LEPTOSPIRAE
Leptospirosis
Definition
Leptospirosis is a
febrile disease (fever) caused by infection with the bacterium Leptospira
interrogans. L. interrogans is sometimes classified as a spirochete because
it has a spiral shape. The disease can range from very mild and symptomless to
a more serious, even life threatening form, that may be associated with kidney
(renal) failure.
Description
An infection by
the bacterium Leptospira interrogans goes by different names in
different regions. Alternate names for leptospirosis include mud fever, swamp
fever, cane cutter's fever, rice field fever, Stuttgart disease, swineherd's
disease, and Fort Bragg fever. More severe cases of leptospirosis are called
Weil's syndrome or icterohemorrhagic fever. This disease is commonly found in
tropical and subtropical climates but occurs worldwide.
According to the
Centers for Disease Control and Prevention (CDC), between 100 and 200 cases of
leptospirosis are reported in the United States each year as of the early
2000s. Almost 75% of cases of leptospirosis in North America occur in males.
About 50% of these cases occur in Hawaii, followed by the southern Atlantic,
Gulf, and Pacific coastal states. However, because of the nonspecific symptoms
of leptospirosis, it is believed that the occurrence in the United States is
actually much higher. Leptospirosis occurs year-round in North America, but
about half of the cases occur between July and October.
Leptospirosis is
called a zoonosis because it is a disease of animals that can be
transmitted to humans. It can be a very serious problem in the livestock
industry. Leptospira bacteria have been found in dogs, rats, livestock,
mice, voles, rabbits, hedgehogs, skunks, possums, frogs, fish, snakes, and
certain birds and insects. Infected animals will pass the bacteria in their
urine for months, or even years. In the United States, rats and dogs are more
commonly linked with human leptospirosis than other animals.
Humans are
considered accidental hosts and become infected with Leptospira interrogans
by coming into contact with urine from infected animals. Transmission of the
organism occurs either through direct contact with urine, or through contact
with soil, water, or plants that have been contaminated by animal urine. Leptospira
interrogans can survive for as long as six months outdoors under favorable
conditions. Leptospira bacteria can enter the body through cuts or other skin
damage or through mucous membranes (such as the inside of the mouth and nose).
It is believed that the bacteria may be able to pass through intact skin, but
this is not known.
Once past the skin
barrier, the bacteria enter the blood stream and rapidly spread throughout the
body. The infection causes damage to the inner lining of blood vessels. The
liver, kidneys, heart, lungs, central nervous system, and eyes may be affected.
There are two
stages in the disease process. The first stage is during the active Leptospira
infection and is called the bacteremic or septicemic phase. The bacteremic
phase lasts from three to seven days and presents as typical flu-like symptoms.
During this phase, bacteria can be found in the patient's blood and
cerebrospinal fluid. The second stage, or immune phase, occurs either
immediately after the bacteremic stage or after a 1-3 day symptom-free period.
The immune phase can last up to one month. During the immune phase, symptoms
are milder but meningitis (inflammation of spinal cord and brain tissues) is
common. Bacteria can be isolated only from the urine during this second phase.
Causes and symptoms
Leptospirosis is
caused by an infection with the bacterium Leptospira interrogans. The
bacteria are spread through contact with urine from infected animals. Persons at
an increased risk for leptospirosis include farmers, miners, animal health care
workers, fish farmers and processors, sewage and canal workers, cane
harvesters, and soldiers. High-risk activities include care of pets, hunting,
trail biking, freshwater swimming, rafting, canoeing, kayaking, and
participating in sports in muddy fields. One recent outbreak occurred in
Ireland following a canoe competition on a local river.
Symptoms of Leptospira
infection occur within 7-12 days following exposure to the bacteria. Because
the symptoms can be nonspecific, most people who have antibodies to Leptospira
do not remember having had an illness. Eighty-five to 90% of the cases are not
serious and clear up on their own. Symptoms of the first stage of leptospirosis
last three to seven days and are: fever (100-105 °F [37.8-40.6 °C]), severe headache, muscle pain, stomach pain, chills, nausea, vomiting, back pain,
joint pain, neck stiffness, and extreme exhaustion. Cough and body rash sometimes occur.
Following the
first stage of disease, a brief symptomfree period occurs for most patients.
The symptoms of the second stage vary in each patient. Most patients have a
low-grade fever, headache, vomiting, and rash. Aseptic meningitis is common in
the second stage, symptoms of which include headache and photosensitivity (sensitivity of the eye to light). Leptospira
can affect the eyes and make them cloudy and yellow to orange colored. Vision
may be blurred.
Ten percent of the
persons infected with Leptospira develop a serious disease called Weil's
syndrome. The symptoms of Weil's syndrome are more severe than those described
above and there is no distinction between the first and second stages of
disease. The hallmark of Weil's syndrome is liver, kidney, and blood vessel
disease. The signs of severe disease are apparent after 3-7 days of illness. In
addition to those listed above, symptoms of Weil's syndrome include jaundice (yellow skin and eyes), decreased or no urine output,
hypotension (low blood pressure), rash, anemia (decreased number
of red blood cells), shock, and severe mental status changes. Red spots on the
skin, "blood shot" eyes, and bloody sputum signal that blood vessel
damage and hemorrhage have occurred.
Diagnosis
Leptospirosis can
be diagnosed and treated by doctors who specialize in infectious diseases.
During the bacteremic phase of the disease, the symptoms are relatively
nonspecific. This often causes an initial misdiagnosis because many diseases
have similar symptoms to leptospirosis. The later symptoms of jaundice and
kidney failure together with the bacteremic phase symptoms suggest
leptospirosis. Blood samples will be tested to look for antibodies to Leptospira
interrogans. Blood samples taken over a period of a few days would show an increase
in the number of antibodies. Isolating Leptospira bacteria from blood,
cerebrospinal fluid (performed by spinal tap), and urine samples is diagnostic
of leptospirosis. It may take six weeks for Leptospira to grow in
laboratory media. Most insurance companies would cover the diagnosis and
treatment of this infection.
Several diagnostic
tests for leptospirosis have been devised in the early 2000s that are more
accurate as well as faster than standard culture. One test uses flow cytometry
light scatter analysis; this method can evaluate a sample of infected serum in
as little as 90 minutes. A second technique is an IgM-enzyme-linked
immunosorbent assay (ELISA), which detects the presence of IgM antibodies to L.
interrogans in blood serum samples.
Treatment
Leptospirosis is
treated with antibiotics, penicillin (Bicillin, Wycillin), doxycycline
(Monodox), ibramycin, or erythromycin (E-mycin, Ery-Tab). As of the early
2000s, however, many doctors prefer to treat patients with ceftriaxone, which
is easier to use than intravenous penicillin. Ciprofloxacin may be combined
with other drugs in treating patients who develop uveitis. It is generally agreed that antibiotic treatment
during the first few days of illness is helpful. However, leptospirosis is
often not diagnosed until the later stages of illness. The benefit of
antibiotic treatment in the later stages of disease, however, is controversial.
A rare complication of antibiotic therapy for leptospirosis is the occurrence
of the Jarisch-Herxheimer reaction, which is characterized by fever, chills,
headache, and muscle pain.
Patients with
severe illness will require hospitalization for treatment and monitoring.
Medication or other treatment for pain, fever, vomiting, fluid loss, bleeding,
mental changes, and low blood pressure may be provided. Patients with kidney
failure will require hemodialysis to remove waste products from the blood.
Prognosis
The majority of
patients infected with Leptospira interrogans experience a complete
recovery. Ten percent of the patients will develop eye inflammation (uveitis)
up to one year after the illness. In the United States, about one out of every
100 patients will die from leptospirosis. Death is usually caused by kidney failure, but has also
been caused by myocarditis (inflammation of heart tissue), septic shock (reduced blood flow to the organs because of the bacterial
infection), organ failure, and/or poorly functioning lungs. Mortality is
highest in patients over 60 years of age.
Prevention
Persons who are at
an extremely high risk (such as soldiers who are training in wetlands) can be pretreated
with 200 mg of doxycycline once a week. As of the early 2000s, there are no
vaccines available to prevent leptospirosis in humans, although such vaccines
have been formulated by veterinarians for dogs, swine, and cattle.
There are many
ways to decrease the chances of being infected by Leptospira. These include:
·
Avoid swimming or wading in freshwater ponds and slowly moving streams,
especially those located near farms.
·
Do not conduct canoe or kayak capsizing drills in freshwater ponds. Use a swimming pool instead.
·
Boil or chemically treat pond or stream water before drinking it or cooking
with it.
·
Control rats and mice around the home.
·
Have pets and farm animals vaccinated against Leptospira.
·
Wear protective clothing (gloves, boots, long pants, and long-sleeved
shirts) when working with wet soil or plants.
Although numerous techniques are available for analysing the proteins
present in a cell, the vast majority of them aren't visual. And if there's one
thing that scientists like, it's to see what they're analysing. The spectra
produced by liquid chromatography-tandem mass spectrometry (LC-MS/MS) just
don't hit the spot, even if they do reveal a great deal about the proteins
present.
But it's not just a matter of personal preference, because it would also
be really useful for scientists to see where specific proteins are located in
the cell and how they're interacting with each other.
Unfortunately, at the moment, individual proteins in a cell are just too
small to be imaged. But large protein complexes, made up of a number of
proteins working as a single unit, can be viewed using a technique known as
cryo-electron tomography (cryoET).
CryoET is a form of electron microscopy, which produces detailed images
of cells and cellular components by firing streams of electrons at them. ET
involves using the electron beam to view the cell at various different angles,
producing numerous two-dimensional sections through the cell. These sections,
known as tomograms, are then combined in a computer to produce a composite
three-dimensional image of the cell and its components.
In cryoET, the cell being imaged is first frozen to cryogenic
temperatures (around the temperature of liquid nitrogen). This is required to
stabilise and protect the cell prior to exposure to the electron beam, which is
a fairly harsh process. Other means of protecting the cell are also available,
such as fixing the cell in formaldehyde (see Set my proteins free), but
freezing allows the cell to be studied in a more natural state.
The problem with cryoET is that it suffers from a high level of
background noise and so produces quite fuzzy images. Finding and identifying
specific protein complexes in these fuzzy images can often prove quite tricky.
This process involves comparing objects in the images with the structure of
known protein complexes in template libraries, but the poor resolution often
leads to incorrect identifications.
So to try to improve the accuracy, a team of Swiss and US scientists led
by Ruedi Aebersold from the Swiss Federal Institute of Technology's Institute
of Molecular Systems Biology in Zurich decided to combine cryoEC with LC-MS/MS.
The idea is to analyse the proteins in a cell with LC-MS/MS, in order to
identify specific protein complexes with accuracy, and then to try to find
these particular complexes in the three-dimensional images generated by cryoET.
So rather than searching essentially blind, scientists can focus on finding
those protein complexes that should definitely be present.
Aebersold and his team also developed a novel scoring function for
matching images generated by cryoET with the structures in a template library,
which they hoped would also improve the accuracy of the identification process.
They tested their novel technique on Leptospira interrogans, a bacterium
that causes the disease leptospirosis, which they chose primarily because it is
long and thin (see image), making it fairly easy to image with cryoET. First,
though, they analysed the bacterium with LC-MS/MS, detecting over 2,000 separate
proteins and identifying 26 protein complexes.
Of these 26 complexes, Aebersold and his team chose nine to find in the
images of L. interrogans produced by cryoET. These nine complexes were present
in L. interrogans at a range of different abundances, from over 1,000 copies
per cell to less than 100. They included complexes such as RNA polymerase II
and the ribosome, which are critical to the proper functioning of the cell, and
a number of less critical complexes involved in protein folding and degradation.
They found that although they could accurately identify the more abundant
complexes in the cryoET images, such as the ribosome and RNA polymerase II,
they still struggled with the less abundant complexes. So they conclude that
although combining cryoET with LC-MS/MS does go some way to improving the
accuracy of the identification process, a full solution will require advances
in cryoET technology.
The views represented in this article are solely those of the author and
do not necessarily represent those of John Wiley and Sons, Ltd.
Scanning electron micrograph of Leptospira interrogans.
Image courtesy of CDC/NCID/Rob Weyant
Morphology and Identification
A. Typical Organisms: Tightly coiled, thin, flexible spirochetes 5-15 JU,m long, with very fine
spirals 0.1-0.2 jU-m wide. One end of the organism is often bent, forming a
hook. There is active rotational motion, but no flagella have been discovered.
Electron micrographs show a thin axial filament and a delicate membrane. The spirochete
is so delicate that in the dark field it may appear only as a chain of minute
cocci. It does not stain readily but can be impregnated with silver.
Leptospira interrogans
B. Culture: Leptospirae grow best aerobically at
C. Growth
Requirements: Leptospirae derive energy
from oxidation of long chain fatty acids and cannot use amino acids or
carbohydrates as major energy sources- Ammonium salts are a main source of
nitrogen. Leptospirae can survive for weeks in water, particularly at alkaline
pH.
Antigenic Structure. The main strains of leptospirae isolated from humans or animals in
different parts of the world are all serologically related and exhibit marked
cross-reactivity in serologic tests. This indicates considerable overlapping
in antigenic structure, and quantitative tests and antibody absorption studies
are necessary for a specific serologic diagnosis. From many strains of
leptospirae, a serologically reactive lipopolysaccharide has been extracted
that has group reactivity.
Pathogenesis and Clinical Findings. Human infection results usually from ingestion of water or food
contaminated with leptospirae. More rarely, the organisms may enter through
mucous membranes or breaks in the skin. After an incubation period of 1-2
weeks, there is a variable febrile onset during which spirochetes are present
in the bloodstream. They then establish themselves in the paren-chymatous
organs (particularly liver and kidneys), producing hemorrhage and necrosis of
tissue and resulting in dysfunction of those organs (jaundice, hemorrhage,
nitrogen retention). The central nervous system is frequently invaded, and this
results in a clinical picture of ' 'aseptic meningitis. There may be lesions in skin and muscles
also. Often there is episcleral injection of the eye. The degree and distribution
of organ involvement vary in the different diseases produced by different
leptospirae in various parts of the world. Many infections are mild or
subclinical. Hepatitis is particularly frequent in patients with
leptospirosis. It is often associated with elevation of serum creatine
phosphokinase, whereas that enzyme is present in normal concentrations in viral
hepatitis.
Kidney
involvement in many animal species is chronic and results in the elimination of
large numbers of leptospirae in the urine; this is probably the main source of
contamination and infection of humans. Human urine also may contain spirochetes
in the second and third weeks of disease.
Agglutinating,
complement-fixing, and lytic antibodies develop during the infection. Serum
from convalescent patients protects experimental animals against an otherwise
fatal infection. Immunity resulting from infection in humans and animals
appears to be specific for leptospirae. Dogs have been artificially immunized
with killed cultures of leptospirae.
