INFECTIOUS DISEASES
DIAGNOSTICs (a)
prokaryotes and eukaryotes
(unicellular agents)
Malaria
(6 CE Hours)
Learning objectives
!! Gain an understanding of the initial
diagnostic overview.
!! Explain the significance of the local
epidemiological background and
considerations.
!! Explain how and where humans and animals
can become infected.
!! Explain how to diagnose and monitor the
progress of infectious diseases.
!! List the contraindications and differential
diagnostic considerations.
Introduction
After an initial triage establishing an animal’s
ease of breathing, fever, anemia (color of the
gums, conjunctivae), debilitation, weakness, loss
of appetite, level of awareness, gait, posture and
mobility, gastrointestinal difficulties (vomiting,
diarrhea, appearance of vomit and stool),
distension of abdomen, ability to urinate (volume
and coloration) and medication given, the initial
impression a diseased animal presents must be
placed in the today’s regional epidemiological
context. This includes diseases in the
neighborhood and the presence and prevalence of
vectors or carriers for potential infectious agents.
Initial sample collection for diagnosis should
include samples for a study of its microbiology,
serology, pathology, hematology, and clinical
biochemistry aspects. Samples should be kept at
refrigerator temperature rather than frozen.
After categorizing the disease presentation based
on its history and initial triage, our assumptions
must be confirmed and verified: Isolate and
identify the causative pathogens, unicellular
eukaryotes and prokaryotes, their antigens, the
antibody produced by these antigens and the
general immune responsiveness of the patient and
the likely effectiveness of available treatment.
Because the number of global zoonoses is
overwhelming, only pathogens considered by the
WHO or CDC to be of interest are included here.
Safety concerns
More than 60 percent of emerging human
diseases are zoonoses, i.e., derived from animals.
It is, therefore, critical that all laboratory workers
concerned with the early diagnostic processes be
aware and trained in proper safety procedures.
Facilities should be provided with airlocks and
negative internal air pressure. All surfaces should
be nonporous and easily sterilizable.
Protective clothing, including booties, when
entering and the removal of protective covers
when exiting the work area should be required.
All protective laboratory clothing, unless
disposable, should be autoclaved before going
through the standard laundering process.
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The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female
Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells  and
mature into schizonts , which rupture and release merozoites . (Of note, in P. vivax and P. ovale
a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream
A ), the
weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony 
B ). Merozoites
parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony 
infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing
merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . Blood
stage parasites are responsible for the clinical manifestations of the disease.
The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an
Anopheles mosquito during a blood meal . The parasites’ multiplication in the mosquito is
C . While in the mosquito’s mid-gut, the microgametes penetrate the
known as the sporogonic cycle 
macrogametes generating zygotes . The zygotes in turn become motile and elongated (ookinetes)
11 . The oocysts
which invade the midgut wall of the mosquito where they develop into oocysts 
12 , which make their way to the mosquito’s salivary glands.
grow, rupture, and release sporozoites 
Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle .
Technicians should wear face masks, goggles,
head cover and gloves, and eating, drinking or
mouth-pipetting should not be allowed.
parasitism, both host and parasite benefit and help
each other to survive. If the parasite lives to kill
its host, it terminates its own chance of survival.
Ideally, laminar flow hoods under negative
pressure should be available to protect from
aerosols produced by homogenization and
sonication of samples being processed.
This review only covers pathogenic parasites that
will harm their hosts, weaken and occasionally
kill them.
Particular care should be taken with the
manipulation, shipment, storage and disposal of
fresh and often highly infectious test samples
stored for cataloguing and reference purposes. If it
does not interfere with testing purposes, samples
should be preserved in disinfecting preservatives,
such as 10 percent buffered formalin or similar.
Pathogens
Parasites here considered are unicellular
eukaryotes and intracellular prokaryotes. The
parasite lives in symbiosis with its host organism
and benefits from it by exploiting it for food,
habitat and spreading its kind. In the ideal
Intracellular parasites are prokaryotic and include
bacteria, viruses and other subcellular organisms.
Bacteria are prokaryotes, single-cell organisms
without nuclear membrane or intracellular
membrane-bound structures. Procaryotes
make up the most abundant biomass on earth,
representing 90 percent of the total weight
of all biological organisms put together. The
prokaryotic cell is made up of:
■■ A cytoplasmic body, containing ribosomes,
one genome (DNA) and various other bodies
and inclusions.
■■ A cell capsule, cell membrane and plasma
membrane.
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■■ Multiple or single external appendages giving
it motility.
Amebiasis
Eukaryotes are more organized; they have a nuclear
membrane, the Golgi apparatus, mitochondria and
chloroplasts, and several chromosomes. They are
much larger than prokaryotes.
Unicellular eukaryotes (endoparasites)
Plasmodium falciparum requires a vector,
Anopheles mosquito, to pass on malaria. Effective
mosquito control is the best preventive measure
available. The disease produces a characteristic
symptomatology: recurring cycles of sudden
coldness, stiffness, then fever and sweats in four- to
six-hour intervals; shivering; joint pain; vomiting;
anemia and hemoglobinuria; retinal damage; and
convulsions. Children with malaria show signs
of severe brain damage, abnormal posturing and
cognitive impairment. Cerebral malaria, to which
children seem to be more vulnerable, is often
associated with retinal whitening.
Blood smear of Plasmodium falciparum
(gametocytes - sexual forms).
Blood smear from a P. falciparum culture (K1
strain - asexual forms) - several red blood cells
have ring stages inside them. Close to the center,
there is a schizont and on the left a trophozoite.
In the U.S., about 1,500 cases are reported
each year, usually through travelers, source
of 63 outbreaks of malaria during the past 50
years. Worldwide, the WHO reports 750,000
to 1 million deaths due to malaria, and about
90 percent of them in Africa. After respiratory
infections, HIV/AIDS, diarrheal diseases,
and tuberculosis, it is the fifth most important
infectious disease in the world.
Malaria may remain dormant for two to four
years and then be reactivated anytime.
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Cysts and trophozoites are passed in feces . Cysts are typically found in formed stool, whereas
trophozoites are typically found in diarrheal stool. Infection by Entamoeba histolytica occurs by
ingestion of mature cysts  in fecally contaminated food, water, or hands. Excystation occurs
in the small intestine and trophozoites  are released, which migrate to the large intestine. The
trophozoites multiply by binary fission and produce cysts , and both stages are passed in the
feces . Because of the protection conferred by their walls, the cysts can survive days to weeks
in the external environment and are responsible for transmission. Trophozoites passed in the stool
are rapidly destroyed once outside the body, and if ingested would not survive exposure to the
A:
gastric environment. In many cases, the trophozoites remain confined to the intestinal lumen (
noninvasive infection) of individuals who are asymptomatic carriers, passing cysts in their stool.
B : intestinal disease), or, through
In some patients the trophozoites invade the intestinal mucosa (
C : extraintestinal disease),
the bloodstream, extraintestinal sites such as the liver, brain, and lungs (
with resultant pathologic manifestations. It has been established that the invasive and noninvasive
forms represent two separate species, respectively E. histolytica and E. dispar. These two species
are morphologically indistinguishable unless E. histolytica is observed with ingested red blood cells
(erythrophagocystosis). Transmission can also occur through exposure to fecal matter during sexual
contact (in which case not only cysts, but also trophozoites could prove infective).
Amebiasis, Entameba histolytica, a singlecelled, protozoan parasite, has been found in
monkeys and man, in dogs, cats and rarely in
other mammals. Usually in a commensalist
relationship, it lives in the large intestine and
cecum asymptomatically. But it may enter the
intestinal mucosa and produce acute or chronic
colitis, severe persistent enteritis and diarrhea,
which is not necessarily characteristic for this
disease. It can turn into a fulminant disease,
causing amebic dysentery with blood and mucus
in the stool.
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By entering the bloodstream and being dispersed
throughout the entire body, it may end up in the
liver with amebic abscesses. Surviving hosts will
continue to shed the infectious ameba, and the
disease may turn chronic or spontaneously resolve.
Giardia
In the chronic stage, there will be weight loss,
anorexia, tenesmus, and chronic continuous
or intermittent diarrhea or dysentery. In
immunocompromised individuals, the organism
may disperse throughout the body, invade
perianal skin, genitalia, liver, brain, lungs,
kidneys, and other organs.
