions  and  establishing  a  new  genre  of  disease-causing agents. According  to Prusiner’s theory,  prion  proteins  can exist in two distinct  conformations, one of which is quite harmless.  Prion  proteins can  also  exist in altered  conformations that  act  as rogue  proteins  or

‘‘evil twins.’’ In the altered conformation, prion proteins are apparently capable  of  inducing  their  benign  counterparts to  undergo  the  same transformation. As the transformed proteins accumulate  and aggregate, they form thread-like  structures  that  ultimately  destroy  nerve cells and result in fatal brain diseases. Despite many uncertainties about the way in which prions  cause brain  disease, Prusiner  suggests that  understanding the three-dimensional structure  of prion  proteins  might  lead to useful therapeutic interventions. Moreover,  the success of the prion hypothesis in explaining infectious, heredity, and sporadic forms of scrapie-like diseases  suggests  that  similar  mechanisms  might  play  a role  in other disorders, including Alzheimer’s disease, Parkinson’s disease, and amyo- trophic lateral sclerosis.

In  1972, Sir Frank Macfarlane Burnet,  the Australian virologist

who  shared  the  Nobel  Prize  with  Peter  Medawar  in  1960, famously declared that ‘‘the most likely forecast about the future of infectious dis- ease is that  it will be very dull.’’ Since the 1960s, many physicians and health  policy  analysts  shared  the  assumption that  microbial  diseases could   be  essentially  ignored   because   of  the  power   of  antibiotics, vaccines,  and  other  therapeutic agents.  By the  end  of the  century,  it was clear  that  predictions  about  the  demise of infectious  disease had been grossly exaggerated.  Infectious organisms—known and previously unknown—continued to  evolve and  find  ways to  exploit  new oppor- tunities.   By  the  end  of  the  twentieth   century,   approximately  five hundred  million illnesses and six million deaths  each year were caused by AIDS,  tuberculosis,  and  malaria.  One  out  of every two  deaths  in developing  countries  is  due  to  infectious  diseases,  but  globalization and rapid  transportation link all parts  of the world.

The ‘‘catalog’’ of human  diseases is likely to grow as new diseases

appear  and old categories, such as ‘‘fevers’’ or ‘‘fevers of unknown  ori- gin,’’ are  re-examined  and  broken  down  into  specific ‘‘new’’ diseases. The appearance of West Nile virus in New York  City in 1999 and  its subsequent  spread into other states demonstrated how easily pathogens could establish themselves in new regions. Previously unknown diseases, such as AIDS,  Legionnaires’  disease, Lyme disease, mad  cow disease, Ebola  fever,  Rift  Valley fever,  SARS,  avian  influenza,  monkey  pox, Nipah virus, Lyssavirus, Chandipura virus, and so forth, have appeared and old diseases have spread  to new areas while many pathogens  have become antibiotic-resistant. For  example, antibiotic-resistant strains  of Staphylococcus aureus have caused fatal  pneumonias, heart  infections, toxic shock syndrome,  and necrotizing  fasciitis (flesh-eating bacteria).

Scientists have identified  many  factors  that  may affect the distri- bution  and  emergence  of infectious  diseases, including  environmental factors,  population growth  and age distribution, migration,  war, inter- national   travel  and  commerce,  technological   and  industrial   factors, and national  and international commitment to disease control and pub- lic health measures. Climate changes produced by global warming might have  significant  consequences  for  the  global  distribution of  diseases, especially water-borne  diseases and diseases transmitted by mosquitoes. Although  the chronic, degenerative diseases of old age have become the major  concern  of the wealthy, industrialized  countries,  in much of the world,  poverty  and  the  lack  of  basic  sanitary  facilities contribute to the continuing  burden  of infectious  diseases. According  to the United Nations, at  the  beginning  of  the  twenty-first  century  more  than  one billion  people  lack  clean  drinking  water  and  more  than  two  million people die each year from illnesses associated with dirty water and poor sanitation.

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