Diagnostic Laboratory Tests. Specimens consist of blood for microscopic examination, culture, and
inoculation of young hamsters or guinea pigs; and serum for agglutination
tests.
A. Microscopic Examination: Darkfield examination or thick smears stained by Giemsa's technique
occasionally show leptospirae in fresh blood from early infections. Darkfield
examination of centrifuged urine may also be positive.
B. Culture: Whole fresh blood can be cultured in diluted serum or on Fletcher's
semisolid medium or Stuart Leptospira
broth (each of which contains 10% rabbit serum).
C. Animal Inoculation: A sensitive technique for the isolation of leptospirae consists of the
intraperi-toneal inoculation of young hamsters or guinea pigs with fresh plasma
or urine. Within a few days, spirochetes become demonstrable in the peritoneal
cavity; on the death of the animal (8-14 days), hemor-rhagic lesions with
spirochetes are found in many organs.
D. Serology: Agglutinating antibodies attaining very high liters (1:10,000 or higher)
develop slowly in leptospiral infection, reaching a peak at 5-8 weeks after
infection. For agglutination tests, cultured leptospirae are used live and are
observed microscopically for clumping. Cross-absorption of sera may permit
identification of a species-specific antibody response. With live suspensions,
agglutination may be followed by lysis. Leptospiral cultures can adsorb to red
blood cells. These will clump in the presence of antibody. These
hemagglutination reactions are group-specific.
Immunity. A solid species-specific immunity (directed against individual serotypes)
follows leptospiral infection.
Treatment. In very early infection, antibiotics (penicillin, tetracyclines) have some
therapeutic effect but do not eradicate the infection.
Epidemiology, Prevention, and Control. The leptosplroses are essentially animal infections; human infection is
only accidental, following contact with water or other materials contaminated
with the excreta of animal hosts. Rats, mice, wild rodents, dogs, swine, and
cattle are the principal sources of human infection. They excrete leptospirae
in urine and feces both during the active illness and during the asymptomatic
carrier state. Leptospirae remain viable in stagnant water for several weeks;
drinking, swimming, bathing, or food contamination may lead to human
infection. Persons most likely to come in contact with water contaminated by
rats (eg, miners, sewer workers, farmers, fishermen) run the greatest risk of
infection. Children acquire the infection from dogs more frequently than do
adults. Control consists of preventing exposure to potentially contaminated
water and reducing contamination by rodent control. Dogs can receive
distemper-hepatitis-leptospirosis vaccinations.
Additional
material about laboratory diagnosis
LEPTOSPIROSIS. Leptospirosis is an acute infectious zoonotic
disease characterized by the primary involvement of the kidneys, liver,
nervous system, and circulatory organs.
The causative agent of leptospirosis is Leptospira interrogans.
Bacterioscopic,
bacteriological, biological, and serological methods of investigation arc
employed for the laboratory diagnosis of the disease. Tlie material to be
studied is blood, urine, and cerebrospinal fluid obtained from the patient.
Depending on the epidemiological peculiarities, one might additionally study
water, foodsluus, and animal excreta.
Bacterioscopic examination. Within 5 days after the onset of the disease Leptospira can be detected in
the blood, and later on, in the urine, cerebrospinal fluid, and parenchymatoiis
organs.
Withdraw 2-3
ml of blood from the vein and mix it with 2-3 ml of 2 per cent sodium citrate.
Isolate the plasma by 30-min centrifuga-Lion at 3000 X g and examine the
pellet.
Samples of
urine, cerebrospinal fluid, and suspension of postmortem organs are examined
immediately after tlieir collection, and their deposit is studied after 2-hour
centriHigation at. 4000 X g.
The material to be investigated is placed on a thin glass slide (no thicker
than
Leptospira
in a dark microscopic field
Bacteriological examination. Samples of blood, urine, and cerebrospinal fluid are inoculated (in an
amount of 5-20 drops) into 3-5 test tubes with a nutrient medium or distilled
water.
To obtain a
nutrient medium, to 3-5 ml of sterile distilled water (pH 7.2-7.4) add, under
sterile conditions, 30 per cent of normal rabbit serum inactivated for 30 min
at 56 °C.
A non-serum
Vervoort-WoIff medium (900 ml of distilled water,
Leptospira propagate in a
nutrient medium without affecting its appearance. The medium mimiiis
transparent throughout the period of observation. To detect the growth of
Leptospira. make a wet-mount preparation from each specimen 10 days after
inoculation and examine it by dark-field microscopy.
From the
test tube where the growth of Leptospira lias been noted transfer 0.5 ml
portions of the medium into 3 test tubes conlaining 5 ml of fresh nutrient.
medium each. The cultures are incubated for 7-10 days at 28-30 °C.
To
differentiate between pathogenic and saprophytic Leptospira, study their
biological, cultural, and biochemical properties. The most demonstrative in
this respect is tlie bicarbonate test which consists in the following: add
Isolated Leptospira are
identilied by determining their serological groups witli the lielp of a kit of
agglutinating sera. To date, 18 sero-groups have
been identified.
Serological diagnosis. Antibodies in the patient's blood serum appear beginning from tlie second
week of the disease. Their number grows and reaches tlie peak on the 14th-17th
day of the disease. To reveal antibodies, one employs the reactions of microscopic agglutination and lysis of Leptospira.
Dilute the patient' serum in multiple ratios varying from 1 : 10 to 1 : 1000.
Into a series of test tubes introduce 0.2~ml amounts of diluted serum and the
same quantity of a live culture of the diagnostic kit of Leptospira strains.
Following one-hour incubation at 37 °C, prepare a wet-mount preparation from the contents of each tube and
examine it by dark-field microscopy.
Serum antibodies in the first
dilutions induce lysis (complete dissolvement of Leptospira), partial lysis or
granular swelling of Leptospira. In subsequent dilutions of the serum one can
see agglutination (appearance of agglomerates). A positive reaction in a serum
dilution of at least 1 : 1000 is diagnostically significant.
A positive serological result
may be observed not only with Leptospira of a serogroup responsible for the
disease but also with Leptospira belonging to other serological groups. To
clinch the accurate serological diagnosis, one should reveal IgG which, unlike
IgM, are strictly specific. For this purpose, utilize 2-merkaptoethanol and
cysteine which selectively split IgM without affecting the serological activity
of IgG.
Inactivation
of IgM by cysteine is carried out by Chernokhvostova's technique. Cysteine
(314 mg) is diluted in 10 ml of
Compare the
results of the reaction in cysteine-treated and native sera. If there is no
difference in concentrations of antibodies in the two samples, they arc
believed to beiong to class G. The presence of IgM is indicated by the complete
absence of antibodies in the treated serum or by at least a four-fold decrease
in their titres in the test versus control sera.
Biological examination is carried out on guinea pigs, rabbits, and 2-4-week-old puppies. Citrate
blood, urine residue, and the examined culture are administered to animals
intraperitoneally or subcutaneously. If
the material contains Leptospira, the test animals develop fever in 5-7 days;
their visible mucous membranes become yellowish. Several days after inoculation
the animals die with manifestations of'hypothermia. All post-mortem organs are
yellowish in colour. Leptospira are detected in the liver, kidneys, lungs, and
adrenals, less frequently in other organs and tissues.
Puppies infected with
Leptospira-containing material develop a chronic process during which
Leptospira can be detected in the urine for a period of 3 months.
Borrelia
BORRELIA RECURRENTIS
Morphology and Identification
A. Typical Organisms: Borrelia recurrentis is an irregular spiral 10-30 mcm long and 0.3 mcm
wide. The distance between turns varies from 2 to 4 mcm (fig.1). The organisms
are highly flexible and move both by rotation and by twisting. B recurrentis stains readily with
bacteriologic dyes as well as with blood stains such as Giemsa's or Wright's
stain.
Borrelia recurrentis in blood smear.
B. Culture: The organism can be cultured in fluid media containing blood, serum, or
tissue; but it rapidly loses its pathogenicity for animals when transferred
repeatedly in vitro. Multiplication is rapid in chick embryos when blood from
patients is inoculated into the chorioallantoic membrane.
C. Growth Characteristics: Virtually nothing is known of the metabolic requirements or activity of
borreliae. At
D. Variation: The only significant variation of Borrelia
is with respect to its antigenic structure.
Antigenic Structure. Isolates of Borrelia from
different parts of the world, from different hosts, and from different vectors
(ticks or lice) either have been given different species names or have been
designated strains of B recurrenris.
Biologic differences between these strains or species do not appear to be
stable.
Agglutinins,
complement-fixing antibodies, and lytic antibodies develop in high titer after
infection with borreliae. Apparently the antigenic structure of the organisms
changes in the course of a single infection. The antibodies produced initially
may act as a selective factor that permits the survival only of anti genically
distinct variants. The relapsing course of the disease appears to be due to the
multiplication of such antigenic variants, against which the host must then
develop new antibodies. Ultimate recovery (after 3-10 relapses) is associated
with the presence of antibodies against several antigenic variants.
Pathology. Fatal cases show spirochetes in great numbers in the spleen and liver,
necrotic foci in other parenchyma-tous organs, and hemorrhagic lesions in the
kidneys and the gastrointestinal tract. Spirochetes have been occasionally
demonstrated in the spinal fluid and brains of persons who have had meningitis.
In experimental animals (guinea pigs, rats), the brain may serve as a
reservoir of borreliae after they have disappeared from the blood.
Pathogenesis and Clinical Findings. The incubation period is 3-10 days. The onset is sudden, with chills and an
abrupt rise of temperature. During this time spirochetes abound in the blood.
The fever persists for 3-5 days and then declines, leaving the patient weak but
not ill. The afebrile period lasts 4-10 days and is followed by a second attack
of chills, fever, intense headache, and malaise. There are from 3 to 10 such
recurrences, generally of diminishing severity. During the febrile stages
(especially when the temperature is rising), organisms are present in the
blood; during the afebrile periods they are absent. Organisms appear less
frequently in the urine.
Antibodies
against the spirochetes appear during the febrile stage, and it is possible
that the attack is terminated by their agglutinating and lytic effects. These
antibodies may select out antigenically distinct variants that multiply and
cause a relapse. Several distinct antigenic varieties of borreliae may be isolated
from a single patient's several relapses, even following experimental
inoculation with a single organism.
Diagnostic Laboratory Tests
A. Specimens: Blood obtained during the rise in fever, for smears and animal
inoculation.
B. Stained Smears: Thin or thick blood smears stained with Wright's or Giemsa's stain reveal
large, loosely coiled spirochetes among the red cells.
C. Animal Inoculation: White mice or young rats are inoculated intraperituneally with
blood-Stained films of tail blood are examined for spirochetes 2-4 days later.
D. Serology: Spirochetes grown in culture can serve as antigens for CF tests, but the
preparation of satisfactory antigens is difficult. Patients suffering from
epidemic (louse-borne) relapsing fever may develop agglutinins for Proteus OXK and also a positive VDRL.
Immunity. Immunity following infection is usually of short duration.
Treatment. The great variability of the spontaneous remissions of relapsing fever
makes evaluation of chemotherapeutic effectiveness difficult. Tetracy-clines,
erythromycin, and penicillin are all believed to be effective. Treatment for a
single day may be sufficient to terminate an individual attack.
Epidemiology, Prevention, and Control. Relapsing fever is endemic in many parts of the world. Its main reservoir
is the rodent population, which serves as a source of infection for ticks of
the genus Ornithodorus. The distribution
of endemic foci and the seasonal incidence of the disease are largely
determined by the ecology of the ticks in different areas. In the USA, infected
ticks are found throughout the West, especially in mountainous areas, but
clinical cases are rare. In the tick, Borrelia
may be transmitted transovarially from generation to generation.
Spirochetes
are present in all tissues of the tick and may be transmitted by the bite or by
crushing the tick. The tick-home disease is not epidemic. However, when an
infected individual harbors lice, the lice become infected by sucking blood;
4-5 days later, they may serve as a source of infection for other individuals
. The infection of lice is not transmitted to the next generation, and the
disease is the result of rubbing crushed lice into bite wounds. Severe
epidemics may occur in louse-infested populations, and transmission is favored
by crowding, malnutrition, and cold climate.
In endemic
areas human infection may occasionally result from contact with the blood and
tissues of infected rodents. The mortality rate of the endemic disease is low,
but in epidemics it may reach 30%.
Prevention
is based on avoidance of exposure to ticks and lice and on delousing
(cleanliness, insecticides). No vaccines are available.
Lyme disease. Lyme disease was named for the small Connecticut community in which the
disease was first recognized in 1975 It is an inflammatory disorder caused by
the spirochete Borrelia burgdorferi.
FILM: Lyme
bacteria Cyst formation with detail http://www.youtube.com/watch?v=lVmCa70bAxE
The
disease is transmitted to humans by Ixodes ticks The whitetailed deer and
white-tailed mouse are the reservoirs of B
burgdorfen. The distribution of the disease is restricted to regions with
these animal reservoirs and Ixodes
ticks. Increases in deer populations and human activities such as clearance of
woodland and hiking and camping in wooded areas have increased the likelihood
of being bitten by a deer tick. This has resulted in an increased number of
cases of Lyme disease Although the territory over which this disease has
occurred is increasing, the majority of the approximately 7,000 reported cases
of Lymc disease have occurred in the Northeast, upper Midwest, and California
Lyme disease
usually begins with a distinctive skin lesion. The circularly expanding annular
skin lesions are hardened (indurated) with wide borders and central clearing
Although these lesions may reach diameters of
Lyme disease
Recent
epidcmiologk and laboratory investigations indicate that Lyme disease may be a
different manifestation of erythema
chronicum miggrans, a syndrome long recognized in Europe. Both diseases arc
caused by the spirochete, Borrelia
burgdorferi, transmitted to humans by the bites of Ixodes ticks Similar spirochetes have been recovered from the blood
of Lvme disease patients and cultured from ixodes
ticks, and patients with Lyme disease have antibodies to the cultured
spirochetes. When this disease is diagnosed, penicillin is an effective
treatment. There is also a new vaccine that appears to be effective for
preventing Lyme disease
Epidemiology of Lyme disease. In October 1975 the Connucticut Department received independent calls
reporting multtpk eases of what speared to be arthritis m chiMren in lyme and
Old Lyme, rural towns in that state. Despite being assured by their physicians
that arthritis was not infectious, the callers were not satisfied An epidemic
investigation ensued in which the extent, characteristicis, mode of
transmission, and etiology of the cluster of cases were studied. As
characteristic of many epidemidiogical investigations, public health officiate
began by trying to locate all individuals who had sudden onset of swelling and
pain in the knee or other large joints lasting a weelk to several months. An
old, large skin rash, repeated attacks at intervals of a few months, fever, and
fatigue were the reported symptoms. State epidemiologists questioned parents
and physidans – asking were the cases related, were there other similar cases,
wasthis an infectious form of arthritis, and what forms of arthritis are
infectious? Next the epidemiologists determined the time, place, and personal
characteristics of these cases. The incidence of onset of disease seemed to
cluster in late spring and summer and lasted from a week to a few months The
cases were concentrated in three adjacent towns on the eastern side of the Connecticut
River and most patients lived in wooded areas near lakes and streams. Of the 51
cases, 39 were children about evenly split between boys and girls There were no
familial patterns. Epidemiologists created an epidemic curve, listing the cases
by the time of onset, and began calling the disease "Lyme arthritis".