Amebiasis causes annually about 70,000 deaths
worldwide, with many of them in developing
countries. Particular risk groups are travelers,
recent immigrants, male homosexuals and
institutionalized populations.
Giardiasis: Giardia lamblia may produce weight
loss, diarrhea and steatorrhea in dogs and cats.
The disease may not present itself at all or it may
appear only intermittently. Puppies and kittens
are more vulnerable. Calves can be affected,
producing soft malodorous stools containing
mucus and fatty matter, vomiting occasionally.
Cysts are resistant forms and are responsible for transmission of giardiasis. Both cysts and
trophozoites can be found in the feces (diagnostic stages) . The cysts are hardy and can survive
several months in cold water. Infection occurs by the ingestion of cysts in contaminated water,
food, or by the fecal-oral route (hands or fomites) . In the small intestine, excystation releases
trophozoites (each cyst produces two trophozoites) . Trophozoites multiply by longitudinal binary
fission, remaining in the lumen of the proximal small bowel where they can be free or attached to
the mucosa by a ventral sucking disk . Encystation occurs as the parasites transit toward the colon.
The cyst is the stage found most commonly in nondiarrheal feces . Because the cysts are infectious
when passed in the stool or shortly afterward, person-to-person transmission is possible. While
animals are infected with Giardia, their importance as a reservoir is unclear.
In man there are about 20,000 cases reported
annually in the United States, but there may be
as many as 2 million infections. The infection
causes symptoms only in about half of those
infected. It ranges from asymptomatic to severe
diarrhea and malabsorption. It is more severe in
children than in adults.
Acute giardiasis develops after an incubation
period of 1 to 14 days (average of seven days)
and usually lasts one to three weeks. Symptoms
include explosive diarrhea, malaise, excessive
flatulence or belching, often nausea causing
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Intermediate magnification micrograph of a
small bowel mucosa (duodenum) biopsy with
giardiasis. H&E stain.
High magnification micrograph of a small bowel
mucosa (duodenum) biopsy with giardiasis.
H&E stain.
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Toxoplasmosis
they are immunocompromised or weakened by
some other disease. Look for signs reflecting
affectation of muscles and the central nervous
system, such as cranial nerve deficit, seizures,
ataxia, stiffness of gait and lameness, and paresis.
The only known definitive hosts for Toxoplasma gondii are members of family Felidae (domestic
cats and their relatives). Unsporulated oocysts are shed in the cat’s feces . Although oocysts are
usually only shed for 1-2 weeks, large numbers may be shed. Oocysts take 1-5 days to sporulate
in the environment and become infective. Intermediate hosts in nature (including birds and
rodents) become infected after ingesting soil, water or plant material contaminated with oocysts .
Oocysts transform into tachyzoites shortly after ingestion. These tachyzoites localize in neural and
muscle tissue and develop into tissue cyst bradyzoites. Cats become infected after consuming
intermediate hosts harboring tissue cysts . Cats may also become infected directly by ingestion
of sporulated oocysts. Animals bred for human consumption and wild game may also become
infected with tissue cysts after ingestion of sporulated oocysts in the environment . Humans can
become infected by any of several routes:
■■ Eating undercooked meat of animals harboring tissue cysts .
■■ Consuming food or water contaminated with cat feces or by contaminated environmental samples
(such as fecal-contaminated soil or changing the litter box of a pet cat) .
■■ Blood transfusion or organ transplantation .
■■ Transplacentally from mother to fetus .
In the human host, the parasites form tissue cysts, most commonly in skeletal muscle, myocardium,
brain, and eyes; these cysts may remain throughout the life of the host. Diagnosis is usually achieved
by serology, although tissue cysts may be observed in stained biopsy specimens . Diagnosis of
congenital infections can be achieved by detecting T. gondii DNA in amniotic fluid using molecular
11 .
methods such as PCR 
vomit, and steatorrhoea (pale, foul smelling,
greasy stools). In the immunocompetent
individual, the disease is often self-limiting. In
chronic giardiasis, the symptoms are recurrent,
and malabsorption and debilitation may occur. In
the immunocompromised patient the disease may
become prolonged or recurring. The disease is
found worldwide, especially in warmer climates.
Toxoplasmosis: In the cat, Toxoplasma gondii
produces stillbirth, weak debilitated newborns,
jaundiced appearance, and enlarged abdomen.
Contiuous sleeping or crying of the newborn
suggests congenital toxoplasmosis. As a rule,
toxoplasma-infected cats show little evidence of
disease, although there may be some fever and
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In man, especially when immunocompetent, the
infection remains usually without symptoms.
Toxoplasmosis is the third leading cause of
death due to food-borne illness in the United
States. One to two out of 10 infected cases may
present an influenza-like picture with cervical
lymphadenopathy that usually resolves itself within
a couple of weeks or months. On rare occasions,
there may be an eye infection and loss of vision.
In the immunocompromised person you may
find systemic infections, retinochorioditis,
pneumonitis and central nervous system disease.
Congenital toxoplasmosis may have serious
implications for children, including premature
birth, damage to the central nervous system and
the eyes, skin and ears. Sometimes symptoms
appear later on in the young adult 20 to 30 years
of age. Retinochoroiditis is a frequent sequel.
Early diagnosis is of the essence because early
treatment of the mother may mitigate the severity
of the disease later on.
Cryptosporidiosis: Cryptosporidium parvum
is common in ruminants, producing intestinal
injury and neonatal diarrheas in young calves,
lambs, kids, foals and piglets, and in man, often
in connection with other enteropathogens.
About 70 percent of baby dairy calves more
than 5 days old have cryptosporidia. Alone or
swollen lymph nodes. While there may be no sign in combination with other enteropathogens, it
of the disease, there may be sudden death in cats. may appear as minor enteric infection or produce
outbreaks of severe diarrhea and high fatality
In more severe cases fever, lymphadenopathy,
rates in the very young, up to 20 days of age.
lethargy and loss of appetite, suggestions of
myositis, encephalitis, hepatitis, uveitis, iritis,
In pigs, we find the agent past the neonatal
retinitis, chorioretinitis and other damage to
stage up to marketing age but usually without
the eye may direct your diagnosis. There may
producing symptoms other than occasional postbe vomiting, diarrhea, occasional lameness and
weaning malabsorptive diarrhea.
signs of neurological disease. When concomitant
In foals, the organism appears less frequently and
with other diseases, especially diseases that
is seen at a later age, with excretion rates peaking
suppress the immune system, toxoplasmosis can
at 5 to 8 weeks of age. No infection is detected in
be severely exacerbated and lead to quick death.
yearlings or older animals. In Arabian foals with
Dogs are less frequently infected. They seem
inherited combined immunodeficiency, you may
to be much more resistant to the disease, unless
detect persistent infection but no severe disease.
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Cryptosporidiosis
Cryptosporidium muris oocysts found in human
feces.
Cryptosporidia have been found in young deer,
causing diarrhea, and also in turkeys and chickens,
producing inflammation of the air sacs, coughing
and gasping for air and, sometimes, death.
In man, you may find a range from no symptoms
to voluminous watery diarrhea, dehydration,
weight loss, abdominal pain, fever, nausea and
vomiting. Although mostly in the small intestine,
symptoms may be found disseminated to other
organs. Symptoms are usually of short duration.
Trichomoniasis: This is a venereal disease,
and is found worldwide in animal and man.
Trichomonas foetus produces infertility in cattle,
early fetal death and extending calving intervals.
It can be found in the genital canal of cattle. An
infected bull may infect as many as 90 percent of
the cows he inseminates naturally. Semen from
infected bulls used for artificial insemination
may also pass on the infection. Bulls may remain
infective indefinitely. Cows will recover within
three months after breeding, however, they are
likely to be re-infected because their immunity
does not last.
Trichomonas gallinae causes canker, rapid
caseous buildup in oral mucosa, throat, weight
loss in domestic fowl and other birds. With a
watery discharge from mouth and eyes, they may
die within eight to 10 days or turn chronic and
continue to shed the organism.
Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host through feces and
possibly other routes such as respiratory secretions . Transmission of Cryptosporidium parvum and
C. hominis occurs mainly through contact with contaminated water (e.g., drinking or recreational
water). Occasionally food sources, such as chicken salad, may serve as vehicles for transmission.
