I. Introduction: Koch's postulates of disease causation were devised to verify the cause of an infectious disease since simple association of an organism with a disease is insufficient evidence that it is the causal agent. They are known as the Rules of Experimental Proof. In plant pathology they are used as follows:
The pathogen must be found associated with the disease in all the plants examined.
For nonobligate parasites, the pathogen must be isolated, grown and characterized on nutrient media. For obligate parasites, the pathogen must be isolated and grown on a susceptible host plant and its appearance and effects on the plant recorded.
The pathogen from pure culture must be inoculated onto healthy plants of the same species or cultivar on which the disease occurs and it must produce the same disease on the inoculated plants.
The pathogen must be isolated in pure culture from the inoculated plants and its characteristics must be exactly like those observed in step b.
The disease Fusarium wilt of tomato is caused by the soilborne fungus Fusarium oxysporum form species lycopersici. This fungus invades the roots of the tomato plants and grows through the xylem of the plant plugging the vessels. Water can no longer be efficiently transported in the plant and the plant becomes stunted, yellowed and wilted.
The purpose of this exercise is to study the fungus Fusarium oxysporum f. sp. Iycopersici, its colony morphology, hyphae, and spores, to inoculate Fusarium into tomato and observe for symptoms, to isolate Fusarium from tomato and to compare the isolated fungus with the one used as inoculum.
Tomato plants cultivar New Yorker or a cultivar that is NOT RESISTANT to Fusarium wilt (resistance is usually denoted by the letter F on the seed packet, seed catalog, or plant label).
Sterile potting mix. 4" or 6" pots
Cultures of Fusarium oxysporum f. sp. Iycopersici growing on potato dextrose agar (PDA) plates
Potato dextrose agar (PDA) plates
Microscope slides and cover slips
Sterile water blanks
Sterile rubber policemen or bent glass rods.
Surface disinfectant (10% bleach or 70% ethanol solutions).
A 250 or 500 ml beaker.
Grow tomato plants for 4 weeks or until they are 3-5 inches tall. Use clean, surface disinfected (soak in 10% bleach 30 minutes and air dry) pots and sterile potting mix. Tomato bedding plants may be purchased at this stage just prior to the local gardening season.
Prepare the potato dextrose agar and pour into the petri plates. Allow plates to solidify in a clean area.
Transfer pure cultures of F. oxysporum f. sp. Iycopersici to the agar plates. Place the transfers under fluorescent lights. This will stimulate sporulation of the fungus and also enhance the development of characteristic colony morphology. Allow the fungus to grow on the plates for 10-14 days.
Preparation of the fungus for inoculation
Place bits of the mycelium into a small drop of water on a slide and gently tease them apart with dissecting needles. Cover with a cover slip and examine under the microscope. Look for spores of the fungus and describe them. Illustrate the spores of the fungus. Note whether there are two types of spores. Describe the appearance of the colony growing on the PDA.
If spores have not been produced, grow the colonies for two more days and check them again. If spores are observed, flood the plates with sterile water by pouring about 10 ml onto the surface of the fungus growing on the plate. Harvest the spores by rubbing the surface of the colony with either a rubber policeman or a bent glass rod. Pour the water containing the spore suspension into a 250 ml or a 500 ml beaker.
Inoculation of the plants
Gently remove the tomato plants from the soil in which they are growing and carefully wash their roots in running water to remove most of the potting mix.
Dip and swirl the root system of the tomato plants into the spore suspension in the beaker.
Dip and swirl the roots of at least 2 tomato plants in sterile water only. These will serve as controls.
Immediately replant the inoculated plants one to a pot in fresh potting mix.
Grow the tomatoes in a sunny location. Observe the plants 2-3 times per week for the development of symptoms.
Isolation of pathogen from plants.
Before symptoms develop prepare additional PDA plates on which to isolate from the inoculated plants.
When symptoms develop (about 2-3 weeks) remove symptomatic and control plants from the soil and wash the roots in running water.
Compare the healthy and diseased plants and describe the symptoms.
Carefully dissect one healthy and one diseased plant. Cut into the base of the stem lengthwise to reveal the xylem just below the epidermis. Note the difference between the healthy and diseased xylem tissues.
Select 3 or more intact, diseased tomato plants from which to isolate the pathogen. Also isolate from an intact healthy tomato plant.
Trim off all the leaves and secondary roots leaving only the main stem and the hypocotyl and main root. Mark the healthy stem and place all of the stems into a 10% bleach solution to soak for 5 minutes. Remove the pieces and place on paper towels to dry. Using sterile technique, cut thin (2-4 mm thick) wedges out of one side of the stem near the root/stem junction making sure to include xylem tissue with each wedge. As each wedge is cut, place it onto or slightly into the agar. Place 5-6 wedges on each plate with one in the middle and the others spaced evenly around the center piece.