The
clustering of cases, the fact that most began in late spring or summer and that
they were most frequently located in wooded areas along lakes or streams
suggested a disease transmitted by an arthropod A study was undertaken to
determine if this was a communicable disease. Cases of the disease were matched
with a similar group of control or unaffected persons for age, sex, and other
relevant factors. It was found that affected people were more likely to have a
household pet than those who were unaffected. Pet owners are more likely to
come in contact with ticks that their dogs and cats might pick up m me woods.
The importance of this finding was emphasized when combined with the fact that
one fourth of the patients reported that their arthritic symptoms were preceded
by an unusual skin rash that started as a red spot that spread to a 6-inch
ring. A dermatology consultant recalled a similar skin outbreak reported in
Switzerland in 1910 that was attributed to tick bites.
This was
only suggestive evidence that a tick bite might initiate an infectious disease.
The connection between the rash and the disease had to be strengthed.
Now public
health authorities had to ask if patients with such a rash always progress to
develop Lyme arthritis. A prospective study lookcd for new patients with a
rash. Of 32 new cases of the characteiastic skin rash, 19 progressed to show
signs and symptoms of Lyme disease. The tick connection was strengthened after
an entomological study found that adult ticks were 16 times more abundant on
the east side of the Connecticut River than the west This corresponded to the proportion of incidence of the disease on
each side of the river. Also, more tick bites were reported by the arthritis
sufferers then by their unaffected neighbors A surveillance network was set up
in. Connecticut and surrounding stales to gather information about other cases.
These investigations showed more adult victims than children and also more
serious manifestations, including neurological and heart diseases.
The Rocky
Mountain Public Health Laboratory in Montana was asked to assist in the
investigation because of its expertise in the area of tickborne disease. They
found unusual spirochetes in the guts of many of the ticks sent from
Connecticute. Spirochetes, which are bacteria with curved cells wound around a
central filament, are often difficult to culture and so it would be difficult
to prove that these were the causative organisms of Lyme disease Therefore they
first tried to infect laboratoiy animals with the infected ticks The rabbits
developed rashes resembling those seen in humans. A spirochete was isolated
ftom the ticks and when pure cultures were inoculated into rabbite, the rabbits developed the characteristic
rash. The infected rabbits contained antispirochetal antibodies in their
serum. The identification was complete
when the spirochete was isolated from human cases. The spirochete was
classified as a member of the Borrelia genus
and named Borrelia burgdorferi after
the entomologist who discovered the organisms in the ticks.
Lyme disease accounted for more
than 90 % of the vector-home
infectious diseases in the United States in 1992. The distribution of the disease
was highly correlated with the distribution, of the principal tick vectors. The
nearly twerrtyfold increase in cases since the early 1980s may be a consequense
of increased surveillance and improved diagnostic methods, or a real increase
in disease prevalence due to increases in deer and tick populations and closer
human contact with these animals.
Summary. Lyme disease is characterized by the development of arthritis and
neurological symptoms that result when the body's immune defenses react to infections
with the spirochete Borrelid burgdorferi.
The
occurrence of Lyme disease has been concentrated in the Northeast and other
areas where Ixodes ticks carry Borrelia burgdorferi.
Additionala
material abour laboratory diagnosis
RECURRENT FEVER (EPIDEMIC). The causative agent of epidemic (louse-borne) relapsing fever (Borrelia recurrentis), of endemic
(tick-borne) typhus (Borrelia persica) and
some other microorganisms induce clinically similar acute infectious diseases
whose course is characterized by fever attacks, general intoxication, and
liver and spleen enlargement.
The main method of
investigation in recurrent types of typhus is bacterioscopic examination, that
is visualization of the causal organism in the patient's blood.
Bacterioscopic examination. During or before an attack of relapsing fever take a sample of blood from
the patient and make a thick-drop preparation and a smear. First, stain a
thick-drop preparation, since its examination provides better possibilities to
obtain a positive result. On a dried unfixed preparation of a blood drop pour
the Romanowsky-Giemsa dye and allow it to act for 35-45 min. Haemo-lysis occurs
in a drop (the dye is diluted with distilled water), so 5 min later replace the
dye with a fresh portion which is kept in place for 30-40 min. Then, the
preparation is carefully washed with water and dried. Under the microscope
Borrelia look like thin blue-violet threads with several large coils (secondary
coils). The length of Borrelia varies from 10 to 30 u-m or more. Sometimes
their size is several times larger than the diameter of leucocytes (the
preparation contains only leucocytes because erythrocytes are lysed). When
Borrelia are present in large numbers, which is characteristic of relapsing fever,
their aggregates may pose difliculty with regard to their differentiation from
fibrin threads. In this case examine micro-scopically a smear which is dried,
fixed with alcohol or Nikiforov's mixture, and stained with the
Romanowsky-Giemsa dye or Pfeiffer's fuchsine. Borrelia are readily visualized
by microscopy. They are at least twice as long as the diameter of red blood
cells. If no Borrelia are found in a thick blood film, smears are not subjected
to examination.
When the patient is afebrile,
Borrelia cannot he detected by the ordinary methods of investigation. Hence,
enrichment methods are recommended to be used in such cases. Withdraw from the
vein 8-10 ml of blood, wait until it has coagulated, separate the serum, and
centrifuge it at 3000 X g for 45-60 min. Then, quickly pour off the fluid by
inverting the test tube, make wet-mount preparations from the sediment (which
are examined by dark-field microscopy), and thick smears (which are fixed,
stained, and studied in the manner used with blood smears).
Biological examination. To confirm the diagnosis of tick-borne recurrent typhus, inject,
pubciitaneously 0.5-1 ml of citrate blood to guinea pigs. Five-six day? later
one can observe large numbers of Borrelia in the blood of the infected animals.
This method permits the differentiation of the causative agents of epidemic
versus tick-borne recurrent typhus fever. The latter induce the disease in
guinea pigs, whereas B. recurrentis
is non-pathogenic for them.
Rickettsiae
Rickettsiae are small bacteria that are obligate intracellular parasites
and – except for Q fever – are transmitted to humans by arthropods. At least 4
rickettsiae (Rickettsia rickettsii,
Rickettsia conorii, Rickettsia
tsuisugamushi. Rickettsia akari) – and perhaps others – are
transmitted transovarially in the ar-thropod, which serves as both vector and
reservoir. Rickettsial diseases (except Q
fever) typically exhibit fever, rashes, and vasculitis. They are grouped on the
basis of clinical features, epidemiologic aspects, and immunologic
characteristics (see Table).
Table.
Rickettsial diseases
|
Disease |
Rickettsia |
Geographic Area of Prevalence |
Insect Vector |
Mammalian Reservoir |
Weil-Felix
Agglutination |
||
|
0X19 |
0X2 |
OXK |
|||||
|
Typhus
group Epidemic typhus |
Rickettsia
prowazekii |
South America, Africa, Asia |
Louse |
Humans |
++ |
+ |
- |
|
Murine typhus |
Rickettsia
typhi |
Worldwide; small |
Flea |
Rodents |
++ |
- |
- |
|
Scrub typhus |
Rickettsia
tsutsugamushi |
Southeast Asia, Japan |
Mite* |
Rodents |
- |
- |
++ |
|
Spotted fever group Rocky
Mountain spotted fever (RMSF) |
Rickettsia
rickettsii |
Western hemisphere |
Tick* |
Rodents, dogs |
+ |
+ |
- |
|
Fievre
boutonneuse Kenya tick Typhus South African tick fever Indian tick typhus |
Rickettsia
conorii |
Africa, India, Mediterranean |
Tick* |
Rodents, dogs |
+ |
+ |
– |
|
Queensland tick typhus |
Rickettsia
australis |
Australia |
Tick* |
Rodents, marsupials |
+ |
+ |
– |
|
North Asian tick typhus |
Rickettsia
sibirica |
Siberia, Mongolia |
Tick* |
Rodents |
4 |
+ |
- |
|
Rickettsial pox |
Rickettsia
akari |
USA. Korea, Russia |
Mite* |
Mice |
- |
- |
- |
|
RMSF-like |
Rickettsia
Canada |
North America |
Tick* |
Rodents |
? |
? |
- |
|
Other Q fever |
Coxiella
burnetii |
Worldwide |
None** |
Cattle, sheep, goats |
– |
– |
-– |
|
Trench fever |
Rochalimaea
quintana |
Rare |
Louse |
Humans |
|
|
|
*Also serve as arthropod reservoir, by maintaining the rickettsiae through transovarian
transmission.
**Human
infection results from inhalation of dust.
Properties of Rickettsiae. Rickettsiae are pleomorphic, appearing either as short rods, 600 x 300 nm in
size, or as cocci, and they occur singly, in pairs, in short chains, or in
filaments. When stained, they are readily visible under the optical microscope.
With Giemsa's stain they stain blue; with Macchiavello's stain they stain red
and contrast with the blue-staining cytoplasm in which they appear.
A wide range
of animals are susceptible to infection with rickettsial organisms-
Rickettsiae grow readily in the yolk sac of the embryonated egg (yolk sac
suspensions contain up to 109 rickettsial particles per milliliter).
Pure preparations of rickettsiae can be obtained by differential centrifugation
of yolk sac suspensions. Many rickettsial strains also grow in cell culture.
Purified
rickettsiae contain both RNA and DNA
in a ratio of 3.5:1 (similar to the ratio in bacteria). Rickettsiae have cell
walls made up of peptidoglycans containing muramic acid, resembling cell walls
of gram-negative bacteria, and they divide like bacteria. In cell culture, the
generation time is 8-10 hours at
Purified
rickettsiae contain various enzymes concerned with metabolism. Thus they
oxidize intermediate metabolites like pyruvic, succinic, and glutamic acids
and can convert glutamic acid into aspartic acid. Rickettsiae lose their
biologic activities when they are stored at
Rickettsiae
may grow in different parts of the celt. Those of the typhus group are usually
found in the cytoplasm; those of the spotted fever group, in the nucleus. Thus
far, one of the rickettsiae, Rochalimaea
quintana, has been grown on cell-free media. It has been suggested that
rickettsiae grow best when the metabolism of the host cells is low. Thus, their
growth is enhanced when the temperature of infected chick embryos is lowered to
Rickettsial
growth is enhanced in the presence of sulfonamides, and rickettsial diseases
are made more severe by these drugs. Para-aminobenzoic acid (PABA), the
structural analog of the sulfonamides, inhibits the growth of rickettsial
organisms. Tetracy-clines or chloramphenicol inhibits the growth of rickettsiae
and can be therapeutically effective.
In general,
rickettsiae are quickly destroyed by heat, drying, and bactericidal chemicals.
Although rickettsiae are usually killed by storage at room temperature, dried
feces of infected lice may remain infective for months at room temperature.
The organism
of Q fever is the rickettsial agent most resistant to drying. This organism may
survive pasteurization at
Rickettsial Antigens and Antibodies. A variety of rickettsial antibodies are known; all of them participate in
the reactions discussed below. The antibodies that develop in humans after vaccination
generally are more type-specific than the antibodies developing after natural
infection.
A. Agglutination of Proteus vulgaris (Weil-Felix Reaction): The Weil-Felix reaction is commonly used in diagnostic work. Rickettsiae
and Proteus organisms appear to
share certain antigens. Thus, during the course of rickettsial infections,
patients develop antibodies that agglutinate certain strains ofP vulgaris. For example, the Proteus strain 0X19 is agglutinated
strongly by sera from persons infected with epidemic or endemic typhus; weakly
by sera from those infected with Rocky Mountain spotted fever; and not at all
by those infected with Q fever. Convalescent sera from scrub typhus patients
react most strongly with the Proteus
strain OXK (Table 1).
B. Agglutination of Rickettsiae: Rickettsiae are agglutinated by specific antibodies. This reaction is very
sensitive and can he diagnostically useful when heavy rickettsial suspensions
are available for mi-croagglutination tests.
C. Complement Fixation With Rickettsial
Antigens: Complement-fixing antibodies
are commonly used in diagnostic laboratories. A 4-fold or greater antibody
titer rise is usually required as laboratory support for the diagnosis of
acute rickettsial infection. Convalescent liters often exceed 1:64.
Group-reactive soluble antigens are available for the typhus group, the spotted
fever group, and Q fever. They originate in the cell wall. Some insoluble
antigens may give species-specific reactions.
D. Immunonuorescence Test With Rickettsial
Antigens: Suspensions of rickettsiae
can be partially purified from infected yolk sac material and used as antigens
in indirect immunofluorescence tests (see p 168) with patient's serum and a
fluore see in-labeled antihuman globulin. The results indicate the presence of
partly species-specific antibodies, but some cross-reactions are observed.
Antibodies after vaccination are IgG; early after infection, IgM.
E. Passive Hemagglutination Test: Treated red blood cells adsorb soluble antigens and can then be
agglutinated by antibody.
F. Neutralization of Rickettsial Toxins: Rickettsiae contain toxins that produce death in animals within a few
hours after injection. Toxin-neutralizing antibodies appear during infection,
and these are specific for the toxins of the typhus group, the spotted fever
group, and scrub typhus rickettsiae. Toxins exist only in viable
rickettsiae—inactivated rickettsiae are nontoxic.
Pathology
Rickettsiae
multiply in endothelial cells of small blood vessels and produce vasculitis.
The ceils become swollen and necrotic; there is thrombosis of the vessel,
leading to rupture and necrosis. Vascular lesions are prominent in the skin,
but vasculitis occurs in many organs and appears to be the basis of hemostatic
disturbances. In the brain, aggregations of lym-phocytes, polymorphonuclear
leukocytes, and mac-rophages are associated with the blood vessels of the gray
matter; these are called typhus nodules. The heart shows similar lesions of the
small bloodvessels. Other organs may also be involved.
Immunity. In cell cultures of macrophages, rickettsiae are phagocytosed and replicate
intracellularly even in the presence of antibody. The addition of lymphocytes
from immune animals stops this multiplication in vitro. Infection in humans is
followed by partial immunity to reinfection from external sources, but relapses
occur.