Many outbreaks in the United States have occurred in waterparks, community swimming pools,
and day care centers. Zoonotic and anthroponotic transmission of C. parvum and anthroponotic
transmission of C. hominis occur through exposure to infected animals or exposure to water
contaminated by feces of infected animals . Following ingestion (and possibly inhalation) by a
a occurs. The sporozoites are released and parasitize epithelial cells
suitable host , excystation 
c ) of the gastrointestinal tract or other tissues such as the respiratory tract. In these cells, the
b,
(
c ,
f ) and then sexual
d,
parasites undergo asexual multiplication (schizogony or merogony) (
g
h.
multiplication (gametogony) producing microgamonts (male)  and macrogamonts (female) 
j ,
i ), oocysts (
k ) develop that
Upon fertilization of the macrogamonts by the microgametes (
sporulate in the infected host. Two different types of oocysts are produced, the thick-walled, which
j , and the thin-walled oocyst 
k , which is primarily involved
is commonly excreted from the host 
in autoinfection. Oocysts are infective upon excretion, thus permitting direct and immediate fecaloral transmission.
Note that oocysts of Cyclospora cayetanensis, another important coccidian parasite, are unsporulated
at the time of excretion and do not become infective until sporulation is completed. Refer to the life
cycle of Cyclospora cayentanensis for further details.
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In man, Trichomonas vaginalis is one of the
most common sexually transmitted diseases
worldwide. About 1 million new cases are
reported in North America every year. Symptoms
range from none to vaginal itching and frothy
green discharge from the vagina. Sexual
intercourse may be painful with lower abdominal
pain and the urge to urinate. Men may have no
Micrograph showing a positive result for
trichomoniasis. A trichomonas organism is seen
on the top-right of the image.
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Cutaneous leishmaniasis in the hand.
Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the
infective stage, promastigotes, during blood meals . Promastigotes that reach the puncture wound are
phagocytized by macrophages  and transform into amastigotes . Amastigotes multiply in infected
cells and affect different tissues, depending in part on the Leishmania species . This originates the
clinical manifestations of leishmaniasis. Sandflies become infected during blood meals on an infected
host when they ingest macrophages infected with amastigotes (, ). In the sandfly’s midgut, the
parasites differentiate into promastigotes , which multiply and migrate to the proboscis .
Cutaneous leishmaniasis ulcer on left forearm.
symptoms, or they may show discharge from the
urethra, urges to urinate and a burning sensation
when urinating.
During a blood meal on the mammalian host, an infected tsetse fly (genus Glossina) injects metacyclic
trypomastigotes into skin tissue. The parasites enter the lymphatic system and pass into the
bloodstream . Inside the host, they transform into bloodstream trypomastigotes , are carried
to other sites throughout the body, reach other blood fluids (e.g., lymph, spinal fluid), and continue
the replication by binary fission . The entire life cycle of African Trypanosomes is represented by
extracellular stages. The tsetse fly becomes infected with bloodstream trypomastigotes when taking
a blood meal on an infected mammalian host (, ). In the fly’s midgut, the parasites transform
into procyclic trypomastigotes, multiply by binary fission , leave the midgut, and transform into
epimastigotes . The epimastigotes reach the fly’s salivary glands and continue multiplication by
binary fission . The cycle in the fly takes approximately 3 weeks. Humans are the main reservoir
for Trypanosoma brucei gambiense, but this species can also be found in animals. Wild game animals
are the main reservoir of T. b. rhodesiense.
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Leishmaniasis: Leishmania belongs to the
genus of trypanosomatida. It is represented by
two forms, a flagellate promastigote carried by
the insect vector and an aflagellate amastigote
producing the disease in their vertebrate hosts.
It is transmitted by sand flies (phlebotomus)
to dogs, rarely cats, rodents and man. It
affects about 12 million people worldwide.
The incubation period is variable, from three
months to years. The dog is the reservoir for this
organism wherever there is the carrier insect in
tropical and subtropical regions of the globe.
The disease it causes includes cutaneous (the
“Oriental sore”), mucocutaneous and visceral
symptomatology. The visceral disease is a
chronic, severe, protozoal disease of humans,
dogs, and certain rodents causing lesions on skin
and mucosae, nose bleed, ocular lesions, local or
generalized lymphadenopathy, anemia, weight
loss, lameness and renal failure.
Sleeping sickness: Trypanosoma come in
four forms: amastigote (short flagellum,
basal body before nucleus); promastigote
(long detached flagellum, basal body before
nucleus), epimastigote (long flagellum
attached along cell body, basal body before
nucleus), and trypomastigote (long flagellum,
attached along body, basal body posterior of
nucleus). Trypanosoma brucei brucei and other
trypanosoma infect cattle, sheep, goats, pigs,
horses and camels. The tsetse fly is required for
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transmission, and the geographic distribution of
the disease is a function of their presence in a
given region.
Domestic animals are considered the reservoir
of the pathogen for man. The tsetse fly inserts
the pathogenic organisms into the skin of the
host from which it is feeding. The pathogen will
grow locally and produce local swelling, enter
the lymphatic system and the blood stream.
Clinical signs include intermittent fever, anemia,
weight loss.
The chronic course in cattle causes swollen
lymphnodes, fat atrophy and anemia and may
eventually lead to death.
In man, Trypanosoma brucei rhodesiense and
Trypanosoma brucei gambiense infection
starts with a fly bite, which produces a painful,
reddened swelling, from there entering the
lymphatic system and the blood stream and
causing daytime drowsiness, anxiety, fever,
headache, fear and mood swings, sweating,
increased sleepiness and the urge to sleep in spite
of sleeplessness at night. There may be swollen
lymph nodes all over the body, and myocarditis
may develop. Avoidance of tsetse fly habitat
territories will avoid the disease.
Prokaryotes
Prokaryotes are single celled organisms that
do not have a nuclear membrane. Bacteria are
prokaryotes.
Anthrax is caused by sporeforming Bacillus
anthracis. Their spores are resistant to external
influences and will survive infective in the
soil for many years. Spores may be ingested
with hay or by grazing or via the inhalation of
contaminated dust. It is common in herbivores all
over the world. Depending on how it enters the
body, it will produce different clinical forms. In
cattle, sheep and other herbivores, it will cause
acute septicemia, hemorrhagic lymphadenitis and
often death. It appears less severe in dogs, pigs,
horses and man.
The mechanical transport from animal to animal
by biting flies has been suggested. Contaminated
foodstuffs, bone meal and meat from infected
animals have been shown to infect omnivores and
carnivores. Humans get it from handling infected
animals or their products or by direct contact.
Depending on the manner of contact with
the pathogen, the disease shows as primarily
a cutaneous disease in man. Pharyngeal or
gastrointestinal anthrax must be expected after
the consumption of undercooked contaminated
meat. Inhalation anthrax, also called woolsorter’s
disease, produces acute hemorrhagic
lymphadenitis and often death.
Brucellosis in cattle is caused by Brucella
abortus (Brucella suis or Brucella melitensisa
are usually also involved, playing a lesser role).
They are facultative intracellular Gram-negative
coccobacilli (0.5 to 0.7 by 0.6 to 1.5 µm in size),
non-motile, non-encapsulated. They are found
worldwide. Brucellosis affects all herbivores as
well as dogs and man.
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This diagram shows the lifecycle of blacklegged ticks that can transmit Lyme disease.
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The disease in cows is characterized by abortion
and placenta retention. In bulls, it produces orchitis
and infection of the accessory sex glands. In the
horse, it appears infrequently, and when it does,
it produces open suppurative bursitis.The disease
in man, not very common in the U.S. (100 to 200
cases annually), is usually caused by Brucella
melitensisa. It is referred to as undulant fever or
Malta fever as it usually occurs throughout the
Mediterranean region. It is considered a potentially
serious public health problem.
Leptospirosis: Leptospira interrogans appears
worldwide in domestic animals and wildlife,
and the clinical signs are similar for all of
them, ranging from asymptomatic to fever,
hemoglubinuria, icterus, abortion, infertility and
death. Eventually, the pathogen may localize in
the kidneys and reproductive system and be shed
in large numbers through the urinary tract for
many months, if not years.