Incubate the plates under fluorescent lights. Check the plates every 2 days for the presence of the fungus growing out of the xylem pieces.
Once the fungus has grown sufficiently from the pieces, transfer isolates to PDA plates. Allow the transfers to grow until sporulation has occurred (10-14 days).
Examine the transfers to see if the fungus matches the original cultures that were used to inoculate the plants.
III. Results: Wilt symptoms should occur on plants inoculated with Fusarium oxysporum f. sp. lycopersici, but not on plants inoculated with water. F. oxysporum f. sp. lycopersici should be recovered from diseased plants but not from the control plants. The recovered fungus should be the same as the one used to inoculate the plants.
Agrios, G. N. 1988. Plant Pathology, 3d ed. pp. 34-35, 411-415.
DeKruif, P. 1926. Microbe Hunters. Harcourt, Brace, and Co., NY. 363 pp.
Grogan, R G. 1981. The science and art of plant disease diagnosis. Annual Review of Phytopathology 19:333-351.
Nelson, P. E., Tousson, T. A., and Cook, R J. 1981. Fusarium: Diseases, Biology, and Taxonomy. Penn. State Univ. Press, University Park. 457 pp.
Toyoda, H., Hashimoto, H., Utsumi, R, Kobayashi, H., and Ouchi, S. 1988. Detoxification of fusaric acid by a fusaric acid-resistant mutant of Pseudomonas solanacearum and its application to biological control of fusarium wilt of tomato. Phytopathology 78:1307-1311.
Walker, J. C. 1971. Fusarium wilt of tomato. American Phytopathological Society Monograph 6, St. Paul, MN. 56 pp.
Koch's Postulates http://www.phage.org/biol2040.htmSupplemental Lecture (98/05/09 update) by Stephen T. Abedon (email@example.com)
Chapter title: Principles of Disease
A list of vocabulary words is found toward the end of this document
"A rather delicate balance exists between our defenses and the disease-producing mechanisms of microorganisms. When our defenses resist these disease-producing capabilities, we maintain our health. But when the disease-producing capability overcomes our defenses, disease results. After the disease has become established, an infected person may recover completely, suffer temporary or permanent damage, or die." (p. 366, Tortora et al., 1995)
A change away from a normal state of health to an abnormal state in which health is diminished.
A sign is an objective change in body function (e.g., health) that may be observed and measured by an individual in addition to the patient.
Contrast with symptom.
A symptom is a changes in body function felt by the patient.
Symptoms are not measurable by a physician.
Contrast with sign.
Residual effects of the disease process. Essentially the unrepaired wear and tear caused by a disease.
A collection of Error! Hyperlink reference not valid. and Error! Hyperlink reference not valid. that are characteristic of a disease or abnormality.
Pathogen [parasite, pathogenesis, pathogenicity, pathology]
A pathogen is a symbiont that does more harm then good.
A pathogen also may be described as a parasite.
Pathogens are the cause of microbial (i.e., infectious) disease.
Pathogenesis is the course of disease.
Pathogencity is the degree to which a pathogen is capable of causing disease.
Pathology is the study of disease.
Demonstrates pathogen causes disease:
Koch's postulates were established by Koch in the late 1800s.
They are also a means by which microorganisms may be established directly as causes of diseases in general (aseptic technique as a means of disease prevention is only circumstantial evidence that microorganisms cause disease).
Koch's postulates are used particularly for demonstration that a specific pathogen is the cause of a specific disease.
The following criteria must hold for a given microorganism to be considered the cause of a disease:
the pathogen must be present in all hosts diagnosed with the disease
the pathogen must be isolatable from the diseased host
the pathogen must be purifiable
the purified pathogen must cause the specific disease
the pathogen must be isolated from the host used in step 4
the pathogen in step 5 must be shown to be the same pathogen purified in step 3
Problems with Koch's postulates
There are a number of infectious diseases which adhere to the spirit of Koch's postulates but for which Koch's postulates, applied strictly, do not hold.
The most common exceptions have to do with:
an inability to culture the pathogen outside of the normal host
a lack of suitable, especially non-human hosts that display the same symptoms
Etiology of disease [etiologic agent]
The etiology of a disease is a description of the cause of that disease (e.g., as addressed by Koch's postulates).
The agent responsible for the occurrence of a of microbial disease.
Pathogens are the etiological agents of microbial disease.
Infection is typically considered in terms of the colonization of the body by a disease-causing microorganism.