Clinical Findings. Except for Q fever, in which there is no skin lesion, rickettsial
infections are characterized by fever, headache, malaise, prostration, skin
rash. and enlargement of the spleen and liver.
A. Typhus Group:
1. Epidemic typhus – In epidemic typhus, systemic infection and prostration are severe, and
fever lasts for about 2 weeks. The disease is more severe and is more often
fatal in patients over 40 years of age. During epidemics, the case mortality
rate has been 6-30%.
2. Endemic typhus-The clinical picture of endemic typhus has many features in common with that
of epidemic typhus, but the disease is milder and is rarely fatal except in
elderly patients.
B. Spotted Fever Group: The spotted fever group resembles typhus clinically; however, unlike the
rash in other rickettsial diseases, the rash of the spotted fever group usually
appears first on the extremities, moves centripetally, and involves the palms
and soles. Some, like Brazilian spotted fever, may produce severe infections;
others, like Mediterranean fever, are mild. The case mortality rate varies
greatly. In untreated Rocky Mountain spotted fever, it is usually much greater
in older age groups (up to 60%) than in younger people.
Rickettsialpox
is a mild disease with a rash resembling that of varicella. About a week
before onset of fever, a firm red papule appears at the site of the mite bite
and develops into a deep-seated vesicle that in turn forms a black eschar (see
below).
C. Scrub Typhus: This disease resembles epidemic typhus clinically. One feature is the
eschar, the punched-out ulcer covered with a blackened scab that indicates the
location of the mite bite. Generalized lymphadenopathy and lymphocytosis are
common. Localized eschars may also be present in the spotted fever group.
D. Q Fever: This disease resembles influenza, nonbacterial pneumonia, hepatitis, or
encephalopathy rather than typhus. There is no rash or local lesion. The Weil-Felix
test is negative. Transmission results from inhalation of dust contaminated
with rickettsiae from dried feces, urine, or milk.
E. Trench Fever: The disease is characterized by the headache, exhaustion, pain, sweating,
coldness of the extremities, and fever associated with aroseolar rash. Relapses
occur. Trench fever has been known only among armies during wars in central
Europe.
Laboratory Findings. Isolation of rickettsiae is technically quite difficult and so is of only
limited usefulness in diagnosis. Whole blood (or emulsified blood clot) is
inoculated into guinea pigs, mice, or eggs. Rickettsiae are recovered most
frequently from blood drawn soon after onset, but they have been found as late
as the 12th day of the disease.
If the guinea pigs fail to show disease .(fever, scrotal swellings,
hemorrhagic necrosis, death), serum is collected for antibody tests to
determine if the animal has had an inapparent infection.
Some
rickettsiae can infect mice, and rickettsiae are seen in smears of peritoneal
exudate. In Rocky Mountain spotted fever, skin biopsies taken from patients
between the fourth and eighth days of illness reveal rickettsiae by
immunofluorescence stain.
The most
sensitive and specific serologic tests are
microimmunofluorescence, microagglutination, and complement fixation. An
antibody rise should be demonstrated during the course of the illness.
Treatment. Tetracyclines and chloramphenicol are effective provided treatment is
started early. Tetracycline, 2-
Epidemiology. A variety of arthropods, especially ticks and mites, harbor Rickettsia organisms in the cells that
line the alimentary tract. Many such organisms are not evidently pathogenic for
humans.
The life
cycles of different rickettsiae vary:
(1) Ricketlsia prowazekii has a life cycle
limited to humans and to the human louse (Pediculus
corporis and Pediculus capitis).
The louse obtains the organism by biting infected human beings and transmits
the agent by fecal excretion on the surface of the skin of another person.
Whenever a louse bites, it defecates at the same time. The scratching of the
area of the bite allows the rickettsiae excreted in the feces to penetrate the
skin. As a result of the infection the louse dies, but the organisms remain
viable for some time in the dried feces of the louse. Rickettsiae are not
transmitted from one generation of lice to another. Typhus epidemics have been
controlled by delousing large proportions of the population with insecticides.
Brill's
disease is a recrudescence of an old typhus infection. The rickettsiae can
persist for many years in the lymph nodes of an individual without any symptoms
being manifest. The rickettsiae isolated from such cases behave like classic R prowaz.ekii; this suggests that humans
themselves are the reservoir of the rickettsiae of epidemic typhus. Flying
squirrels in the USA may provide an extrahiiman reservoir, and human cases have
occurred after bites by ectopara-sites. Epidemic typhus epidemics have been
associated with war and the lowering of standards of personal hygiene, which in
turn have increased the opportunities for human lice to flourish. If this
occurs at the time of recrudescence of an old typhus infection, an epidemic may
be set off. Brill's disease occurs in local populations of typhus areas as well
as in persons who migrate from such areas to places where the disease does not
exist. Serologic characteristics readily distinguish Brill's disease from
primary epidemic typhus. Antibodies arise earlier and are IgG rather than the
IgM detected after primary infection. They reach a maximum by the tenth day of
disease. The Weil-Felix reaction is usually negative. This early IgG antibody
response and the mild course of the disease suggest that partial immunity is
still present from the primary infection.
(2) Rickettsia typhi has its reservoir in
the rat, in which the infection is inapparent and long-lasting. Rat fleas carry
the rickettsiae from rat to rat and sometimes from rat to humans, who develop
endemic typhus. Cat fleas can serve as vectors. In endemic typhus, the flea
cannot transmit the rickettsiae transovarially.
(3) Rickettsia tsuisugumushi has its true
reservoir in the mites that infest rodents. Rickettsiae can persist in rats for
over a year after infection. Mites transmit the infection transovariaily.
Occasionally, infected mites or rat fleas bite humans, and scrub typhus
results. The rickettsiae persist in the mite-rat-mite cycle in the scrub or
secondary jungle vegetation that has replaced virgin Jungle in areas of partial
cultivation. Such areas may become infested with rats and trombiculid mites.
(4) Rickettsia rickettsii may be found in
healthy wood ticks (Dermacentor
andersoni) and is passed transovarially. Vertebrates such as rodents, deer,
and humans are occasionally bitten by infected ticks in the western USA. In
order to be infectious, the tick carrying the rickettsiae must be engorged
with blood, for this increases the number of rickettsiae in the tick. Thus,
there is a delay of 45-90 minutes between the time of the attachment of the
tick and its becoming infective. In the eastern USA, Rocky Mountain spotted
fever is transmitted by the dog tick Dermacentor
variahiiis. Dogs are hosts to dog ticks but rarely, if ever. serve as a
continuing source of tick infection. Most Rocky Mountain spotted fever in the
USA now occurs in the eastern and the southeastern regions.
(5) Rickettsia akari has its vector in
blood-sucking mites of the species Allodermanyssus
san-guineus. These mites may be found on the mice (Mus musculus) trapped in apartment houses in the USA where
rickettsialpox has occurred. Transovarial transmission of the rickettsiae
occurs in the mite. Thus the mite may act as a true reservoir as well as a
vector. R akari has also been
isolated in Korea.
(6) Rochalimaea quintana is the causative
agent of trench fever; it is found in lice and in humans, and its life cycle is
like that of R prowazekii. The
disease has been limited to fighting armies. This organism can be grown on
blood agar in 10% COa.
(7) Coxiella burnetii is found in ticks,
which transmit the agent to sheep, goats, and cattle. Workers in
slaughterhouses and in plants that process wool and cattle hides have
contracted the disease as a result of handling infected animal tissues. C burnetii is transmitted by the
respiratory pathway rather than through the skin. There may be a chronic
infection of the udder of the cow. In such cases the rickettsiae are excreted
in the milk and occasionally may be transmitted to humans by ingestion or
inhalation.
Infected
sheep may excrete C burnetii in the
feces and urine. The placentas of infected cows and sheep contain the
rickettsiae, and parturition creates infectious aerosols, The soil may be
heavily contaminated from one of the above sources, and the inhalation of
infected dust leads to infection of humans and livestock. It has been proposed
that endospores formed by C burnetii
contribute to its persistence and dissemina-tion. Coxiella infection is now widespread among sheep and cattle in the
USA. Coxiella can cause endocarditis
in humans in addition to pneumonitis and hepatitis.
Geographic Occurrence. A. Epidemic Typhus: Potentially worldwide, it has disappeared from the USA, Britain, and Scandinavia.
It is still present in the Balkans, Asia, Africa, Mexico, and the Andes. In
view of its long duration in humans as a latent infection (Brill's disease), it
can flourish quickly under proper environmental conditions, as it did in
Europe during World War II as a result of the deterioration of community sanitation.
B. Endemic, Murine Typhus: Worldwide, especially in areas of high rat infestation. It may exist in
the same areas as—and may be confused with— epidemic typhus or scrub typhus.
C. Scrub Typhus: Far East, especially Burma, India, Ceylon, New Guinea, Japan, and Taiwan. Trombicula pallida, the chigger most
often found in Korea, maintains the infection among wild rodents of Korea (Apodemus agrarius), but only
infrequently does it transfer scrub typhus to humans.
D. Spotted Fever Group: These infections occur around the globe, exhibiting as a rule some
epidemiologic and immunologic differences in different areas. Transmission by
a tick of the Ixodidae family is common to the group. The diseases that are
grouped together include Rocky Mountain spotted fever (western and eastern
RMSF), Colombian, Brazilian, and Mexican spotted fevers; Mediterranean
(boutonneuse), South African tick, and Kenya fevers;
North
Queensland tick typhus; and North Asian tick-borne rickettsiosis.
E. Rickettsialpox: The human disease has been found among inhabitants of apartment houses in
the northern USA. However, the infection also occurs in Russia, Africa, and
Korea.
F. Q Fever: The disease is recognized around the world and occurs mainly in persons
associated with goats, sheep, or dairy cattle. It has attracted attention
because of outbreaks in veterinary and medical centers where large numbers of
people were exposed to animals shedding Coxiella.
Seasonal Occurrence. Epidemic typhus is more common in cool climates, reaching its peak in
winter and waning in the spring. This is probably a reflection of crowding,
lack of fuel, and low standards of personal hygiene, which favor louse
infestation.
Rickettsial
infections that must be transmitted to the human host by vector reach their
peak incidence at the time the vector is most prevalent—the summer and fall
months.
Control. Control is achieved by breaking the infection chain or by immunizing and
treating with antibiotics.
A. Prevention of Transmission by Breaking the Chain
of Infection:
1. Epidemic typhus-Delousing with insecticide.
2. Murine typhus-Rat-proofing buildings
and using rat poisons.
3. Scrub typhus-Clearing from campsites the secondary jungle vegetation in which rats and mites
live.
4. Spotted fever-Similar measures for the spotted fevers may be used; clearing of
infested land;
personal
prophylaxis in the form of protective clothing such as high boots, socks worn
over trousers; tick repellents; and frequent removal of attached ticks.
5.
Rickettsial pox-Elimination of rodents and their parasites from human
domiciles.
B. Prevention of Transmission of Q Fever by
Adequate Pasteurization of Milk: The
presently recommended conditions of "high-temperature, short-time
"pasteurization at
C.
Prevention by Vaccination: Active
immunization may be carried out using formalinized antigens prepared from the
yolk sacs of infected chick embryos or from cell cultures. Such vaccines have
been prepared for epidemic typhus (R
prowazekii), Rocky Mountain spotted fever (R ricketlsii), and Q fever (C
burnelii). However, commercially produced vaccines are not available in the
USA in 1982. Cell culture-grown , inactivated suspensions of rickettsiae are
under study as vaccines. A live vaccine (strain E) for epidemic typhus is
effective and used experimentally but produces a self-limited disease.
D. Chemoprophylaxis: Chloramphenicol has been used as a chemoprophylactic agent against scrub
typhus in endemic areas. Oral administration of 3-g doses at weekly intervals
controls infection so that no disease occurs even though rickettsiae appear in
the blood. The antibiotic must be continued for a month after the initiation of
infection to keep the person well. Tetracyclines may be equally effective.
Diagnosis
of RICKETTSIAL INFECTIONS in details
Rickettsioses
present a group of infectious diseases wliicli are induced by Rickettsia and
affect both humans and animals.
The laboratory
diagnosis of rickettsioses largely relies on the use of serological methods of
investigation, including such reactions as A, CF, IHA, IF, and some others.
Some peculiarities of conducting-these reactions in individual rickettsioses
are described in the corresponding sections. Rickettsia are isolated from the
patient’s blood and other material only for scientific purposes.
Isolation is performed in special laboratories by infecting experimental
animals, chicken embryos, or insects.
Epidemic Typhus Fever. The causal organism of epidemic typhus and Brill-Symmers disease (Rickettsia prowazekii) induces in man
an acute illness characterized by a sudden onset, liigli pyrexia, and
roseolopetechial rash. For the laboratory diagnosis of these diseases one
usually employs such tests as CF, IHA, A with R. prowazeku, indirect haemolysis, and IF.
Serological diagnosis. The CF reaction allows to
diagnose both an active pathological process and a history of the illness
(retrospective diagnosis). The reaction is performed according to the
conventional technique employed for isolating antibodies in the blood serum of
patients and individuals with a history of the disease. Blood samples are
withdrawn from the patient on the 5th-7lh day of the illness. The serum is
diluted in ratios ranging from 1 : 10 to 1 : 320. Pickettsial suspension (a
corpuscular antigen), well purified and vacuum-dried, is usually used as an
antigen.
Complement
fixation (phase 1 of the reaction) is carried out at +
The agglutination reaction with R. prowazekii is positive in patients
with epidemic typhus fever in almost all cases. The reaction is diagnostically
significant in serum dilutions of 1:40-1:80 but increasing litres of antibodies
in paired sera are a more reliable diagnostic indicator (tables).
Table
Agglutination reaction with Rickettsia
prowazekii antigen or Rickettsia typhi antigen
|
Ingredient, ml |
Number
of the test tubes |
|||||||||
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
Isotonic sodium chloride
solution |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
|
Patient's serum diluted 1:5 |
0,2 |
® |
® |
® |
® |
® |
® |
¯ |
0,2 |
– |
|
Dilution |
1:10 |
1:20 |
1:40 |
1:80 |
1:160 |
1:320 |
1:640 |
1:1280 |
– |
– |
|
Rickettsia
prowazekii antigen or Rickettsia typhi antigen |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
– |
0,2 |
|
|
Temperature 37 °C, 18-20 h |
|||||||||
|
Results |
|
|
|
|
|
|
|
|
|
|
The IHA reaction is considered positive if
haemagglutination is observed in a 1:250 serum dilution (tabl.).