The leptospira survive for a long time in surface
waters, which seem to be the major source of reinfestation. Floods usually result in an increase of
the disease incidence.
In its thoracic form it may enter the abdominal
cavity as well, producing suppurative pleuritis
and peritonitis with pericardial, pleural and
peritoneal effusions. Abscesses can be found in
all the visceral organs as well as stomatitis and
ulcerative gingivitis.
Borrelia bacteria, the causative agent of Lyme
disease, magnified.
Q Fever: Its cause, Coxiella burnetii, is
an obligate intracellular Gram-negative
coccobacillus. In the wild and domestic ruminant
it localizes in the placenta, uterus, mammary
glands and supramammary lymph nodes. It is
shed with milk, urine, feces and in placental
tissue and amniotic fluid. Cats, arthropods and
birds are known to have been infected.
Leptospira enter the body through the skin,
mucous membranes by direct contact with
infected urine, or urine-contaminated surface
water. Sexual transmission is possible. They enter
the lymphatic system and bloodstream and are
readily disseminated to all visceral organs.
Listeriosis: Listeria monocytogenes, a Grampositive pleomorphic coccobacillus, nonsporeforming, motile by means of flagellae, will infect
a variety of domestic and wild animals, birds,
fish, arthropods and humans. As many as 10
percent of humans are thought to be intestinal
carriers. The disease is found primarily in
the elderly, pregnant women, newborns and
immunocompromised adults. About 2,500 cases
are reported annually, and 500 of them may die;
pregnant women are 20 times more likely than
other healthy adults to get listeriosis. About onethird of listeriosis cases happen during pregnancy.
It is found in at least 37 mammalian species and
17 species of birds as well as fish and shellfish.
Existing worldwide, it is saprophytic and prefers
temperate or cooler climates. Aside from the
mammalian gastrointestinal tract as reservoir,
it can be found in sewage, surface water, soil
animal food products (milk, cheese) and feces.
Thus the main source of infection for ruminants
is contaminated vegetation, hay, silage and soil.
The initial signs of infection include loss of
appetite, lack of responsiveness, disorientation
and circling. Abortion may occur in the later
stage of pregnancy. The fetus may die in utero,
arrive stillborn or die soon after birth. In sheep,
the abortion rate may go as high as 20 percent.
There will be septicemia, encephalitis and
meningoencephalitis. In poultry, you may see
septicemia, myocardial and hepatic necrosis.
Sheep and goats may die within one or two days
after the onset of the disease.
Borreliosis: Borrelia burgdorferi sensu lato
(there are at least 11 members of this group)
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In cattle, it produces acute or chronic mastitis,
granulomatous lesions and fistulous drainages.
Horses are affected infrequently and usually
only when immunocompromised. There are
about 500 to 1,000 new cases in man every year
in the U.S. It produces an invasive pulmonary
infection, generalized infection or brain abscess
in about 80 percent of its cases and cellulitis in
20 percent. The pulmonary infection comes with
fever, cough, chest pain and a 10 percent fatality
rate, which increases with generalization of the
disease and the development of brain abscesses.
The central nervous system is accompanied by
lethargy, confusion, headache, seizures and other
neurological deficits.
Bull's eye pattern from a tick bite.
is a Gram-negative spirochetal bacterium
that requires a tick (Ixodes scapularis, Ixodes
pacificus) for transmission from its reservoir
host (small rodents, dogs) to its next host. In the
host animal (dogs), you will notice anorexia,
lameness, fever, lethargy, lymphadenopathy and
painful swollen joints. Infection of the kidneys,
signaled by uremia, hyperphosphatemia, proteinlosing nephropathy and peripheral edema, is
usually fatal. Bradycardia, facial paralysis and
seizures are additional indicators of the disease.
In man, in produces Lyme disease. The deer
tick (Ixodes scapularis) drops on the individual
wandering in the woods, bites and passes on the
pathogen. The bite is marked by a red spot that
enlarges and produces several red rings, leading
to fever, muscle pain and swollen joints. It may
affect the brain and the nervous system. The
disease is found in almost all the states of the
U.S. and elsewhere in the world.
Nocardiosis: Nocardia asteroides (there is a
number of different strains in the Nocardia
family) is a non-motile, non-sporeforming,
aerobic, Gram-positive rod, partially acid-fast,
splitting sugar by oxidation. Being saprophytic
and soil-borne, the pathogen enters the body
through wounds or by inhalation, produces
suppurative pustules in dogs, less often in cats,
usually with lymph node involvement.
Distributed worldwide except for New Zealand,
it is so infectious that a single particle inhaled
will cause the disease. The pathogen is resistant
to heat, drying and most disinfectants and may
remain infective for months. Its main route of
transmission is from animal to animal either
directly or via ticks, which may serve as reservoir
was well.
In the U.S., the highest incidence is in sheep
(41.6 percent), followed by goats (16.5 percent),
and cattle (3.4 percent ).
Shigellosis is caused by the Gram-negative
bacteria Shigella which, worldwide, sickens
164.7 million people and kills 1.1 million. More
than 99.9 percent of the people affected live in
developing countries, and more than 60 percent of
the deaths involve children under 5 years of age.
In particular, it produces dysentery, frequent
watery diarrhea, small amounts of stool, blood,
pus and mucus. The pathogen is passed in
the stool and is source of continued spread,
especially with poor sanitation and hygiene.
Fluid replacement is a critical part of therapy.
Shigella is common throughout the world; in the
United States, 18,000 people develop shigellosis
each year. It is highly infectious: it takes only a
few of the organisms to cause infection.
In the large intestine, the bacteria cause
inflammation and are then excreted in stool.
Tularemia is produced by Francisella tularensis,
a facultative intracellular non-sporulating, Gramnegative coccobacillus that infects more than 250
species of mammalian wild life, rodents, fish,
birds, reptiles and man. Sheep are the primary
host, but cats, dogs, pigs and horses have been
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found infected. Transmission is via ticks and
biting flies, which serve not only as vector
but also as reservoir and is via direct contact,
inhalation, ingestion.
Malaria
Clinical signs of infection suggest the pathway
of entry, and regional lymphnodes are usually
implicated. The general picture includes high
fever, lethargy, anorexia, stiffness and reduced
mobility, increased pulse and respiratory rates,
coughing, diarrhea, pollakiuria (abnormally
frequent urination), and eventually, septicemia
and prostration death.
The pathogen is susceptible to proper disinfection
and heat, yet can survive for months in a moist
environment.
Amplification and identification
Pathogens must be isolated and brought up to
workable quantities for identification. Samples
must be collected under aseptic conditions, with
sterilized instruments and containers. Growth
media, cell cultures, laboratory animals may have
to be employed to enrich these pathogens as an
important first step. A matrix of culture media
and controlled growth conditions will facilitate an
approximation to the identity of the agent under
investigation. Samples are taken from an infected
area or tissue, taking a scraping or swab, from
blood or other body fluids.
Plasmodium falciparum
Plasmodium falciparum: Understanding the
life cycle of this parasite facilitates the choice
of diagnosistic steps. There are approximately
156 named species of Plasmodium, which infect
various species of vertebrates. Four species are
mosquito inoculates sporozoites into the human
host  Sporozoites infect liver cells  and
mature into schizonts,  which rupture and
release merozoites . (Of note, in P. vivax and P.
ovale, a dormant stage [hypnozoites] can persist
in the liver and cause relapses by invading the
bloodstream weeks, or even years later.)
sporozoites into a new human host perpetuates
the malaria life cycle .
Diagnosis: During the symptomatic phase of
the disease, the parasite can be found in the
peripheral blood. At this time, blood samples
can be taken and subjected to microscopic
examination. Finger prick blood is placed on a
microscopic slide, stained with Romanowsky
stain and examined microscopically under oil
immersion. At least 200 fields must be checked.
Because the parasite may be sequestered in
deeper tissues and tissue capillaries, a low
count, expressed in number of organisms found
per number of red cells examined, does not
necessarily imply a low level of the disease.