(Note, however, that the term colonization actually has a specific meaning, i.e., the microorganism growth on epithelial tissues, and most pathogens cause disease by invading tissues, rather than growing on their surfaces.)
"Infection refers to the multiplication of any parasitic organism within or upon the host's body. (Sometimes the term infestation is used to refer to the presence of larger parasites, such as worms or arthropods, in or on the body.) If an infection disrupts the normal functioning of the host, disease occurs." (p. 394, Black, 1996)
However, there actually exist numerous variations on this infection theme including:
An infection by a microorganism which results in some sort of expression of lack of health (i.e., disease) may be described as a symptomatic infection.
Typically an infection is only apparent symptomatically if noticeable disease results.
A symptomatic infection may lead to an asymptomatic infection or to a lack of infection.
No disease, yet:
Colonization of the body by a microorganism that does not cause symptoms may be described as an asymptomatic infection.
An asymptomatic infection may lead to a symptomatic infection or to a lack of infection.
If the asymptomatic infection does not change in status over time, the microorganisms involved essentially have taken on a role of normal flora.
Subclinical [inapparent] infection
Subclinical or inapparent infections are simply asymptomatic infection by different names.
An opportunistic infection involves the pathogenic colonization of one part of the body with a normally benign microorganism that usually resides in a different part of the body.
See disease caused by opportunists below.
septicemia may be caused by a bacterium that normally resides, for example, benignly in the large intestine or on the skin.
A local infection is an infection that is limited to a small area of the body.
A systemic infection is an infection that is found throughout the body.
Systemic infection/local origin:
A focal infection is a systemic infection that originates from a local infection.
In one sense, all systemic infections are focal infections since they have to start somewhere.
However, focal infection implies some amount of temporal difference between the start of the local and the systemic infections.
Local infection/global symptoms:
Focal infection is also another way of saying that a local infection is responsible for symptoms occurring in some other locality in the body.
For example, tetanus is caused by the release of exotoxin from a local infection.
One infecting microorganism might sufficiently weaken a host that the host is consequently susceptible to infection by additional microorganisms (e.g., opportunistic infection).
The originally infecting microorganism in such a circumstance would be described as the etiological agent of the initial (i.e., primary) infection.
Typically the primary infection is also an acute infection.
Secondary infection is an infection that occurs because some initial (primary) infection has weakened the host.
Some diseases are caused by the infection by more than one type of organism simultaneously. Such infections are called mixed infections.
An acute infection is an infection that develops rapidly and only lasts a short time.
A chronic infection is an infection that develops slowly and lasts a long time.
A subacute infection is an infection intermediate to acute and chronic.
A latent infection is inactive though continuing to infect, and which remains capable of producing symptoms.
"A latent disease is characterized by periods of inactivity either before signs and symptoms appear or between attacks." (p. 407, Black, 1996)
Herpes viruses are examples of pathogens which readily enter a latent stage during which symptoms disappear, only to reappear at a later time upon the reactivation of the latent infection.
Bacteremia refers to the presence of bacteria in the blood.
Septicemia refers to the presence of replicating bacteria in the blood.
Viremia refers to the presence of viruses in the blood.
Note that since viruses are obligate intracellular pathogents, it is often possible for viruses which do not infect blood cells to be present in the blood, but not actively replicating.
The word infection also refers to the colonization of individual cells, e.g., a viral infection of an individual cell refers to a virus replicating intracellularly (or at least having that potential).
Types of microbial diseases
Microbes can cause a number of different types of infections as outlined above.
Microbes can also cause a number of categorically distinct (though not necessarily non-overlapping) types of diseases including:
disease caused by opportunists
An infectious disease is a disease caused by a microorganism and therefore potentially infinitely transferable to new individuals.
Note that the reason for my using the qualifier infinitely is that organisms are different from inanimate entities, such as poisons, because they have a way of fighting off dilution: they replicate.
A communicable disease is an infectious disease that readily spreads from person to person.
That is, just because a disease is infectious does not mean that it is easy to catch.
A communicable disease is readily easily caught, especially from an infected person.
A contagious disease is a very communicable disease, i.e., an infectious disease that very readily spreads from person to person.
Not typically spread from person to person:
A noncommunicable disease is an infectious disease (i.e., with a microorganismal etiology) that is not typically spread from person to person.
"Such diseases may result from (1) infections caused by an individual's normal microbiota, such as an inflammation of the abdominal cavity lining following rupture of the appendix; (2) poisoning following the ingestion of preformed toxins, such as staphylococcal enterotoxin, a common cause of food poisoning; and (3) infections caused by certain organisms found in the environment, such as tetanus, a bacterial infection resulting from spores in the soil gaining access to a wound." (p. 400, Black, 1996)
A noncommunicable disease represents one end of the spectrum of communicability of infectious diseases.