Table
Indirect hemagglutination test
|
Ingredient, ml |
Number
of the test tubes |
||||||||||
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
|
Isotonic sodium chloride
solution |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
– |
0,4 |
0,4 |
|
Patient's serum diluted 1:62,5 |
0,4 |
® |
® |
® |
® |
® |
® |
¯ |
– |
– |
– |
|
Dilution |
1:125 |
1:250 |
1:500 |
1:1000 |
1:2000 |
1:4000 |
1:8000 |
1:16000 |
Control of serum |
Control of antigen |
Control of erythrocytess |
|
Erythrocytic diagnosticum
with Rickettsial antigens |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
– |
0,1 |
– |
|
Sheep erythrocytes 1 % |
– |
– |
– |
– |
– |
– |
– |
– |
0,1 |
– |
0,1 |
|
|
Temperature 30 °C 18 h |
||||||||||
|
Results |
|
|
|
|
|
|
|
|
|
|
|
The reaction of indirect haemolysis is distinguished
by high sensitivity and speciticity. Moreover, it allows the detection of
antibodies at early stages and can be used as a rapid method of diagnosis since
one can read the presumptive results already 30 min after its performance.
A typhus antigen
required for the reaction is adsorbed on sheep erythrocytes (to 4 ml of the
antigen add 0.15 ml of washed off sheep red blood cells). The mixture is
incubated for 1 h and then centrifuged for 8-10 min at 2500 X s. The residue of erythrocytes is suspended
in 10 ml of isotonic sodium chloride solution. The serum tested is inactivated
at
A drop modification of the reaction of
indirect haemolysis. The patient's serum preliminarily diluted 1:50 is
introduced into an agglutination tube containing 4 drops of isotonic sodium
chloride solution to obtain a 1:100 dilution. From this test tube transfer
4 drops into
the next test tube with 4 drops of saline solution
(dilution 1:200) and so on until a 1:3200 dilution is achieved. Then, to each
test tube add 1 drop of the typhus antigen and 1 drop of complement diluted
1:10. Shake the tubes and incubate them for 1 h at
The IF reaction is conducted in the
following manner: on fixed Rickettsia smears place sequential dilutions of the
tested serum and incubate them in a moist chamber at 37 "C for 15-20 min.
Then, wash the smears for 1 min with a light stream of water and place for 10
min into a cuvette with phosphate buffer solution, wash with distilled water,
and dry in the air. After that, apply on the smears antiglobulin luminescent
serum taken in the working dilution indicated on the ampoule label, incubate
them for 20 min in a moist chamber, wash with water, dry, and study by
luminescent microscopy.
Endemic Typhus Fever. The causal organism of endemic (murine) typhus fever (Rickettsia typhi) induces a disease whose clinical manifestations
are similar to those of epidemic typhus fever.
Serological diagnosis. To differentiate between endemic and epidemic typhus fever, one sets up
at the same time agglutination reactions
with antigens from R. prowazekii
and R. mooseri, as well as theCF and
IHA tests. In case of endemic typhus fever the diagnostic titre of the serum
with R. mooseri is 3-4 times as high as that of the serum with R. prowazekii.
Biological examination. Male guinea pigs are infected intra-peritoneally with patients' blood. The
appearance of a scrotal reaction, fever, and also detection of Rickettsia in
scrapings from the testi-cular membranes verifies the diagnosis of endemic
typhus fever.
Q Fever. The causative agent of Q fever (Coxiella
burnetii) induces an acute infectious disease characterized by a
polymorphic clinical picture, sometimes by a subacute and chronic course. In
the laboratory diagnosis of this type of rickettsiosis one utilizes serological
reactions (A, CF, IHA), allergy, and biological tests.
Serological diagnosis. The agglutination reaction is
made at the second week of the disease (on the 10th-12th day). The patient's
blood serum, because of its low levels of agglutinins, is diluted from 1:4 to
1:64 and decanted into the test tubes in 0.25-ml portions. Then, 0.25 ml of the
antigen is added to each test tube. The results of the reaction are read after
18-20-hour incubation of the test tubes. The presence of the reaction in
dilutions 1:4 and over is considered positive. The agglutination reaction
should be repeated to establish a growth in the antibody titre (the principle
of paired sera).
The CF test may be positive at the end of
the first week of the disease. Complement-fixating antibodies reach their
highest concentration in the serum during the third week of the disease. The
procedure of the reaction is the same as in typhus fever. The IHA test is more sensitive than
complement fixation.
The allergy cutaneous test is carried out
according to the standard procedure. Corpuscular antigen from Rickettsia burnetii is used as an
allergen. The results of the test are read 24-48 hrs after the administration
of 0.1 ml of the allergen and assessed by the size of the infiltrate and
hyperaemia which is occasionally attended by oedema. The reaction is specific
but can be used only for retrospective diagnosis. In individuals with a history of Q fever
the test remains positive for 9-10 years.
Biological examination. Inoculation of guinea pigs makes it possible to isolate the causal
organism of Q fever. Take 3-5 ml of blood from patients in the febrile period and administer to a guinea pig by either intraperitoneal or
intratesticular route (0.3-0.5 ml of the infective blood deep into the
testicles). With material from the infected guinea pig inoculate chicken
embryos by the Cox procedure. In repeated passages
Rickettsia transform from filtrable to visible forms and are detected by the
Romanowsky-Giemsa and Zdrodovsky techniques.
Tsuteugamushi Disease (Scrub
Typhus). Scrub typhus is an acute
anthropozoonotic rickettsiosis characterized by multiple vasculites, fever,
and involvement of the nervous system and circulatory organs.
The causal organism of this infection is Rickettsia tsutsugamushi. For
the diagnostic purpose, one uses blood and serum obtained from febrile
patients.
Scrological diagnosis is carried out beginning from the second week of the disease and involves
the agglutination and complement-fixation tests. Proteus OX-K is utilized as an
antigen in the agglutination test.
R. tsutsugamushi may be
found by infecting with the material examined the subculture of L cells and
primary trypsin-treated fibroblasts of the chicken embryo.
Biological examination consists of the intraperitoneal inoculation of white mice with the
patient's blood. The animal dies 6-14 days after the inoculation. Prepare
impression-smears of the internal organs of the animal and stain them by the
Romanowsky-Giemsa method. As a result, the cytoplasm of Rickettsia stains blue,
while the nuclei are red. Following Zdrodovsky's staining, Rickettsia are
ruby-red.
Rickettsia
may also be revealed with the help of the IF
test.
Rickettsioses of the Spotted
Fever Group. This group of rickettsioses
includes the following diseases: Marseilles (Boutonneuse) fever caused by Rickettsia conorii; East-African
tick-borne fever caused by Rickettsia
pi]perii\ Rickettsialpox induced by Rickettsia
akari; Rocky Mountain spotted fever induced by Rickettsia rickettsii; and Australian (Queensland) tick-borne fever
caused by Rickettsia australis.
The
laboratory diagnosis of these rickettsioses is based on the isolation of the
causative agent from patients' blood with the aid of a biological test, its
cultivation in the yolk sac of the chicken embryo, and also on the
determination of specific antibodies in the patient's blood.
Biological examination. Withdraw 3-5 ml of blood from the patient's vein and inject it
intraperitoneally to male guinea pigs. Take some vectors (ticks), treat them
with alcohol, wash, grind in a mortar, and prepare a suspension in isotonic
sodium chloride solution, which is administered intraperitoneally to male
guinea pigs. In 6-14 days after the inoculation the animals develop
periorchitis. The causal organisms of Rocky Mountain spotted fever induce a
scro-tal phenomenon characterized by scrotal necrosis.
Intraperitoneal
administration to white mice of blood from a patient with rickettsialpox
(vesicular rickettsiosis) induces (in 8-10 days) rickettsial peritonitis with a
dramatic spleen enlargement.
The causal
organisms of many rickettsioses can be relatively easily isolated by
inoculating chicken embryos according to Cox's technique.
Serological diagnosis. Sufficient amounts of antibodies in patients' blood sera are accumulated
on the second week of the disease. The CF
and IffA reactions are typically employed for
their detection. Determination of an increase in the antibody titres makes the
diagnosis unquestionable.
Specific
antigens from Rickettsia inducing a given disease are used for serological
reactions.
Chlamydiae
Chlamydiae are a large group of obligate intracellular parasites closely
related to gram-negative bacteria. They are
divided into 2 species, Chlamydia
psittaci and Chlamydia trachomatix,
on the basis of antigenic composition, intracellular inclusions, sulfonamide
susceptibility, and disease production (see below). All chlamydiae exhibit
similar morphologic features, share a common group antigen, and multiply in the
cytoplasm of their host cells by a distinctive developmental cycle.
Because of
their obligate intracellular parasitism, chlamydiae were once considered viruses.
Chlamydiae differ from viruses in the following important characteristics:
(l) Like bacteria, they possess both RNA and DNA.
(2) They multiply by binary fission; viruses never do.
(3) They possess bacterial type cell walls with peptidoglycans probably
containing muramic acid.
(4) They possess ribosomes; viruses never do.
(5) They have a variety of metabolically active enzymes, eg, they can
liberate C02 from glucose. Some can synthesize folates.
(6) Their growth can be inhibited by many antimicrobial drugs,
Chlamydiae
can be viewed as gram-negative bacteria that lack some important mechanisms
for the production of metabolic energy- This defect restricts them to an
intracellular existence, where the host cell furnishes energy-rich
intermediates.
Developmental Cycle. All chlamydiae share a general sequence of events in their reproduction.
The infectious particle is a small cell ("elementary body") with an
electron-dense nucleoid. It is taken into the host cell by phagocytosis. A
vacuole derived from the host cell surface membranes, forms around the small
particle. This small particle is reorganized into a large one ("initial
body") measuring about 0.5-1 Jiim and devoid of an electron-dense
nucleoid. Within the membrane-bound vacuole, the large particle grows in size
and divides repeatedly by binary fission. Eventually the entire vacuole becomes
filled with small particles derived by binary fission from large bodies to form
an "inclusion" in the host cell cytoplasm. The newly formed small particles
may be liberated from the host cell to
infect new cells. The developmental cycle takes 24-48 hours.
Structure and Chemical Composition. Examination of highly purified suspensions of chlamydiae, washed free of
host cell materials, indicates the following: the outer cell wall resembles the cell wall of gram-negative bacteria. It
has a relatively high Upid content, and the peptidoglycan perhaps contains
muramic acid. Cell wall formation is inhibited by penicillins and cycloserine,
substances that inhibit peptidoglycan synthesis in bacteria. Both DNA and RNA
are present in both small and large particles, In small particles, most DNA is
concentrated in the electron-dense central nucleoid. In large particles, the
DNA is distributed irregularly throughout the cytoplasm. Most RNA probably
exists in ribosomes, in the cytoplasm. The large particles contain about 4
times as much RNA as DNA, whereas the small, infective particles contain about
equal amounts of RNA and DNA.
The circular
genome of chlamydiae (MW 7 x 108) is similar to bacterial
chromosomes. Chlamydiae contain large amounts of lipids, especially
phos-pholipids, which are well characterized. A toxic principle is intimately
associated with infectious chlamydiae. It kills mice after the intravenous
administration of more than 108 particles. Toxicity is destroyed by
heat but not by ultraviolet light.
Staining Properties. Chlamydiae have distinctive staining properties (similar to those
ofrickettsiae) that differ somewhat at different stages of development. Single
mature particles (elementary bodies) stain purple with Giemsa's stain and red
with Macchiavello's stain, in contrast to the blue of host cell cytoplasm. The
larger, noninfec-live bodies (initial bodies) stain blue with Giemsa's stain.
The Gram reaction of chlamydiae is negative or variable, and Gram's stain is
not useful in the identification of the agents.
Fully
formed, mature intracellular inclusions are compact masses near the nucleus
which are dark purple when stained with Giemsa's stain because of the densely
packed mature particles. If stained with dilute Lugol's iodine solution, the
inclusions formed by some chlamydiae (mouse pneumonitis, lym-phogranuloma
venereum [LGV], trachoma, inclusion conjunctivitis) appear brown because of the
glycogen-like matrix that surrounds the particles.
Antigens. Chlamydiae possess 2 types of antigens. Both are probably located in the
cell wall. Group antigens are shared by all chlamydiae. These are heat-stable
lipoprotein-carbohydrate complexes, with 2-keto-3-deoxy-octonic acid as an
immunodominant component. Antibody to these group antigens can be detected by
complement fixation and immunofluorescence. Specific antigens (species-specific
or immunotype-specific) remain attached to cell walls after group antigens
have been largely removed by treatment with fluorocarbon or deoxycholate. Some
specific antigens are membrane proteins that have been purified by
immunoadsorption. Specific antigens can best be detected by
immunofluorescence. Specific antigens are shared by only a limited number of
chlamydiae, but a given organism may contain several specific antigens. Fifteen
immunotypes of C trachomatis have
been identified (A, B, Ba, C-K, L1-L3), and the last 3 are LGV immunotypes. The
toxic effects of chlamydiae are associated with antigens. Specific
neutralization of these toxic effects by antiserum permits similar an-tigenic
grouping of organisms.
A very
unstable hemagglutinin capable of clumping some chicken and mouse erythrocytes
is present in chlamydiae. This he maggluti nation is blocked by group antibody.
Growth and
Metabolism. Chlamydiae
require an intracellular habitat, presumably because they lack some essential
feature of energy metabolism. All types of chlamydiae proliferate in
embryonated eggs, particularly in the yolk sac. Some also grow in cell cultures
and in various animal tissues. Cells have attachment sites for chlamydiae.
Removal of these sites prevents easy uptake of chlamydiae.
Chlamydiae
appear to have an endogenous metabolism similar to that of some bacteria but
participate only to a limited extent in potentially energy-yielding processes.
They can liberate CO; from glucose, pyru-vate, and glutamate; they also contain
dehydrogen-ases. Nevertheless, they require energy-rich intermediates from the
host cell to carry out their biosynthetic activities.
Reactions to
Physical and Chemical Agents. Chlamydiae are rapidly inactivated by heat. They
lose infectivity completely after 10 minutes at
The
replication of chlamydiae can be inhibited by many antibacterial drugs. Cell
wall inhibitors such as penicillins and cycloserine result in the production of
morphologically" defective forms but are not effective in clinical
diseases. Inhibitors of protein synthesis (tetracyc lines, erythromycins) are
effective in laboratory models and at times in clinical infections. Some
chlamydiae synthesize folates and are susceptible to inhibition by
sulfonamides. Aminoglycosides have little inhibitory activity for chlamydiae.