Microscopic examination provides for species
After this initial replication in the liver (exoidentification as well as for a measure of
A ), the parasites
erythrocytic schizogony 
undergo asexual multiplication in the erythrocytes the extent of parasitemia and the various
developmental stages of the parasite. Presence of
B ). Merozoites infect
(erythrocytic schizogony 
malarial pigment in neutrophils and monocytes
red blood cells . The ring stage trophozoites
as evidence of plasmodium destruction, is a
mature into schizonts, which rupture releasing
better measure of the severity of the disease.
merozoites . Some parasites differentiate into
The prevalence of late stage, more mature forms
sexual erythrocytic stages (gametocytes) . of the parasite (trophozoites, schizonts), is an
Blood stage parasites are responsible for the
additional indicator of disease progression and
clinical manifestations of the disease. The
prognosis. Immunocompromised individuals
gametocytes, male (microgametocytes) and
frequently have much larger numbers in the blood
female (macrogametocytes), are ingested by an
(“hyperparasitemia”).
Anopheles mosquito during a blood meal .
The absence of parasitized erythrocytes in the
The parasites’ multiplication in the mosquito is
blood smear requires several repeat tests over
C . While in the
known as the sporogonic cycle 
the following two days to confirm the absence
mosquito’s stomach, the microgametes penetrate
of malaria. Deep sequestration of infected
the macrogametes generating zygotes . The
erythrocytes and anti-malarial chemotherapy
zygotes in turn become motile and elongated
may explain the absence of organisms from the
(ookinetes)  which invade the midgut wall of
peripheral blood.
the mosquito where they develop into oocysts
11 . The oocysts grow, rupture, and release

The quantitative buffy coat test, concentrating
12 , which make their way to the
sporozoites 
the parasite-carrying red blood cells, can be
mosquito’s salivary glands. Inoculation of the
used to detect and identify the parasite. It
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appears to be slightly more sensitive than the
blood smear procedure. The growing parasite
affects the buoyant density of the infected
erythrocyte, and because it does contain DNA
as opposed to red blood cells, which have no
nucleus, centrifugation will separate the blood
cells of different density and a nucleic acid stain
like acridine orange will identify the parasite
containing cells. The blood sample is centrifuged
in a capillary tube coated with acridine orange
and examined under ultraviolet light; the DNA
will stain green and the RNA orange. This
procedure has been found to increase detection
rates by about 4 percent.
If necessary, the layer between the buffy coat
and red cell pack, containing mostly white
blood cells, can be examined like a blood smear.
Commercially available test systems ParaSight-F
and ICT Malaria Pf have been found useful in
detecting plasmodium falciparum histidine-rich
protein 2, a water-soluble antigen.
Malaria rapid diagnostic devices speed up
screening for malaria but occasionally do require
microscopic verification of results.
The polymerase chain reaction process over
the past years has become the test of choice for
amplification and identification of infectious
pathogens. It is very sensitive and highly specific.
One or a few minute pieces of DNA can be
amplified exponentially to billions of copies
within a very short time span. Essentially the test
consists of (1) the piece of DNA to be amplified,
the “target” or template; (2) heat-stable DNA
polymerase, like Taq polymerase; (3) primers; (4)
deoxynucleoside triphosphates, dNTPs, the DNA
building blocks; (5) buffer; (6) divalent (Mg²)
and (7) monovalent phosphate cations; and (8)
thermal cycler equipment.
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The brew is subjected to 20 to 40 heating and
cooling cycles to allow for the assembly of
the new DNA replicates. Each cycle doubles
the amount of DNA generated, thus giving an
exponential rise of DNA molecules matching the
original target DNA.
Amebiasis
The detection of antibody can confirm the
diagnosis of the disease and be of historical
importance in the study of its epidemiology.
The enzyme-linked immunosorbent assay
(ELISA), also known as immune fluorescent
assay (IFA), has been used in different ways for
different purposes. The double antibody sandwich
procedure requires microtiter plates, bottoms
coated with the capture antibody, test samples
added and incubated for 1 hour at 37 degrees C,
rinsed to remove unfixed matter, and horseradish
peroxydase-linked antibody added to the test
sample, which is allowed to react and then rinsed
to remove unbound antibody. A chromogenic
substrate, 3,3’,5,5’-Tetramethylbenzidine, is then
added to visualize the indicator antibody to be
read on a spectrophotometer.
An indirect hemagglutination test has been used
to measure antibody titers: glutaraldehyde fixed
sheep red blood cells are sensitized with malaria
antigen and agglutinated by antibody to malaria.
Entamoeba histolytica
Entamoeba histolytica life cycle: Cysts and
trophozoites are passed in feces . Cysts
are typically found in formed stool, whereas
trophozoites are typically found in diarrheal stool.
Infection by Entamoeba histolytica occurs by
ingestion of mature cysts  in fecally contaminated
food, water or hands. Excystation  occurs
in the small intestine, and trophozoites  are
released, which migrate to the large intestine. The
trophozoites multiply by binary fission and produce
cysts , and both stages are passed in the feces .
Because of the protection conferred by their
walls, the cysts can survive days to weeks in the
external environment and are responsible for
transmission. Trophozoites passed in the stool are
rapidly destroyed once outside the body, and if
ingested, would not survive exposure to the gastric
environment. In many cases, the trophozoites
A:
remain confined to the intestinal lumen (
noninvasive infection) of individuals who are
asymptomatic carriers, passing cysts in their stool.
In some patients, the trophozoites invade the
B : intestinal disease), or,
intestinal mucosa (
through the bloodstream, extraintestinal sites such
C : extraintestinal
as the liver, brain and lungs (
disease), with resultant pathologic manifestations.
The clinical picture of amebiasis is not much
different from other intestinal diseases.
Morphological, chemical and immunological
means must be employed to identify the
cause. Cysts and trophozoites can be detected
and identified microscopically in fresh or
concentrated stool samples or aspirates from the
colon. Trichrome staining has been found useful
in the fixed samples. Western blotting has been
used to detect Entamoeba histolytica antigen.
Also called enzyme-linked electro-immunotransfer blot assay (EITB), it consists of the
concentration and isolation of the antigen
It has been established that the invasive and
sample by gel-electrophoresis (SDS-PAGE),
noninvasive forms represent two separate species, the transfer to nitrocellulose paper, the marking
respectively E. histolytica and E. dispar. These
of antigen on this paper with specific antibody,
two species are morphologically indistinguishable the identification of that marker-antibody by
unless E. histolytica is observed with ingested red horseradish peroxidase-linked antibody against
blood cells (erythrophagocystosis).
that marker-antibody, and visualizing its presence
by the action of the enzyme on a chromogenic
Transmission can also occur through exposure
substrate (e.g. o-diaminobenzidine or similar).
to fecal matter during sexual contact (in which
case, not only cysts, but also trophozoites could Without evidence of a pathogen in the stool,
prove infective).
antibody indicative of such pathogen should
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be sought. Antibody tests include indirect
hemagglutination, enzyme immune assays and
immunodiffusion.
For indirect hemagglutination, glutaraldehyde
fixed sheep erythrocytes are sensitized with
Entamoeba histolytica antigen and used to
indicate agglutination by entamoeba antibody in
U-shaped microtiter plates.
Enzyme immunoassays (EIA) and the enzymelinked immunosorbent assay (ELISA) are based
on the same principle as the radioimmuneassay
(RIA) except that they are replacing
radioactivity as reporter label with an enzyme.
Instead of measuring radioactivity, you are
measuring the colorimetric signals of enzyme
activity on a chromogenic substrate. This is of
particular value because of the enzyme’s ability
to amplify weak signals.
To measure antibody levels, the bottoms of
microtiter plates are coated with antigen;
dilutions of antibody are added, allowed to
incubate, and rinsed off liberally; and then
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horseradish-bound anti-γglobuline antibody id
added, allowed to incubate, then unbound matter
is rinsed off liberally again and the chromogenic
substrate added. The presence of enzyme will be
indicated by color changes that can be measured
colorimetrically.
Giardia
The sandwich ELISA works essentially the same
way, with the exception that the bottom of the
microtiter plates is first coated with antibody to
capture the antigen, i.e., hold it to the bottom
of the plate, then the sample antibody is added,
followed by the horseradish labeled antiγglobuline antibody and the indicator substrate.
Evidently, the detection and measure of antigen
can be executed in similar fashion by simply
reversing the reaction components.
EIA/ELISA test kits and automated process
systems are now commercially available for
use everywhere for many disease-producing
pathogens, including Entamoeba histolytica.
