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Silkworms
and chickens - Louis Pasteur
The idea that there may be some unseen agent that spread through
the human population and caused disease was not new. Several hypotheses
had been put forward with this core idea. They were not accepted
due again in part because of social immaturity. People were still
very fatalistic about contracting disease. They had not developed
the understanding that the factor that spread disease could be
isolated and identified. The medical establishment was still embryonic.
There was no research that might provide some basic evidence to
support the hypotheses and identify a pathogenic organism.
Several scientific advances had to be made before the idea of
pathogens could be widely accepted. What was required was the
understanding of germs. There was some evidence provided in the
shape of Anton van Leeuwenhoeck's development of the microscope.
With the microscope he was able to describe organisms not visible
to the naked eye, but this still did not trigger the idea that
similar organisms could be the cause of infectious disease.
The equivalent of today's epidemiologists began to emerge and
studied the dynamics of infections and epidemics. They were able
to recognize that such diseases as typhoid and cholera spread
rapidly in densely populated areas and could sometimes be traced
to a physical source. This led to the use of isolation as a defense
against infected people spreading the disease. However there was
still no recognition that this may indicate a physical entity
as the cause of disease.
Lois Pasteur was a scientist interested in fermentation of beer
and wine and meat decay which at the time was also regarded as
fermentation. He was the first to isolate microorganisms from
ferments. He was able to purify them and then introduce the microbes
to fresh material to transfer the fermentation process. He also
demonstrated that this transfer could be stopped by heating (pasteurization).
He later became involved in examining silkworm blight that was
seriously affecting France's silk industry in the 1850s. He was
able to transfer his experiences in fermentation and demonstrate
the presence of a microorganism in affected worms. He could show
that transfer of the microbe from affected to unaffected worms
transferred the condition. Amazingly from our point of view, this
was still not recognised as being a possible mechanism of disease
transfer in humans.
Pasteur started studying anthrax in domestic animals. A significant
cause of death for domesticated animals at the time. By 1840 scientists
were already aware of rod-shaped microbes in the blood of anthrax
infected animals and Pasteur was able to recognise the similarities
between these microbes and the ones he had seen at work in fermentation
and decay processes. Once again Pasteur isolated the microbe and
showed that injection into unaffected animals transferred the
disease.
By 1878 Pasteur had switched to examining chicken cholera. Chicken
cholera is not the equivalent of human cholera but the general
attributes were the same. It was a devastating disease for the
farming industry. Pasteur from his previous experience set to
work isolating the microbe and demonstrating its presence by culturing
the causative agent (what we now call Pasteurella multocida, a
bacterium), and transferring it between affected and unaffected
animals. However, he accidentally took this work a step further.
Pasteur attempted in one experiment to transfer the microbe to
unaffected animals as he had done before. But the culture he used
was old and, unknown to him, the culture was what we describe
as attenuated, weakened and limited in its infective capability.
The chickens got sick but later recovered. Realizing his mistake
in using an attenuated culture he later reinjected the chickens
using a fresh culture. However, the chickens did not die as he
expected. He recognised that the old attenuated culture was a
form of vaccine against chicken cholera.
Koch-Pasteur
germ theorem
By this time Pasteur had a challenger to his crown in the shape
of German scientist Robert Koch. Koch had been the first to isolate
the anthrax microbe although it was Pasteur who demonstrated its
ability to transfer disease. Koch, unaware of Pasteur's work, also
demonstrated the ability of the anthrax microbe to transfer the
disease. The competition between Koch and Pasteur became bitter
and acrimonious. While Pasteur worked from the applied side of microbial
science, Koch was the key theorizer, advancing much of the germ
theory based on analysis of his and Pasteur's work. In 1881, following
on from his experiments on chicken cholera, Pasteur produced an
attenuated form of anthrax to use as a vaccine and added fuel to
the fire. Pasteur went on to produce attenuated vaccines for swine
erysipelas and rabies.
Despite the fast and furious pace of development of a germ theory
of disease by Koch and Pasteur, many were still reluctant to accept
it applied to humans. Koch shook the medical world (and trumped
Pasteur) by being the first to isolate the microbe to cause the
human disease of tuberculosis in 1882. Now there could be no objection
to the germ theory as applied to humans. Koch outlined the parameters
required for identification of an etiologic agent, called "Koch's
postulate", these requirements still stand when identifying
infective organisms.