Characteristics of Host-Parasite Relationship. The outstanding biologic feature of infection by chlamydiae is the balance
that is often reached between host and parasite, resulting in prolonged, often
lifetime persistence. Spread from one species (eg, birds) to another (eg,
humans) more frequently leads to disease. Antibodies to several antigens of
chlamydiae are regularly produced by the infected host. These antibodies have
little protective effect. Commonly the infectious agent persists in the
presence of high antibody titers. Treatment with effective antimicrobial drugs
(eg, tetracyclines) for prolonged periods may eliminate the chlamydiae from the
infected host. Very early, intensive treatment may suppress antibody
formation. Late treatment with antimicrobial dmgs in moderate doses may
suppress disease but permit persistence of the infecting agent in tissues.
The
immunization of susceptible animals with various inactivated or living vaccines
tends to induce protection against death from the toxic effect of living
challenge organisms. However, such immunization in animals or humans has been
singularly unsuccessful in protecting against infection. At best, immunization
or prior infection has induced some resistance and resulted in milder disease
after challenge or reinfec-tion.
Classification. Historically, chlamydiae were arranged according to their pathogenic
potential and their host range. Antigenic differences were defined by
antigen-antibody reactions studied by immunofluorescence, toxin neutralization,
and other methods. The 2 presently accepted species and their characteristics
are as follows:
(1) C psillaci: This species produces diffuse
intracytoplasmic inclusions that lack glycogen; it is usually resistant to
sulfonamides. It includes agents of psittacosis in humans, omithosis in birds,
meningo-pneumomtis, feline pneumonitis, and many other animal pathogens.
(2) C trachnmatis: This species produces compact
intracytoplasmic inclusions that contain glycogen; it is usually inhibited by
sulfonamides, It includes agents of mouse pneumonitis and several human
disorders such as trachoma, inclusion conjunctivitis, nongonococcal urethritis,
salpingitis, cervicitis, pneumonitis of infants, and lymphogranuloma venereum.
In nucleic acid hybridization experiments, the 2 species appear not to be
closely related.
PSITTACOSIS (Ornithosis). Psittacosis is a disease of birds that may be transferred to humans. In humans, the agent, C psillaci, produces a spectrum of
clinical manifestations ranging from severe pneumonia and sepsis with a high mortality
rafe to a mild inapparent infection.
Properties of the Agent. A. Size and Staining
Properties: Similar to other members of
the group (see above).
B. Animal Susceptibility and Growth of Agent: Psittacosis agent can be propagated in em-bryonated eggs, in mice and
other animals, and in some cell cultures. In all these host systems, growth can
be inhibited by tetracyclines and, to a limited extent, by penicillins. In
intact animals and in humans, tetracyclines can suppress illness but may not be
able to eliminate the infectious agent or end the carrier state.
C. Antigenic Properties: The heat-stable group-reactive complement-fixing antigen resists
pro-teolytic enzymes but is destroyed by potassium perio-date. It is probably a
lipopolysaccharide.
Infected
tissue contains a toxic principle, intimately associated with the agent, that
rapidly kills mice upon intravenous or intraperitoneal infection. This toxic
principle is active only in particles that are infective.
Specific
serotypes characteristic for certain mammalian and avian species may be
demonstrated by cross-neutralization tests of toxic effect. Neutralization of
infectivity of the agent by specific antibody or cross-protection of immunized
animals can also be used for serotyping and parallels immunofluorescence.
D. Cell Wall Antigens: Walls of the infecting agent have been prepared by treatment with
deoxycho-late followed by trypsin. The deoxycholate extracts contained
group-reactive complement-fixing antigens, while the cell walls retained the
species-specific antigens. The cell wall antigens were also associated with
toxin neutralization and infectivity neutralization.
Pathogenesis and Pathology. The agent enters through the respiratory tract, is found in the blood
during the first 2 weeks of the disease, and may be found in the sputum at the
time (he lung is involved.
Psittacosis
causes a patchy inflammation of the lungs in which consolidated areas are
sharply demarcated. The exudate is predominantly mononuclear. Only minor
changes occur in the large bronchioles and bronchi. The lesions are similar to
those found in pneumonitis caused by some viruses and mycoplas-mas. Liver,
spleen, heart, and kidney are often enlarged and congested.
Clinical Findings. A sudden onset of illness taking the form of influenza or nonbacterial
pneumonia in a person exposed to birds is suggestive of psittacosis. The
incubation period averages 10 days. The onset is usually sudden, with malaise,
fever, anorexia, sore throat, photophobia, and severe headache. The disease
may progress no further and the patient may improve in a few days. In severe
cases the signs and symptoms of bronchial pneumonia appear at the end of the
first week of the disease. The clinical picture often resembles that of
influenza, nonbacterial pneumonia, or typhoid fever. The fatality rate may be
as high as 20% in untreated cases, especially in the elderly.
Laboratory Diagnosis. A. Recovery of Agent: Laboratory diagnosis is dependent upon the recovery of psittacosis agent
from blood and sputum or, in fatal cases, from lung tissues. Specimens are
inoculated intra-abdominally into mice, into the yolk sacs of embryonated eggs,
and into cell cultures. Infection in the test systems is confirmed by the
serial transmission of the infectious agent, its microscopic demonstration,
and serologic identification of the recovered agent.
B. Serology: A variety of antibodies may develop in the course of infection. In
humans, complement fixation with group antigen is the most widely used
diagnostic test. Acute and later phase sera should be run in the same test in
order to establish an antibody rise. In birds, the indirect CF test may provide
additional diagnostic information. Although antibodies usually develop within
10 days, the use of antibiotics may delay their development for 20-40 days or
suppress it altogether.
Sera of
patients with other chlamydiat infections may fix complement in high liter with
psittacosis antigen. In patients with psittacosis, the high liter persists for
months and, in carriers, even for years. Infection of live birds is suggested
by a positive CF test and by the presence of an enlarged spleen or liver. This
can be confirmed by demonstration of particles in smears or sections of organs
and by passage of the agent in mice and eggs.
Immunity. Immunity in animals and humans is incomplete. A carrier state in humans can
persist for 10 years after recovery. During this period the agent may continue
to be excreted in the sputum.
Skin tests with
group-reactive antigen are positive soon after infection with any member of the
group. Specific dermal reactions may be obtained by the use of some
skin-testing antigens prepared by extracting suspensions of agent with dilute
hydrochloric acid or with detergent. Live or inactivated vaccines induce only
partial resistance in animals.
Treatment. Tetracyclines are the drugs of choice. Psittacosis agents are not sensitive
to aminoglycosides, and most strains are not susceptible to sulfonamides.
Although antibiotic treatment may control the clinical evidence of disease, it
may not free the patient from the agent, ie, the patient may become a carrier.
Intensive antibiotic treatment may also delay the normal course of antibody
development. Strains may become drug-resistant.
With the
introduction of antibiotic therapy, the fatality rate has dropped from 20% to
2%. Death occurs most frequently in patients 40-60 years of age.
Epidemiology. The term psittacosis is applied to the human disease acquired from contact
with birds and also the infection ofpsittacine birds (parrots, parakeets, cockatoos,
etc). The term ornithosis is applied to infection with similar agents in all
types of domestic birds (pigeons, chickens, ducks, geese, turkeys, etc) and
free-living birds (gulls, egrets, petrels, etc). Outbreaks of human disease can
occur whenever there is close and continued contact between humans and infected
birds that excrete or shed large amounts of infectious agent. Birds often
acquire infection as fledglings in the nest; may develop diarrheal illness or
no illness; and often carry the infectious agent for their normal life span.
When subjected to stress (eg, malnutrition, shipping), birds may become sick
and die. The agent is present in tissues (eg, spleen) and is often excreted in
feces by healthy birds. The inhalation of infected dried bird feces is a common
method of human infection. Another source of infection is the handling of
infected tissues (eg, in poultry rendering plants) and inhalation of an
infected aerosol.
Birds kept
as pets have been an important source of human infection. Foremost among these
were the many psittacine birds imported from South America, Australia, and the
Far East and kept in aviaries in the USA. Latent infections often flared up in
these birds during transport and crowding, and sick birds excreted exceedingly
large quantities of infectious agent. Control of bird shipment, quarantine,
testing of imported birds for psittacosis infection, and prophylactic
tetra-cyclines in bird feed help to control this source. Pigeons kept for
racing or as pets or raised for squab meat have been important sources of
infection. Pigeons populating civic buildings and thoroughfares in many cities
are not infrequently infected but shed relatively small quantities of agent.
Among the
personnel of poultry farms involved in the dressing, packing, and shipping of
ducks, geese, turkeys, and chickens, subclinical or clinical infection is
relatively frequent. Outbreaks of disease among birds have at times resulted in
heavy economic losses and have been followed by outbreaks in humans.
Persons who
develop psittacosis may become infectious for other persons if the evolving
pneumonia results in expectoration of large quantities of infectious sputum.
This has been an occupational risk to hospital personnel.
Control. Shipments of psittacine birds should be held in quarantine to ensure that
there are no obviously sick birds in the lot. A proportion of each shipment
should be tested for antibodies and examined for agent. An intradermal test has
been recommended for detecting ornithosis in turkey flocks. The incorporation
of tetra-cyclines into bird feed has been used to reduce the number of
carriers. The source of human infection should be traced, if possible, and
infected birds should be killed.
OCULAR, GENITAL, AND RESPIRATORY INFECTIONS DUE TO CHLAMYDIA TRACHOMATIS. TRACHOMA. Trachoma is an ancient eye
disease, well described in the Ebers Papyrus, which was written in Egypt 3800
years ago. It is a chronic keratoconjunctivitis
that begins with acute inflammatory changes in the conjunctiva and cornea and
progresses to scarring and blindness.
Properties of C trachomatis.
A. Size and
Staining Properties: See chlamydiae
B. Animal Susceptibility and Growth: Humans are the natural host for C
trachomatis. Monkeys and chimpanzees can be infected in the eye and genital
tract. Alt chlamydiae multiply in the yolk sacs of embryonated hens' eggs and
cause death of the embryo when the number of particles becomes sufficiently
high. C trachomatis also replicates
in various cell lines, particularly when cells are treated with cycloheximide,
cytochalasin B, or idoxuridine. C
trachomulis of different immunotypes replicates differently . Isolates
from trachoma do not grow as well as those from LGV or genital infections.
Intracytoplasmic replication results in a developmental cycle that leads to
formation of compact inclusions with a glycogen matrix in which particles are
embedded.
A toxic
factor is associated with C trachomatis provided
the particles are viable. Neutralization of this toxic factor by
immunotype-specific antisera permits typing of isolates that gives results
analogous to those achieved by typing by immunofluorescence. The immunotypes
specifically associated with endemic trachoma are A, B, Ba, and C.
Clinical Findings. In experimental infections, the incubation period is 3-10 days. In endemic
areas, initial infection occurs in early childhood and the onset is insidious.
Chlamyd-ial infection is often mixed with bacterial conjunctivitis in endemic
areas, and the 2 together produce the clinical picture. The earliest symptoms
of trachoma arelacrimation, mucopurulent discharge, conjunctiva! hyperemia, and
follicular hypertrophy. Biomicro-scopic examination of the cornea reveals
epithelial keratitis, subepithelial infiltrates, and extension of limba!
vessels into the cornea (pannus).
As the
pannus extends downward across the cornea, there is scarring of conjunctiva,
lid deformities (entropion, trichiasis), and added insult caused by eyelashes
sweeping across the cornea. With secondary bacterial infection, loss of vision
and blindness occur over a period of years. There are, however, no systemic
symptoms or signs of infection.
Laboratory Diagnosis. A. Recovery of C trachomatis: Typical cyto-plasmic inclusions are found in epithelial cells of conjunctiva!
scrapings stained with fluorescent antibody or by Giemsa's method. These occur
most frequently in the early stages of the disease and on the upper tarsal
conjunctiva.
Inoculation
of conjunctival scrapings into embryonated eggs or cycloheximide-treated cell
cultures permits growth of C trachomatis
if the number of viable infectious particles is sufficiently large.
Cen-trifugation of the inoculum into treated cells increases the sensitivity of
the method. The diagnosis can sometimes be made in the first passage by
looking for inclusions after 2-3 days of incubalion by immunofluorescence,
iodine staining, or staining by Giemsa's method.
B. Serology: Infected individuals often develop both group-reactive and
immunotype-specific antibodies in serum and in eye secretions. Immunofluorescence
is the most sensitive method for their detection. Neither ocular nor serum
antibodies confer significant resistance to reinfection.
Treatment. In endemic areas, sulfonamides, erythromycins, and tetracyclines have been
used to suppress chlamydiae and bacteria that cause eye infections. Periodic
topical application of these drugs to the conjunctivas of all members of the
community is sometimes supplemented with oral doses; the dosage and frequency
of administration vary with the geographic area and the severity of endemic
trachoma. Drug-resistant C Irachomatis
has not been definitely identified except in laboratory experiments. Even a
single monthly dose of 300 mg of doxycycline can result in significant clinical
improvement, reducing the danger of blindness. Topical application of
corticosteroids is not indicated and may reactivate latent trachoma. Chlamydiae
can persist during and after drug treatment. and recurrence of activity is
common.
Epidemiology and
Control. It is believed that over 400
million people throughout the world are infected with trachoma and that 20
million are blinded by it. The disease is most prevalent in Africa, Asia, and
the Mediterranean Basin, where hygienic conditions are poor and water is
scarce. In such hyperendemic areas, childhood infection may be universal, and
severe, blinding disease (resulting from frequent bacterial superinfections) is
common. In the USA, trachoma occurs sporadically in some areas, and endemic
foci persist on Indian reservations.
Control of
trachoma depends mainly upon improvement of hygienic standards and drug
treatment.
When
socioeconomic levels rise in an area, trachoma becomes milder and eventually
may disappear. Experimental trachoma vaccines have not given encouraging
results, Surgical correction of lid deformities may be necessary in advanced
cases.
GENITAL CHLAMYDIAL INFECTIONS
and INCLUSION CONJUNCTIVITIS. C trachomatis, immunotypes D-K, is a common cause of sexually transmitted
diseases that may also produce infection of the eye (inclusion conjunctivitis).
In sexually active adults, particularly in the USA and western Europe—and
especially in higher socioeconomic groups—C trachomatis
is a prominent cause of nongonococcat urethritis and, rarely, epididymitis in
males. In females, C trachomatis causes
urethritis, cervicitis, salpingitis, and pelvic inflammatory disease. Any of
these anatomic sites of infection may give rise to symptoms and signs, or the
infection may remain asymptomatic but communicable to sex partners. Up to 50%
of nongonococcal or postgonococcal urethritis or the urethra! syndrome is
attributed to chlamydiae and produces dysuria, non-purulent discharge, and
frequency of urination.
This
enormous reservoir of infectious chlamydiae in adults can be manifested by
symptomatic genital tract illness in adults or by an ocular infection that
closely resembles trachoma. In adults, this inclusion conjunctivitis results
from self-inoculation of genital secretions and was formerly thought to be
"swimming pool conjunctivitis".