Antigen is produced as a soluble extract
from pure entamoeba cultures. This test
showed antibody in 95 percent of people with
extraintestinal amebiasis, 70 percent with
active intestinal infection, and 10 percent of
asymptomatic shedders of cysts.
Individuals with symptoms for acute amebic liver
abscess and negative antibody response should be
retested seven to 10 days later. A negative repeat
suggests another pathogen as culprit. Antibody
may persist for years after an infection, and
its finding by itself does not indicate a current
or active infection. Antigen determination is
significant in so far as it will help to confirm the
diagnosis and to distinguish pathogenic strains
from less pathogenic ones.
Giardiasis
Giardiasis: Giardia intestinalis is a protozoan
flagellate (Diplomonadida).
Life cycle: Cysts are resistant forms and are
responsible for transmission of giardiasis. Both
cysts and trophozoites can be found in the feces
(diagnostic stages) . The cysts are hardy
and can survive several months in cold water.
Infection occurs by the ingestion of cysts in
contaminated water, food, or by the fecal-oral
route (hands or fomites) .
In the small intestine, excystation releases
trophozoites (each cyst produces two trophozoites)
. Trophozoites multiply by longitudinal binary
fission, remaining in the lumen of the proximal
small bowel where they can be free or attached
to the mucosa by a ventral sucking disk .
Encystation occurs as the parasites transit toward
the colon. The cyst is the stage found most
commonly in nondiarrheal feces .
Because the cysts are infectious when passed
in the stool or shortly afterward, person-toperson transmission is possible. While animals
are infected with Giardia, their importance as a
reservoir is unclear. Diagnosis using fresh or concentrated feces
is fairly straightforward when you find cysts
or trophozoites for identification. If negative,
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sampling should be repeated several times
two days apart or duodenojejunal aspirates
(intubation) should be examined for trophozoites.
The enterotest (“string test”) is another way of
obtaining duodenal and bile samples. A gelatin
capsule attached to 140 cm-long soft nylon yarn (90
cm if for a child) is swallowed, left for four hours
and then the string is being pulled back up through
the mouth. The capsule would have been dissolved
by then. The mucus- and bile-covered string can
then be examined microscopically for trophozoites.
Giardia may appear only intermittently in
the stool of a patient; high excretion patterns
may alternate with low level ones. Sometimes
duodenal samples from infected patients may be
negative as well.
To increase sensitivity and specificity and
the probability of detecting lower numbers of
cysts, the direct fluorescent antibody (DFA)
test should be tried on concentrated stool
samples. Under the fluorescent microscope the
cysts become highlighted.
The detection of copro-antigens by enzyme
immunoassay (EIA) in unconcentrated stool
samples is a well recognized procedure.
Commercial kits are available. The rapid
immuno-chromatographic cartridge assays,
commercially available in different formats, work
on the unconcentrated stool sample as well.
Rapid analyte measurement platform, RAMP for
short, comprises a disposable test cartridge with
an analyte-specific immunochromatographic
nitrocellulose membrane strip and a portable
scanning fluorescence reader to measure
antigen/antibody complexes. Capture antibody
embedded in the test strip will combine with
antigen, if any, and will be identified by
fluorescent-labeled latex particles combined
with analyte-specific antibody. This test may
vary by temperature, viscosity of sample,
capillary speed and time permitted for migration
as well as by nature of test strip, structure,
wettability and other surface characteristics.
Having both test and control sample on the same
strip negates this potential problem.
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is added to demonstrate the presence of the
antigen/horseradish-antibody complex.
Toxoplasma gondii
Blood, cerebrospinal and other body fluids can be
given to mice intraperitoneally for amplification.
One week to 10 days post-inoculation, the
peritoneal fluid of the test mice is examined
microscopically for the presence of the organism.
If negative, check their blood for antibody four to
six weeks after inoculation.
Human fibroblast cell lines have been used to grow
Toxoplasma gondii. The cell cultures are held
for a month or until trophozoites are observed or
plaques develop in the cell sheet. This method of
parasite growth is fairly easy and straightforward.
If isolation attempts produce the live organism,
you are obviously dealing with an acute infection.
If not, the amplification by polymerase chain
reaction can be considered. PCR has become
the test of choice for rapid amplification and
identification of pathogen DNA from body
fluids and affected tissues, vitreous and aqueous
humor, effusions from lung and the central
nervous system, urine, peripheral blood and
amniotic fluid. This is of particular importance
for pregnant women, where early treatment is
essential to reduce effects on the child.
A commercial solid-phase qualitative
immunochromatographic assay allows detection
and identification of Giardia lamblia and
Cryptosporidium parvum within less than a
quarter of an hour on the same fecal sample.
Toxoplasma gondii
Toxoplasma gondii is a protozoan parasite that
infects most species of warm-blooded animals,
including humans, and can cause the disease
toxoplasmosis.
Life cycle: The only known definitive hosts
for Toxoplasma gondii are members of family
Felidae (domestic cats and their relatives).
Unsporulated oocysts are shed in the cat’s feces
. Although oocysts are usually only shed for
1-2 weeks, large numbers may be shed. Oocysts
take 1-5 days to sporulate in the environment and
become infective.
Intermediate hosts in nature (including birds and
rodents) become infected after ingesting soil,
water or plant material contaminated with oocysts
. Oocysts transform into tachyzoites shortly
after ingestion. These tachyzoites localize in
neural and muscle tissue and develop into tissue
cyst bradyzoites .
Cats become infected after consuming
intermediate hosts harboring tissue cysts . Cats
may also become infected directly by ingestion
of sporulated oocysts. Animals bred for human
consumption and wild game may also become
infected with tissue cysts after ingestion of
sporulated oocysts in the environment .
Humans can become infected by any of several
routes: eating undercooked meat of animals
harboring tissue cysts , consuming food
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or water contaminated with cat feces or by
contaminated environmental samples (such as
fecal-contaminated soil or changing the litter
box of a pet cat) , blood transfusion or organ
transplantation , and transplacentally from
mother to fetus .
In the human host, the parasites form tissue cysts,
most commonly in skeletal muscle, myocardium,
brain, and eyes; these cysts may remain throughout
the life of the host. Diagnosis is usually achieved
by serology, although tissue cysts may be observed
in stained biopsy specimens . Diagnosis of
congenital infections can be achieved by detecting
T. gondii DNA in amniotic fluid using molecular
11 .
methods such as PCR 
Toxoplasmosis is one of the most common
infectious diseases in the world and the third
leading cause of death from food-borne disease.
According to the CDC, some 60 million people in
the U.S. could have toxoplasmosis and many are
without symptoms.
The diagnosis of Toxoplasma gondii infection is
based on isolation and direct observation of the
parasite in exudates from the lung (bronchoalveolar
lavage) and lymph node biopsy. Multiple tissue
cysts can be found near inflammatory necrotic
lesions and demonstrate an acute infection or
possibly the reactivation of a past infection.
The immunoperoxidase procedure, utilizing
toxoplasma antibody, is useful in identifying the
organism. In this procedure, antibody conjugated
with horseradish peroxidase is added to the
antigen test sample in question (biopsy sample,
body fluid centrifugate). After appropriate
incubation to allow reaction, a thorough rinsing
off of unattached label, a chromogenic substrate
Toxoplasma antibody tests play an important role
in the diagnosis of the disease, especially if there
has been no direct observation of the pathogen.
IgG and IgM are measured by immunofluorescent
assay (IFA) or enzyme immune assay (EIA). The
presence of IgG proves past infection but does
not necessarily indicate a recent infection. IgM
is more suggestive of an acute infection, but it
also has been found as late as 1½ years after the
initial infection. On the other hand, the absence
of IgM confirms that there has been no recent
infection. The presence of IgM alone suggests a
false positive. The presence of both IgG and IgM
therefore is still no proof of an acute infection.
IgG avidity (binding force to the corresponding
antigen) increases with increasing passage of time
since infection. It is measured as ratio of antibody
titers with normal serum and serum treated
with urea. However, while a high rate of avidity
indicates a past infection, a low rate is still no
proof of an acute infection; low avidity levels may
persist in some individuals for many months.