Thus the field of immunology, and much of the basis for modern
medicine was born from the work of just two people in the 1880s.
Most historians define the turning point as the publication of Pasteur's
work on an attenuated chicken cholera vaccine (Pasteur L. De l'attenuation
du virus du cholera des poules. C R Acad Sci (Paris) 1880: 101;
673-680). Smallpox may have been the spur towards a more analytical
form of science and development of the understanding that infectious
diseases could be manipulated and controlled, but the real driving
force behind development of the immunological field were observations
made on disease in animals.
How
does disease develop?
In the 1880-90s immunization with attenuated vaccines developed
and was taken up across Europe and America for a number of diseases.
However, examination of many more diseases frustrated scientists.
Of course most viral based conditions would not reveal any bacterial
microorganism that could be cultured and used in a vaccine. Even
for some bacteria based conditions such as syphilis, tuberculosis
and salmonellosis development of vaccines was unsuccessful.
Two major questions to be answered was how infection by bacteria
could cause tissue to degrade and how vaccines worked to defend
an individual against death from infection. Pasteur and Koch made
little attempt to explain the mechanisms of disease and the humans
body's defense against it. Vaccines worked and the initial focus
of research was to expand the number of vaccines available.
In 1888 Emile Roux and Alexandre Yersin isolated a soluble toxin
from cultures of diphtheria. The bacterium itself is only found
in the throat but its destructive effects are found throughout the
body. Clearly to us the bacteria must be sending out an invisible
factor, most likely chemical in nature, to cause the body wide destruction.
This idea was the hypothesis of Roux and Yersin. They filtered diphtheria
cultures to remove the bacteria and then used the remaining fluid
filtrate (we call supernatant) to inject into healthy animals. As
expected the animals showed diphtheria lesions but without any obvious
presence of bacteria.
Next on the podium were Emil von Behring and Shibasaburo Kitasato
who took serum from animals infected with diphtheria and injected
it into healthy animals. When these animals were later inoculated
with diphtheria they were found to be resistant to infection. We
now know this method of conferring infection resistance as "passive
immunity". This first demonstration of defense against infection
was revealed and described as mediated by "antitoxin".
It was clear to Behring and Kitasato that the antitoxin was specific
only for diphtheria, it did not confer any defense against other
forms of infection. We now know this antitoxin to be antibodies
produced specifically against the diphtheria microbe. Rudolf Kraus
in 1897 first visualized the reaction of antitoxins to bacteria
by simply adding serum from infected animals to a culture of the
bacteria and seeing a cloudy precipitate develop as the antibodies
bound together the bacteria.
Other scientists took different approaches and revealed serum
based responses towards bacteria and their products. Initially these
serum properties were given a range of different names such as precipitins,
bacteriolysins, and agglutinins. Immunological research would have
to wait until 1930 before these subtly different properties were
unified and recognised as a single entity. Long before antibodies
were actually isolated and identified in serum, Paul Erlich had
put forward his hypothesis for the formation of antibodies. The
words antigen and antibody (intentionally loose umbrella terms)
were first used in 1900. It was clear to Erlich and others that
a specific antigen elicited production of a specific antibody that
apparently did not react to other antigens.
Erlich introduced an number of ideas that were later to be proved
correct. He hypothesized that antibodies were distinct molecular
structures with specialized receptor areas. He believed that specialised
cells encountered antigens and bound to them via receptors on the
cell surface. This binding of antigen then triggered a response
and production of antibodies to be released from the cell to attack
the antigen. He understood that antigen and antibody would fit together
like a "lock and key". A different key would not fit the
same lock and vice versa. However he did get two important points
wrong. First, he suggested that the cells that produced antibody
could make any type of antibody. He saw the cell as capable of reading
the structure of the antigen bound to its surface and then making
an antibody receptor to it in whatever shape was required to bind
the antigen. He also suggested that the antigen-antibody interaction
was by chemical bonding rather than physical like pieces of a jigsaw
puzzle. However these faults in Erlich's theory were quickly clarified
by Karl Landsteiner and others.
So, by 1900 the medical world was aware that the body had a comprehensive
defense system against infection based on the production of antibodies.
They did not know what these antibodies looked like and they knew
little about their molecular interaction with antigens but another
major step on the road had been made. We can see that the antibody
system of defense was ultimately a development of the ancient Greek
system of medicine which believed in imbalances in the body humors.
The antibody response later became known as the "humoral"
arm of the immune system.
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