The neonate
acquires the infection during passage through
an infected birth canal. Inclusion conjunctivitis
of the newborn begins as a mucopurulent conjunctivitis 7-12 days after
delivery. It tends to subside with erythromycin or tetracycline treatment, or
spontaneously after weeks or months. Occasionally, inclusion conjunctivitis
persists as a chronic chlamyd-ial infection with a clinical picture
indistinguishable from subacute or chronic childhood trachoma in nonendemic
areas and usually not associated with bacterial conjunctivitis.
Laboratory Diagnosis. A. Recovery of C trachomatis: Scrapings of epithelial cells from urethra, cervix, vagina, or conjunctiva
and biopsy specimens from salpinx or epididymis can be inoculated into
chemically treated cell cultures for growth of C irachomatis (see above). Isolates can be typed by
microimmunofluorescence wilh specific sera. In neonatal—and sometimes
adult—inclusion conjunctivitis, the cytoplasmic inclusions in epithelial cells
are so dense that they are readily detected in conjunclival exudate and
scrapings examined by immunofluorescence or stained by Giem-sa's method.
B. Serologic Tests: Because of the relatively great antigenic mass of chlamydiae in genital
tract infections, serum antibodies occur much more commonly than in trachoma
and are of higher titer. A liter rise occurs during and after acute chlamydial
infection. In genital secretions (eg, cervical), antibody can be detected
during active infection and is directed against the infecting immunotype.
Treatment. It is essential that chlamydial infections be treated simultaneously in
both sex partners and in offspring to prevent reinfection.
Tetracyclines
(eg, doxycycline, 100 mg/d by mouth for 10-20 days) are commonly used in nongonococcal
urethritis and in nonpregnant infected females. Erythromycin, 250 mg 4-6 times
daily for 2 weeks, is given to pregnant women. Topical tetracycline or
erythromycin is used for inclusion conjunctivitis, sometimes in combination
with a systemic drug.
Epidemiology and Control. Genital chlamydial infection and inclusion conjunctivitis are sexually
transmitted diseases that are spread by indiscriminate contact with multiple
sex partners. Neonatal inclusion conjunctivitis originates in the mother's
infected genital tract. Prevention of neonatal eye disease depends upon
diagnosis and treatment of the pregnant woman and her sex partner. As in all
sexually transmitted diseases, the presence of multiple etiologic agents
(gonococci, treponemcs, Trichomonas,
herpes, mycoplasmas, etc) must be considered. Instillation of 1% silver
nitrate into the newbom 's eyes does not prevent development of chlamydial
conjunctivitis. The ultimate control of this—and all—sexually transmitted
disease depends on reduc-tion in
promiscuity, use of condoms, and early diagnosis and treatment of the
infected reservoir.
RESPIRATORY TRACT INVOLVEMENT
WITH C. TRACHOMATIS
Adults with inclusion
conjunctivitis often manifest upper respiratory tract symptoms (eg, otalgia,
otitis, nasal obstruction, pharyngitis), presumably resulting from drainage of
infectious chlamydiae through the nasolacrimal duct. Pneumonitis is rare in
adults.
Neonates
infected by the mother may develop respiratory tract involvement 2-12 weeks
after birth, culminating in pneumonia. There is striking tachypnea, paroxysmal
cough, absence of fever, and eosinophilia. Consolidation of lungs and
hyperinflation can be seen by x-ray. Diagnosis can be established by isolation
of C tracbomutis from respiratory secretions
and can be suspected if pneumonitis develops in a neonate who has inclusion
conjunctivitis. Systemic erythromycin (40 mg/kg/d) is effective treatment in
severe cases.
LYMPHOGRANULOMA VENEREUM
(LGV). LGV is a sexually transmitted
disease, characterized by suppurative inguinal adenitis, that is common in
tropical and temperate zones. The agent is C
irachomatis of immunotypes L1-L3.
Properties of the Agent. A. Size and Staining
Properties: Similar to other chlamydiae.
B. Animal Susceptibility and Growth of Agent: The agent can be transmitted to monkeys and mice and can be propagated in
tissue cultures or in chick embryos. Most strains grow in cell cultures; their
infectivity for cells is not enhanced by pretreatment with DEAE-dextran.
C. Antigenic Properties: The particles contain complement-fixing heat-stable chlamydial group antigens
that are shared with all other chlamydiae. They also contain one of 3 specific
antigens (L1-L3), which can be defined by immunofluorescence. Infective particles
contain a toxic principle.
Clinical Findings. Several days to several weeks after exposure, a small, evanescent papule or
vesicle develops on any part of the external genitalia, anus, rectum, or
elsewhere. The lesion may ulcerate, but usually— especially in women—it remains
unnoticed and heals in a few days. Soon thereafter, the regional lymph nodes
enlarge and tend to become matted and often painful. In males, inguinal nodes
are most commonly involved both above and below Poupart's ligament, and the
overlying skin often turns purplish as the nodes suppurate and eventually
discharge pus through multiple sinus tracts. In females and in homosexual
males, the perirectal nodes are prominently involved, with proctitis and a
bloody mucopurulent anal discharge. Lymphadenitis may be most marked in the
cervical chains.
During the
stage of active lymphadenitis, there are often marked systemic symptoms
including fever, headaches, meningismus, conjunctivitis, skin rashes, nausea
and vomiting, and arthralgias. Meningitis, arthritis, and pericarditis occur
rarely. Unless effective antimicrobial drug treatment is given at that stage,
the chronic inflammatory process progresses to fibrosis, lymphatic obstruction,
and rectal strictures. The lymphatic obstruction may lead to elephantiasis of
the penis, scrotum, or vulva. The chronic proctitis of women or homosexual
males may lead to progressive rectal strictures, rectosigmoid obstruction, and
fistula formation.
Laboratory Diagnosis. A. Smears: Pus, buboes, or biopsy material may be stained, but particles are rarely
recognized.
B. Isolation of Agent: Suspected material is inoculated into the yolk sacs of embryonated eggs,
into cell cultures, or into the brains of mice. Streptomycin (but not
penicillin or ether) may be incorporated into the inoculum to lessen bacterial
contamination. The agent is identified by morphology and serologic tests.
C. Serologic Tests: The CF reaction is the simplest
serologic test for the presence of antibodies. Antigen is prepared from
infected yolk sac. The test becomes positive 2-4 weeks after onset of illness,
at which time skin hypersensitivity can sometimes also be demonstrated. In a
clinically compatible case, a rising antibody level or a single liter of more
than 1:64 is good evidence of active infection. If treatment has eradicated the
LGV infection, the complement fixation riter falls. Serotogic diagnosis of LGV
can employ immunofluorescence, but the antibody is broadly reactive with many
chlamydial antigens. A more specific antibody can be demonstrated by
counterimmunoelec-trophoresis with a chlamydial protein extracted from LGV.
D. Frei Test: Intradermal injection of
heat-inactivated egg-grown LGV (0.1 mL) is compared to control material
prepared from noninfected yolk sac. The skin test is read in 48-72 hours- An
inflammatory nodule more than
Immunity. Untreated infections tend to be chronic, with persistence of the agent for
many years. Little is known about active immunity. The coexistence of latent
infection, antibodies, and cell-mediated reactions is typical of many
chlamydial infections.
Treatment. The sulfonamides and tetracyclines have been used with good results,
especially in the early stages. In some drug-treated persons there is a marked
decline in complement-fixing antibodies, which may indicate that the infective
agent has been eliminated from the body. Late stages require surgery.
Epidemiology. The disease is most often spread by sexual contact, but not exclusively
so. The portal of entry may sometimes be the eye (conjunctivitis with an
oculo-glandular syndrome). The genital tracts and rectums of chronically
infected (but at times asymptomatic) persons serve as reservoirs of infection.
Although the
highest incidence of LGV has been reported from subtropical and tropical areas,
the infection occurs all over the world.
Control. The measures used for the control of other sexually transmitted diseases
apply also to the control of LGV. Case-finding and early treatment and control
of infected persons are essential.
DIAGNOSIS CHLAMYDIAL
INFECTIONS IN DETAILS
CHLAMYDIAL INFECTIONS. In human beings Chlamydia cause ornithosis, trachoma, lymplio-granuloma
venereum, neonatal inclusion blennorrhoea, adult inclusion conjunctivitis,
urogenital infections, atypical pneumonia, and other illnesses which may run in
the form of acute or chronic infection, or an asymptomatic carrier state.
The
following types of examination are employed for the laboratory diagnosis of
chlamydial infections.
Bacteriological examination involves the isolation of the causative agent by infecting experimental
animals, chicken. embryos (see p. 182) or cell cultures (see p. 16ti). The
causal organism is identified by the presence of elementary particle
accumulation in impression smears from the internal organs of animals,
allantoic fluid, cells of tissue cultures stained with the Romanowsky-Giemsa
dye or acridine orange.
Serological methods of investigation include such tests as CF, HAI,
and IF, which are carried out
according to the ordinary scheme with chlamydial antigens.
The allergy intracutaneous test provides the
earliest and most accessible method of diagnosis.
Since the
laboratory diagnosis of all chlamydial infections is analogous, we w^ll
consider in detail the diagnosis of only trachoma and ornithosis.
Trachoma. Trachoma is a contagious keratoconjunctivitis characterized by a chronic
course. The causal organism of this disease is Chlamydia trachomatis.
To obtain
biological material for the laboratory diagnosis, first anaesthetize the eye,
then remove pus and mucus from the conjunctiva with the help of a cotton swab,
and scrape off tha conjunctival epithelium with a blunt scalpel.
Bacterioscopic and bacteriological examination. Place a scraping onto glass slides, fix the preparations, and stain for
3-4 hrs, using the Romanowsky-Giemsa technique or acridine orange. In positive
cases one finds coccal inclusions in epithelial cells (Plate VIII, 4), which
measure up to 10 u,m (Prowazek-Halberstaedter bodies). To detect antigens by
the IF reaction, preparations of scrapings are fixed in cold acetone for 10-15
min and treated with fluorescent antibodies. Upon luminescent microscopy C. trachomatis appear as fluorescent
inclusions in the cytoplasm of conjunctival cells. Cytoplasmic .inclusions are
also formed in cells of cultures of the human thyroid tissue infected with
material obtained from patients.
It is
recommended that the causative agent of trachoma be grown in the yolk sac of
the chicken embryo. The material is treated with antibiotics for several hours
at -4- 4: °C and introduced in 0.3-ml portions into the yolk sac of chicken
embryos. Microscopic examination of impression smears prepared from the yolk
sac of dead embryos reveals large numbers of Chlamydia.
To detect
antibodies in trachoma patients' sera, an indirect
IF reaction may be used.
Ornithosis. Ornithosis, which is a communicable disease caused by Chlamydia psittaci, is characterized by general intoxication and
lung involvement.
To isolate
the causal organism, take 5-10 ml of blood from the patient within the first
two weeks of the illness. To perform serologi-cal tests, blood samples should
be obtained within the first days and then 30-45 days after the onset of the
disease. Washings from the nasal portion of the throat are made in the same
manner which is utilized in examination for influenza (see p. 190). Sputum and
vomitus are collected in sterile vessels. Post-mortem samples include damaged
sites of the lung, liver, and spleen. The material to be examined is brought to
the laboratory in the frozen form, checked for the absence of other bacteria, and
used to prepare suspensions.
Biological examination. To isolate the causative agents of ornitho-sis, use white mice and chicken
embryos. Inject 0.5 ml of a suspension of the tested material into the brain
of mice. Make three sequential passages. In positive cases prepare
histological sections and impression smears from the brain and stain them by
the Romanowsky-Giemsa dye or acridine orange. C. psittaci are usually seen as accumulations of elementary
particles in the cell cytoplasm.
Upon the
intranasal inoculation of white mice the causative agent of ornithosis induces
the disease and death of the animals from pneumonia in 3-4 days. Examination of
impression smears of the lung tissue reveals cytoplasmic inclusions and
elementary particles in the epithelial cells of alveoles and bronchioles, and
also in phagocytes. Rabbits, young guinea pigs, rats, and Syrian hamsters are
also sensitive to C. psittaci.
Bacteriological examination. Inoculate cliicken embryos via the yolk sac, allantoic cavity, and chorio-allantoic
membrane, and then incubate them at 35-
Using the
examined material, inoculate continuous cultures of cells, for example, L,
IIela, Hep-2, etc.
The efficacy of cell culture
inoculation increases when forced adsorption is utilized. For this purpose,
after streaking of the tested material onto a monolayer of cells the cultures
are centrifuged at 1000-1500 Xg for 20-30 min. Furthermore, it is recommended
that one should use cell cultures which have been either irradiated or treated
withcytostatics. After 24-48-hour incubation, monolayers of . cells on glass
slides are stained with the Romanowsky-Giemsa stain or acridine orange.
Microscopic examination of the resultant preparations reveals cytoplasmic
inclusions of the causal organism in the form of roundish formations.
Serological diagnosis. The CF reaction is made with
patients' paired sera, according to the ordinary technique and using the standard
ornithosis antigen (ornithin) which is commercially available. A two-fold or
greater increase in the antibody titre in the second serum is diagnostically
important.
The indirect IF test is also employed for
recovering antibodies in the patient's serum.
The intracutaneous allergy test with ornithin is assessed in 24 and 48 hrs. The test is positive in the
acute period of the disease (from the 3rd day to approximately the 3rd-4th week
of the disease).
OTHER AGENTS OF THE GROUP. Many mammals are subject to chlamydial infections, mainly with C. pstiaci. Common animal disease entities
are pneumonitis, arthritis, enteritis, and abortion, but infection is often
latent. Some of these agents may also be transmitted to humans and cause
disease in them.
Chlamydiae
have been isolated from Reiter's disease in humans, both from the involved
joints and from the urethra. The causative role of these agents remains
uncertain.
In
nonbacterial regional lymphadenitis (cat-scratch fever), a skin test with
heat-inactivated pus gives a delayed positive reaction. Chlamydiae have been
proposed as a possible cause but without proof. The usefulness of tetracyclines
in this syndrome is dubious
MYCOPLASMAS (PPLO) and
WALL-DEFECTIVE MICROBIAL VARIANTS.
Mycoplasmas
(previously called pleuropneumonia-like organisms or PPLO) are a group of organisms
with the following characteristics: (1) The smallest reproductive units have a
size of 125-250 nm. (2) They are highly pleomorphic because they lack a rigid
cell wall and instead are bounded by a triple-layered "unit
membrane." (3) They are completely resistant to penicillin but inhibited
by tetracycllne or erythromycln. (4) They can reproduce in cell-free media; on
agar the center of the whole colony is characteristically embedded beneath the
surface (“fried egg” appearance). (5) Growth is inhibited by specific antibody.