These considerations are crucial for the pregnant
woman because a high IgG avidity level suggests
that the infection had not been acquired during
gestation. If, however, there are symptoms of
the disease in the pregnant woman and there is
a low IgG antibody titer, and if that titer were to
increase within a week or two, it can be assumed
that you are dealing with acute toxoplasmosis.
Another test developed to confirm presence of an
acute infection is the differential agglutination
test (AC/HS test) by which titers of antibody
agglutinating aceton fixed tachyzoites (AC) are
compared with antibody titers agglutinating
tachyzoites fixed with formalin (HS). Higher
titers of antibody to the AC fixed tachyzoites
suggest a recent, acute infection.
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IgE antibodies do not persist as long as do IgM
and IgA and are considered an added indicator
for a recent infection. Newborns should be tested
for both IgM and IgA using the EIA procedure.
Neutralizing IgG antibody are tested by employing
the Sabin-Feldman dye test. This test is not much
used in the field anymore, because it requires
live tachyzoites as indicator for the presence of
tachyzoite-killing antibody. However, it is very
sensitive and highly specific. Live tachyzoites
take up alkaline methylene blue. Antibody in the
presence of complement will kill tachyzoites and
keep them from taking up the dye.
Cryptosporidiosis
Cryptosporidiosis
Cryptosporidiosis: Many species of
Cryptosporidium exist that infect humans and a
wide range of animals.
Life cycle of Cryptosporidium parvum
and C. hominis. (Cryptosporidium
stages are reproduced from Juranek DD.
Cryptosporidiosis, in: Strickland GT, editor.
Hunter’s Tropical Medicine and Emerging
Infectious Diseases, 8th ed. Philadelphia: WB
Saunders; 2000. Originally adapted from the life
cycle that appears in Current WL, Garcia LS.
Cryptosporidiosis. Clinc Microbiol Rev 1991;
4:325-58.) Sporulated oocysts, containing four
sporozoites, are excreted by the infected host
through feces and possibly other routes such
as respiratory secretions . Transmission of
Cryptosporidium parvum and C. hominis occurs
mainly through contact with contaminated
water (e.g., drinking or recreational water).
Occasionally, food sources, such as chicken
salad, may serve as vehicles for transmission.
Many outbreaks in the United States have
occurred in water parks, community swimming
pools, and day care centers. Zoonotic and
anthroponotic transmission of C. parvum and
anthroponotic transmission of C. hominis
occur through exposure to infected animals or
exposure to water contaminated by feces of
infected animals .
Following ingestion (and possibly inhalation)
a occurs. The
by a suitable host , excystation 
sporozoites are released and parasitize epithelial
c ) of the gastrointestinal tract or
b,
cells (
other tissues, such as the respiratory tract.
In these cells, the parasites undergo asexual
multiplication (schizogony or merogony)
c ,
f ) and then sexual multiplication
d,
(
(gametogony) producing microgamonts (male)
g and macrogamonts (female) 
h.

Upon fertilization of the macrogamonts by the
j ,
i ), oocysts (
k ) develop
microgametes (
that sporulate in the infected host. Two different
types of oocysts are produced, the thick-walled,
j ,
which is commonly excreted from the host 
k , which is primarily
and the thin-walled oocyst 
involved in autoinfection.
Oocysts are infective upon excretion, thus
permitting direct and immediate fecal-oral
transmission. Note that oocysts of Cyclospora
cayetanensis, another important coccidian
parasite, are unsporulated at the time of
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excretion and do not become infective until
sporulation is completed. Because the presence
and number of oocysts may vary from stool
sample to stool sample, it is recommended
that several samples be taken, preferably
concentrated by centrifugation.
For isolation and identification, stool samples,
fresh or concentrated, are examined under
the microscope utilizing acid-fast staining
procedures or immunofluorescence microscopy.
The centrifugate of fresh feces contains a lot of
unnecessary clutter that can be reduced by the
use of formalin/ethyl acetate. After filtration
through wet gauze, the stool sample is mixed
with 10 ml of 10 percent formalin in saline plus
2 ml of ethyl acetate, shaken and centrifuged.
Flotation may produce a cleaner sample than
sedimentation by centrifugation.
Sucrose solutions of a specific gravity of 1.266
will allow the oocysts to float to the top. Utilizing
sucrose solution of a specific gravity of 1.203 and
centrifugation appears to be slightly less efficient.
Antibody to cryptosporidium can be detected
by ELISA and IFA. PCR amplification and
identification is used to detect the presence of
cryptosporidium oocysts in waste and surface
water as well as drinking water.
Trichomoniasis
Trichomoniasis: Trichomonas vaginalis, a
flagellate, is the most common pathogenic
protozoan of humans in industrialized countries.
Life cycle: Trichomonas vaginalis resides in the
female lower genital tract and the male urethra
and prostate , where it replicates by binary
fission . The parasite does not appear to have
a cyst form, and does not survive well in the
external environment. Trichomonas vaginalis is
transmitted among humans, its only known host,
primarily by sexual intercourse .
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Leishmaniasis
Trichomoniasis
Leishmaniasis: Leishmaniasis is a vector-borne
disease that is transmitted by sand flies and
caused by obligate intracellular protozoa of the
genus Leishmania. Human infection is caused
by about 21 of 30 species that infect mammals.
These include the L. donovani complex with
three species (L. donovani, L. infantum, and
L. chagasi); the L. mexicana complex with 3
main species (L. mexicana, L. amazonensis,
and L. venezuelensis); L. tropica; L. major;
L. aethiopica; and the subgenus Viannia with
four main species (L. (V.) braziliensis, L. (V.)
guyanensis, L. (V.) panamensis, and L. (V.)
peruviana).
The different species are morphologically
indistinguishable, but they can be differentiated
by isoenzyme analysis, molecular methods or
monoclonal antibodies.
Geographic distribution: Worldwide. An
estimated 7.4 million new cases occur each year
in women and men, with a higher prevalence
among persons with multiple sexual partners or
other venereal diseases.
In infected women there are small red sores on
vaginal wall and cervix. It can be isolated and
identified from vaginal exudates.
Trichomonas foetus in cattle, also distributed
worldwide, produces infertility, fetal deaths,
abortions, and extended calving periods. Early
signs of the disease are cows that should be
pregnant appear ing in heat, too many cows
that appear open or late, and pyometra after
breeding. The pathogen can be found in vaginal
cultures, and carriers are known to have produced
live calves. Bulls are the main carriers and
distributors of the pathogen.
To confirm the diagnosis, take sample scrapings
from prepuce or lavage, centrifuge and examine
concentrate by dark-field contrast microscopy.
If necessary, culture the test samples in a liquid
medium, such as described by Feinberg and
Whittington (J. Clin. Path. 1967; 10: 327):
proteolysed liver (25 g), NaCl (6.5 g), dextrose
(5.0 g), inactivated horse serum (80 ml), distilled
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water (1,000 ml), Penicillin (1 Mio Units),
Streptomycin (500,000 Units) and incubate for
four to five days at 37 degrees C.
The ELISA sandwich procedure can be used to
detect Trichomonas vaginalis from the vaginal
exudates: trichomonas antibody is attached to test
surface, the test sample is added and allowed to
react, excess is rinsed off and anti-trichomonas
antibody conjugated with horseradish peroxydase
is added as indicator.
The IFA has been found useful for the detection
of trichomonas antibody. A comparison of three
antibody assays gave the following results (Patel,
SR et al. Infectious Diseases in Obstetrics and
Gynecology 2000; 8: 248-257):
Test
Sensitivity Specificity
Polymerase chain 95 percent
reaction
98 percent
Enzyme-linked 82 percent
immunosorbent
assay
Direct
85 percent
fluorescence
antibody test
73 percent
99 percent
Life cycle: Leishmaniasis is transmitted by the
bite of infected female phlebotomine sand flies.
The sandflies inject the infective stage (i.e.,
promastigotes) from their proboscis during blood
meals . Promastigotes that reach the puncture
wound are phagocytized by macrophages  and
other types of mononuclear phagocytic cells.
Progmastigotes transform in these cells into the
tissue stage of the parasite (i.e., amastigotes) ,
which multiply by simple division and proceed
to infect other mononuclear phagocytic cells .
Parasite, host, and other factors affect whether
the infection becomes symptomatic and whether
cutaneous or visceral leishmaniasis results.