The distinction, not spread from person to person, is important since it implies not only that:
individuals carrying the disease are not likely to spread the disease to others
whatever led to the infection in such an individual likely did not include person to person contact
Disease caused by opportunists
A disease caused by an opportunistic infection is an example of a noncommunicable disease.
Opportunist infections may be caused by pathogens that fail to cause disease when living in certain body sites in healthy individuals (i.e., are normal or transient flora), but can cause disease when located elsewhere on the body or under abnormal circumstances in their normal location.
Immunocompromised = highly susceptible:
Opportunists tend to cause disease especially in immunocompromised individuals.
If the immunodepression is the result of an infectious disease then the infection by the opportunist would be called a secondary infection.
Note that this overgrowth of opportunists in immunocompromised individuals suggests a role for the immune system in inhibiting the overgrowth of at least some normal flora.
Periods of infectious disease
Infectious disease tends to progress in broadly similar manners, passing through a number of roughly defined periods typically occurring in the following temporal order:
period of incubation
period of illness (illness phase)
period of decline (decline phase)
period of convalescence
Expression of these periods varies with patient and pathogen.
Note that though this order is typical, it may be circumvented and/or derailed via either:
the occurrence of mortality
as a consequence of an individuals immune response
Period of incubation
Start to symptoms:
The period of incubation is the interval occurring between the start of an infection and the appearance of symptoms.
The patient is infected but does not yet know it.
The length of the period of incubation is dependent on various host and pathogen characteristics.
For many pathogens an infected individual can spread disease to others prior to realizing that they are sick.
Prodromal period (phase)
Beginning of symptoms:
The prodromal period is a short interval in some diseases following the period of incubation.
It is the beginning of appearance of symptoms
The patient is starting to get sick.
Note that in the case of acquired immunity against a pathogen the progress of disease may end during the prodromal period as a consequence of the rapid immune system response to the infection.
For example, acquired immunity might be as a consequence of vaccination or previous natural exposure to the pathogen.
Period of illness [illness phase]
The period of illness, of course, is the time of greatest symptomatic experience (the patient is sick).
The peak of illness intensity is called the acme.
Period of decline [decline phase]
The period of decline occurs as pathogen replication is brought under control either by host immune response or through outside intervention.
A subsidence of symptoms is experienced during this period.
That is, the patient is getting better.
Period of convalescence
With the period of convalescence the pathogen replication has been stopped.
This cessation of pathogen replication occurs either by the killing of all pathogens present or by killing all actively replicating individuals (i.e., all but latently infecting in the latter case).
The body regains its pre-illness strength.
Etiology of disease
Period of convalescence
Period of decline
Period of illness
Period of incubation
Problems with Koch's postulates
Measles is an example of a disease which herd immunity (i.e., protection of a population against the epidemic spread of a disease as a consequence of the fraction of susceptible hosts being sufficiently small) has little effect. This implies that the ability of the measles virus to be transmitted from one host to another is very high. Measles especially is therefore an example of a (circle best answer) [PEEK]
all of the above
none of the above
A microorganism is isolated from a sick animal. The microorganism is grown in the laboratory. The same microorganism is subsequently isolated from two additional animals displaying the similar symptoms. Is this microorganism the cause of the disease afflicting these animals? Why? [PEEK]
Give me an example of a circumstance that would cause problems for the successful application of Koch's postulates.[PEEK]
A disease which is not spread from person to person but nevertheless is caused by a microbe is __________.[PEEK]
A communicable disease that develops rapidly and just as quickly subsides could be called __________.[PEEK]
During which of the following would you expect the host to appear most healthy? (choose best answer) [PEEK]
period of convalescence
period of incubation
period of illness
period of decline
In what way does septicemia differ from both bacteremia and viremia?[PEEK]
In terms of your appearance of health, which of the following would most likely have the least impact? (choose best answer) [PEEK]
an acute infection
a chronic infection
a subacute infection
a subclinical infection
any of the above
Clostridium tetani is replicating within a deep puncture wound. Some exotoxin but no bacteria are entering the blood or lymph. Which of the following best describes this situation? (circle only one correct answer) [PEEK]
all of the above
none of the above
Starting with two animals, one healthy and one sick with anthrax, tell me the steps you would have to go through in order to duplicate, in spirit at least, what Koch went through to prove that Bacillus anthracis is the anthrax etiology. [PEEK]
Matching: Place letter in the appropriate blank: [PEEK]
period of incubation
period of convalescence
period of decline
period of illness
arises and subsides rapidly: __________.
lack of health caused by microorganism which is poorly transmitted from person to person: __________.
presence and replication of cellular microorganism in blood: __________.
opportunistic infection of individual immunodepressed due to effects of additional infection: __________.
synonymous with asymptomatic infection and inapparent infection: __________.
infection very easily spread from person to person: __________.
period during which microorganism replication has just begun but individual is not yet sick or showing symptoms: __________.
an infection which comes on fast but seems to take forever to go away: __________.
presence of acellular microorganism in blood: __________.