(6) Mycoplasmas do not revert to, or originate from, bacterial parental forms. (7) Mycoplasmas have an affinity for cell membranes.
L phase variants are wall-defective microbial forms (WDMF) that can replicate serially as nonrigid
cells and produce colonies on solid media. Some L phase variants are stable;
others are unstable and revert to bacterial parent forms. Wall-defective
microbial forms are not genetically related to mycoplasmas. WDMF can result
from spontaneous mutation or from the effects of chemicals. Treatment of
eubacteria with cell wall-inhibiting drugs or lysozyme can produce WDMF. Protoplasts are WDMF derived from
gram-positive organisms; they are osmotically fragile, with external surfaces
free of cell wall constituents. Spheroplasts
are WDMF derived from gram-negative bacteria; they retain some outer membrane
material.
Morphology
and Identification. A. Typical Organisms: Mycoplasmas cannot be studied by the usual bacteriologic methods because
of the small size of their colonies, the plasticity and delicacy of their
individual cells (due to the lack of a rigid cell wall), and their poor
staining with aniline dyes. The
morphology appears different according to the method of examination (eg,
darkfield, immunoflu-orescence, Giemsa-stained films from solid or liquid
media, agar fixation).
Mycoplasma
pneumoniae
Mycoplasma
hominis
Ureaplasma urealyticum
Growth in
fluid media gives rise to many different forms, including rings, bacillary and
spiral bodies, filaments, and granules. Growth on solid media consists
principally of plastic protoplasmic masses of indefinite shape that are easily
distorted. These structures vary greatly in size, ranging from 50 to 300 nm in
diameter.
B. Culture: Many strains of mycoplasmas grow in heart infusion peptone broth with 2%
agar (pH 7.8) to which about 30% human ascitic fluid or animal serum (horse,
rabbit) has been added. Following incubation at
After 2-6
days on special agar medium incubated in a Petri dish that has been sealed to
prevent evaporation, isolated colonies measuring 20-500 /n.m can be detected
with a hand tens. These colonies are round, with a granular surface and a dark
center nipple typically buried in the agar. They can be subcultured by cutting
out a small square of agar containing one or more colonies and streaking this
material on a fresh plate or dropping it into liquid medium. The organisms can
be stained for microscopic study by placing a similar square on a slide and
covering the colony with a coverglass onto which an alcoholic solution of
methylene blue and azure has been poured and then evaporated (agar fixation).
Such slides can also be stained with specific fluorescent antibody.
Mycoplasma colonies
C. Growth Characteristics: Mycoplasmas are unique in microbiology because of (1) their extremely
small size and (2) their growth on complex but cell-free media.
Mycoplasmas
pass through filters with 450-nm pore size and thus are comparable to
chlamydiae or large vimses. However, parasitic mycoplasmas grow on cell-free
media that contain lipoprotein and sterol. The sterol requirement for growth
and membrane synthesis is unique. Mycoplasmas are resistant to thallium
acetate in a concentration of 1:10,000, which can be used to inhibit bacteria.
Many mycoplasmas use glucose as a source of energy; ureaplasmas require urea.
Many human
mycoplasmas produce peroxides and hemolyze red blood cells. In cell cultures
and in vivo, mycoplasmas develop predominantly at cell surfaces. Many
established cell line cultures carry mycoplasmas as contaminants.
D. Variation: The extreme pleomorphism of mycoplasmas is one of their principal
characteristics. There is no genetic relationship between mycoplasmas and WDMF
or their parent bacteria. The characteristics of WDMF are similar to those of
mycoplasmas, but, by definition, mycoplasmas do not revert to parent bacteria
or originate from them. WDMF continue to synthesize some antigens that are
normally located in the cell wall of the parent bacteria (eg, streptococcal L
forms produce M protein and capsular polysaccharide.
Reversion of
L forms to the parent bacteria is enhanced by growth in the presence of 15-30%
gelatin or 2.5% agar, whereas reversion is inhibited by inhibitors of protein
synthesis.
Antigenic structure. Many antigenicaily distinct species of mycoplasmas
have been isolated from animals (eg, mice, chickens, turkeys).
In humans, at least 11 species can be
identified, including Mycoplasma hominis, Mycoplasma salivarium, Mycoplasma
orale, Mycoplasma fermentans, Mycoplasma pneumoniae, Ureaplasma urealyticum, and
others.
The last 2 species are of pathogenic
significance.
The species
are classified by biochemical and serologic features. The complement-fixing
antigens of mycoplasmas are glycolipids. Some species have more than one
serotype.
It is
doubtful that WDMF cause tissue reactions resulting in disease. They may be
important for the persistence of microorganisms in tissues and recurrence of
infection after antimicrobial treatment.
The parasitic
mycoplasmas appear to be strictly host-specific, being communicable and
potentially pathogenic only within a single host species. In animals ,
mycoplasmas appear to be intracellular parasites with a predilection for
mesothelial cells (pleura, peritoneum, synovia of joints). Several
extracellular products can be elaborated, eg, hemolysins.
A. Diseases of Animals: Bovine pleuropneumonia is a contagious disease of cattle producing pulmonary
consolidation and pleural effusion, with occasional deaths. The disease
probably has an airborne spread. Mycoplasmas are found in inflammatory
exudates.
Agalactia of
sheep and goats i n the Mediterranean area is a generalized infection with
local lesions in the skin, eyes, joints, udder, and scrotum; it leads to atrophy
of lactating glands in females. Mycoplasmas are present in blood early; in milk
and exudates later.
In poultry,
several economically important respiratory diseases are caused by mycoplasmas.
The organisms can be transmitted from hen to egg and chick. Swine, dogs, rats,
mice, and other species harbor mycoplasmas that can produce infection involving
particularly the pleura, peritoneum, joints, respiratory tract, and eye. In
mice, a Mycoplasma of spiral shape (Spiroplasma) can induce cataracts.
B. Diseases of Humans: Mycoplasmas have been cultivated from human mucous membranes and tissues,
particularly from the genital, urinary, and respiratory tracts and from the
mouth. Some mycoplasmas are inhabitants of the normal genitourinary tract,
particularly in females. In pregnant women, carriage of mycoplasmas on the
cervix has been associated with chorioamnionitis and low birth weight of
infants. U urealyticum (formerly
called T strains of mycoplasmas), requiring 10% urea for growth, is found in
some cases of urethritis and prostatitis in men who suffer from
"nongonococcal urethritis. " This organism and M. hominis have also been associated infrequently with salpingitis
and pelvic inflammatory disease. U.
urealyticum may play a causative role and is suppressed by tetracycline or
spectinomycin. However, a majority of cases of “nongonococcal urethritis” are
caused by Chlamydia trachomatis.
Infrequently,
mycoplasmas have been isolated from brain abscesses and pleural or joint
effusions. Mycoplasmas are part of the normal flora of the mouth and can be
grown from normal saliva, oral mucous membranes, sputum, or tonsillar tissue.
M. hominis and M. salivarium can be recovered from the oral cavity of many healthy
adults, but an association with clinical disease is uncertain. Over half of
normal adults have specific antibodies to M.
hominis.
M. pneumoniae is one of
the causative agents of nonbacterial pneumonia (see p 276). In humans, the
effects of infection with M. pneumoniae
range from inapparent infection to mild or severe upper respiratory disease,
ear involvement (myringitis), and bronchial pneumonia (see p 276).
C. Diseases of Plants: Aster yellows, corn stunt, and other plant diseases appear to be caused by
mycoplasmas. They are transmitted by insects and can be suppressed by
tetracyclines.
Specimens
consist of throat swab, sputum, inflammatory exudates, and respiratory,
urethral, or genital secretions.
A. Microscopic Examination: Direct examination of a specimen is useless. Cultures are examined as described
above.
B. Culture: The material is inoculated onto special solid media and incubated for
3-10 days at
C. Serology: Antibodies develop in humans infected with mycoplasmas and can be
demonstrated by several methods. CF tests can be performed with glycolipid
antigens extracted with chloroform-methanol from cultured mycoplasmas. HI tests
can be applied to tanned red cells with adsorbed Mycoplasma antigens. Indirect immunofluorescence may be used. The
test that measures growth inhibition by antibody is quite specific. When
counterimmunoelectrophoresis is used, antigens and antibody migrate toward each
other, and precipitin lines appear in 1 hour. With all these serologic
techniques, there is adequate specificity for different human Mycoplasma species, but a rising
antibody liter is required for diagnostic significance because of the high
incidence of positive serologic tests in normal individuals.
Treatment. Many strains of mycoplasmas are inhibited by a variety of antimicrobial
drugs, but most strains are resistant to penicillins, cephalosporins, and
vancomy-cin. Tetracyclines and erythromycins are effective both in vitro and in
vivo and are, at present, the drugs of choice in mycoplasmal pneumonia.
Epidemiology, Prevention, and Control. Isolation of infected livestock will control the highly contagious
pleuropneumonia and agalactia in limited areas. No vaccines are available.
Mycoplasmal pneumonia behaves like a communicable viral respiratory disease (see
next section).
Acute
nonbacterial pneumonitis may be due to many different infectious agents,
including adeno-viruses, influenza viruses, respiratory syncytia! virus,
parainfluenza type 3 virus, chlamydiae, and Coxiella
burnetii, the etiologic agent of Q fever. However, the single most
prominent causative agent, especially for those between ages 5 and 15, is M pneumoniae. Mycoplasmal pneumonia
appears to be much more common in military recruit populations than in college
populations of comparable age.
The first
step in M pneumoniae infection is the
attachment of the tip of the organism to a receptor on the surface of
respiratory epithelial cells. The clinical spectrum of M pneumoniae ranges from asymptomatic infection to serious
pneumonitis, with occasional neu-rologic and hematologic (ie, hemolytic anemia)
involvement and a variety of possible skin lesions. Bul-lous myringitis occurs
in spontaneous cases and in experimentally inoculated volunteers. Typical cases
during epidemic periods might show the following:
The
incubation period varies from I to 3 weeks. The onset is usually insidious,
with lassitude, fever, headache, sore throat, and cough. Initially, the cough
is nonproductive, but it is occasionally paroxysmal. Later there may be
blood-streaked sputum and chest pain. Early in the course, the patient appears
only moderately ill, and physical signs of pulmonary consolidation are often
negligible compared to the striking consolidation seen on x-rays. Later, when
the infiltration is at a peak, the illness may be severe. Resolution of
pulmonary infiltration and clinical improvement occur slowly for 1—4 weeks.
Although the course of the illness is exceedingly variable, death is very rare
and is usually attributable to cardiac failure. Complications are uncommon,
but hemolytic anemia may occur. The most common pathologic findings are
interstitial and peribronchial pneumonitis and necrotizing bronchiolitis.
The
following laboratory findings apply to M
pneumoniae pneumonia: The white and differential counts are within normal
limits. The causative Myco-plasma can
be recovered by culture early in the disease from the pharynx and from sputum.
Immunofluores-cent stains of mononuclear cells from the throat may reveal the
agent. There is a rise in specific antibodies to M pneumoniae that is demonstrable by complement fixation,
immunofluorescence, passive hemagglutina-tion, and growth inhibition.
A variety of
nonspecific reactions can be observed . Cold hemagglutinins for group O human
eryth-rocytes appear in about 50% of untreated patients, in rising liter, with
the maximum reached in the third or fourth week after onset. A titer of 1:32 or
more supports the diagnosis of M
pneumonias infection.
Tetracyclines
or erythromycins in full systemic doses (
M pneumoniae infections are endemic all
over the world. In populations of children and young adults where close contact
prevails, and in families, the infection rate may be high (50-90%), but the
incidence of pneumonitis is variable (3-30%). For every case of frank
pneumonitis, there exist several cases of milder respiratory illness. M pneumoniae is apparently transmitted
mainly by direct contact involving respiratory secretions. Second attacks are
infrequent. The presence of antibodies to M
pneumoniae has been associated with resistance to infection but may not be
responsible for it. Cell-mediated immune reactions occur. The pneumonic process
may be in part attributed to an immunologic response rather than only to
infection by mycoplasmas. Experimental vaccines have been prepared from
agar-grown M pneumoniae. Several such
killed vaccines have aggravated subsequent disease; a degree of protection has
been claimed with the use of other vaccines.
On rare
occasions, central nervous system involvement has accompanied or followed
mycoplasmal pneumonia.
References:
1. Hadbook on Microbiology. Laboratory diagnosis of
Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 149-165.
2.
Medical Microbiology and Immunology: Examination and Board Rewiew /W.
Levinson, E. Jawetz.– 2003.– P. 151-164.
3.
Review of
Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical
Publication, Los Altos, California, 2002. – P. 285-314.
4.
Essential of
medical microbiology /W. A. Volk.. et al.– 5 ed., 1995
SUPPLEMENT
http://en.wikipedia.org/wiki/Treponema_pallidum
http://www.cehs.siu.edu/fix/medmicro/trepo.htm
http://microbewiki.kenyon.edu/index.php/Treponema
http://www.gsbs.utmb.edu/microbook/ch036.htm
http://www.bacteriamuseum.org/species/Tpallidum.shtml
http://dermatlas.med.jhmi.edu/derm/result.cfm?Diagnosis=1596362672 !!!!!
Atlas
http://en.wikipedia.org/wiki/Bejel
http://en.wikipedia.org/wiki/Pinta_(disease)
http://microbewiki.kenyon.edu/index.php/Leptospira
http://en.wikipedia.org/wiki/Leptospira
http://www.gsbs.utmb.edu/microbook/ch035.htm
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.chapter.1929
http://microbewiki.kenyon.edu/index.php/Borrelia
http://en.wikipedia.org/wiki/Borrelia_burgdorferi
http://textbookofbacteriology.net/Lyme.html
http://www.wadsworth.org/databank/borreli.htm
http://www.kcom.edu/faculty/chamberlain/Website/Lects/RICKETT.HTM !!!
http://en.wikipedia.org/wiki/Coxiella_burnetii
http://www.cdc.gov/ncidod/dvrd/qfever/
http://microbewiki.kenyon.edu/index.php/Coxiella
http://menshealth.about.com/b/a/190382.htm
http://en.wikipedia.org/wiki/Chlamydia
http://www.netdoctor.co.uk/diseases/facts/chlamydia.htm
www.gsbs.utmb.edu/microbook/ch039.htm
http://pathmicro.med.sc.edu/mayer/chlamyd.htm
http://en.wikipedia.org/wiki/Mycoplasma
www.emedicine.com/EMERG/topic467.htm
www.health.state.ny.us/diseases/communicable/mycoplasma/fact_sheet.htm
http://www.gsbs.utmb.edu/microbook/ch037.htm
FILM Lyme
bacteria Cyst formation with detail http://www.youtube.com/watch?v=lVmCa70bAxE