Distinguish sign from symptom. [PEEK]
Define focal infection. [PEEK]
Is an acute infection necessarily a contagious disease? Why or why not? [PEEK]
Describe a typical infectious diseases (from which the host recovers) in terms of its phases or periods, defining each period (or phase) as a function of degree of symptoms experienced and relative numbers of replicating microorganisms. [PEEK]
Define acme. [PEEK]
What is the name of the formal mechanism that would typically be employed to establish the etiology of a novel infectious disease? [PEEK]
You feel sick so you go to a doctor. She tells you that you are sick because one microbe that normally lives in one location in your body (e.g., your large intestine), but does not cause disease there, is now growing in another location within your body and there is causing disease. Which is the best description of this disease? [PEEK]
Contrast "period of incubation" with "prodromal period/phase," other than in terms of the order in which they tend to occur. [PEEK]
A pimple is to septicemia as a local infection is to a(n) __________ infection. [PEEK]
A(n) __________ infection is an infection that develops slowly and then lasts a long time. [PEEK]
Distinguish mixed infection from secondary infection. [PEEK]
Practice question answers
i, contagious disease
No, or, at least, not necessarily. Just because a microorganism may be isolated from a number of sick animals has little bearing on whether that microorganism is the etiological agent unless and until it can be demonstrated that that microorganism causes the disease by Koch's postulates. As far as we can tell, that hasn't been attempted in the example. For all we know, given the information provided, the microorganism isolated is a nonpathogenic member of the animal's normal flora.
disease only affects humans, or microbes that can't be cultured in vitro.
a noncommunicable disease
an acute infection
iii, period of incubation since by definition the host does not display any signs or symptoms of the infection, i.e., they are healthy.
Septicemia refers to pathogen replication in the blood while the latter two refer only to the presence of the pathogen in the blood.
iv, a subclinical infection since this by definition shows only minimal symptoms.
iii, focal infection. This is a local infection the effects of which are manifest in other locations within the body (i.e., tetanus)
(i) Isolate microbe from sick animal, (ii) purify microbe, (iii) identify characteristics associated with the purified microbe, (iv) infect healthy animal with purified microbe, (v) if animal gets sick (i.e., same disease), attempt to isolate and purify the same microbe from this second animal. If it is the same microbe as was purified and identified in steps (ii) and (iii), then you have successfully identified and purified the anthrax etiology. Basically this works out making sure that there exists a single organism, which you can isolate, identify, and then show that it not only can make a second animal sick with the same disease, but can also show that this same organism has replicated and/or is still present in the second animal.
xv, xiv, , xviii, xiii, xi, xxiii, iii, xxi, xxiv.
A sign can be measured by a physician while a symptom can be felt only by a patient, i.e., not measured by a physician.
A local---i.e., contained, infection---which becomes systemic---i.e., enters the blood or lymph, either as a toxin or as a whole organism. In less technical terms, a local infection gone very bad.
No! An acute infection is one which comes on fast and subsides, also rapidly. It need not be caused by a contagious pathogen. Indeed, it need not be a communicable disease since these terms concern only how the disease is acquired and not its time course.
Period of incubation: relatively small numbers of replicating microorganisms and no symptoms; Prodromal period: more replicating microorganisms and some symptoms; Period of illness: Increasing numbers of replicating microorganisms, reaching a peak called the acme; Period of decline: decline in numbers of replicating microorganisms, decline in the symptomatic expression of the illness; Period of convalescence: no replicating microorganism, recovery from symptoms to state of health.
The point of maximum symptomatic expression of a disease.
iv, opportunistic infection
symptoms are lacking during the period of incubation but are present, to some extent at least, during the prodromal period
A mixed infection is a disease caused by two microorganisms simultaneously infecting, for example, the vaginitis caused, in part, by Gardnerella vaginalis. A secondary infection is a second disease caused by a second microorganism.
Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 392-412.
Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 369-373. http://www.troy.k12.ny.us/schools/ths/ths_biology/labs_online/school_labs/koch_lab_school.html