Sandflies become infected by ingesting infected
cells during blood meals (, ). In sandflies,
amastigotes transform into promastigotes,
develop in the gut  (in the hindgut for
leishmanial organisms in the Viannia subgenus;
in the midgut for organisms in the Leishmania
subgenus), and migrate to the proboscis .
Geographic distribution: Leishmaniasis is found
in parts of about 88 countries. Approximately
350 million people live in these areas. Most
of the affected countries are in the tropics and
subtropics. The settings in which leishmaniasis
is found range from rain forests in Central and
South America to deserts in West Asia. More
than 90 percent of the world’s cases of visceral
leishmaniasis are in India, Bangladesh, Nepal,
Sudan, and Brazil. Leishmaniasis is found in
Mexico, Central America and South America
– from northern Argentina to Texas (not in
Uruguay, Chile, or Canada), southern Europe
(but Leishmaniasis is not common in travelers
to southern Europe), Asia (not Southeast Asia),
the Middle East, and Africa (particularly East
and North Africa, with some cases elsewhere).
The diagnosis of Leishmaniasis is based upon its
symptomatology: in dogs, skin ulceration, loss
of appetite, weight loss, localized or systemic
lymphadenopathy, anemia, renal lesions,
increased levels of plasma urea, creatinine,
protein urea and hematuria, ocular damage,
epistaxis, lameness and sometimes chronic
diarrhea and liver failure. There may be slowly
progressive chronic ulceration usually around the
head and extremities.
Elite
the host, they transform into bloodstream 
trypomastigotes , are carried to other sites
throughout the body, reach other blood fluids
(e.g., lymph, spinal fluid), and continue the
replication by binary fission .
The entire life cycle of African Trypanosomes
is represented by extracellular stages. The
tsetse fly becomes infected with bloodstream
trypomastigotes when taking a blood meal
on an infected mammalian host (, ). In
the fly’s midgut, the parasites transform into
procyclic trypomastigotes and multiply by binary
fission , leave the midgut, and transform into
epimastigotes . The epimastigotes reach the
fly’s salivary glands and continue multiplication
by binary fission .
The cycle in the fly takes approximately three
weeks. Humans are the main reservoir for
Trypanosoma brucei gambiense, but this species
can also be found in animals. Wild game animals
are the main reservoir of T. b. rhodesiense.
Geographic distribution: T. b. gambiense is
found in foci in large areas of West and Central
Africa. The distribution of T. b. rhodesiense is
much more limited, with the species found in
East and Southeast Africa.
Diagnosis: Initial symptoms to look out for are
the outward signs of the disease: intermittent
fever; anemia; and weight loss, complicated by
poor nutrition and stress. In addition. you will
see chancres in the skin of animals infected by a
feeding tsetse fly.
The immune response induced by the pathogen
exacerbates inflammation and the damage caused
in the host animal. The variability of surface-coat
glycoproteins of the trypansoma organism – there
are hundreds of them – makes it difficult to
eliminate the pathogen.
While matching antibody will kill the
organism, carrying the equivalent surface-coat
glycoproteins, many with different surface-coat
glycoproteins will continue to exist and multiply.
This reality makes the production of an effective
vaccine difficult, and the recovered patient
vulnerable to reinfection.
In man, cutaneous leishmaniasis expresses
itself in one to multiple lesions developing to
sores where the sand fly bit. These sores can be
painful, a central crater surrounded by raised
edges. A scab may cover them. Usually, the
draining lymph nodes are swollen. Persons with
visceral leishmaniasis exhibit fever, weight loss,
an enlarged liver, a greatly enlarged spleen and
lymphadenopathy.
Sometimes cutaneous lesions appear as
“Oriental sore” or “Aleppo boil,” which may
appear as many as 20 years after the initial
disease. Blood work shows anemia, and low
white cell and platelet counts. Leishmania
amastigotes, basophilic ovals, are usually found
in macrophages obtained from lymh node or bone
marrow biopsies. They can be seen under the
light microscope utilizing the Giemsa stain.
Elite
Culturing the organism and hamster
inoculations are the most sensitive and
effective way of isolating the pathogen. Indirect
immunofluorescence, ELISA as well as PCR
can also be used to detect the presence of
Leishmania. While antibody can usually be found
in the visceral disease, the cutaneous disease may
produce very little or no antibody.
Sleeping sickness
Sleeping sickness: To understand the disease,
it is necessary to understand the Trypanosoma
brucei life cycle.
Life cycle: During a blood meal on the
mammalian host, an infected tsetse fly (genus
Glossina) injects metacyclic trypomastigotes
into skin tissue. The parasites enter the lymphatic
system and pass into the bloodstream . Inside
Fever, weakness, debilitation and anemia in an
endemic area are suggestive of trypanosome
infection. In man you will notice:
■■ Chancre at site of tsetse bite.
■■ Fever, pruritus, lymphadenopathy
(hemolymphatic stage).
■■ Headaches, behavior changes,
sleepiness, loss of consciousness, coma
(meningoencephalitic stage).
Taking a blood sample and examining a wet
mount for motility or a Giemsa-stained blood
smear under the light microscope would be the
first step. There is only an intermittent presence
of trypomastigotes in the blood stream, and there
may not be enough organisms to be detectable
all the time. Examination of the buffy coat after
centrifugation for the presence of the organism
might be more efficient.
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Trypanosomes can also be found in samples
taken from the skin lesion, lymph node,
bone marrow, cerebrospinal fluid during the
meningoencephalitic stage. Sometimes it may be
necessary to employ the mini anion-exchange/
centrifugation procedure to detect trypanosomes.
It consists of a two-step process:
■■ Separation of parasites from the blood
in a gel column by anion exchange
chromatography.
■■ Concentration by centrifugation. The centrifugate
is then examined under the microscope.
The quantitative buffy coat (QBC) technique will
detect blood parasites other than trypanosomes
(leptospira, plasmodium, babesia). A capillary
tube containing EDTA, a floating glass cylinder
and acridine orange is filled with fresh blood and
centrifuged. Ultraviolet light microscopy will
then detect the fluorescing parasites between red
blood cells and buffy coat.
A last resort to detect trypanosomes would be the
inoculation of rats or mice, which is probably the
most sensitive of tests. The presence of antibody
is of questionable diagnostic value because of
the time required for antibody development.
However, in an endemic area it may be advisable,
especially with symptoms present in the absence
of a pathogen. The IFA and ELISA assays for
circulating antibody have both been found useful.
The card agglutination test for trypanosomiasis
(CATT) permits the efficient screening of large
populations for the presence of the infection.
This test consists of a white plastic card to
which is added a finger prick drop of blood to be
combined and mixed with a drop of blue colored,
inactivated bloodstream forms of trypanosomes.
The card is shaken for five minutes, and in the
presence of antibody, the indicator parasites will
be agglutinated in a blue clot
In the micro CATT, the sample drop can be
replaced by a chip of filter paper soaked with
blood or serum of the subject under test. Another
modification of the CATT is the use of antigencoated latex beads for visualizing agglutination.
In surviving patients, antibody can be found for
years, meaning the presence of antibody per se
does not suggest an acute disease, nor does it
protect from reinfection with antigenic variants of
the same strain of parasite.
Anthrax
Anthrax: An aerobic, sporeforming bacterium,
Bacillus anthracis is found commonly in wild
and domestic herbivores and man. The disease it
causes is usually fatal. Early signs include sudden
onset of high fever (41.5 degrees C), excitability
alternating with stupor, cardiac distress, dyspnea,
staggering, trembling, lack of appetite, lack of
rumination, drop in milk production and abortion
quickly followed by bloody discharges from all
orifices, collapse, convulsions, death. Often death
occurs before any signs are noticed.
The occasional chronic infection is expressed by
extensive localized edema and swelling of the
neck, chest and shoulder region. In the horse,
you will notice fever, colic, loss of appetite,
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Form of
anthrax
Symptoms
Mortality
Inhalational
Influenza-like symptoms, headache, fever,
malaise, chills, night sweats, dry cough, dyspnea
and chest pain, fatigue and muscle aches, shock.
>90 percent even if treated
Gastrointestinal
Affecting mouth and upper parts of
gastrointestinal tract, headache, sore throat and
difficulty swallowing, anorexia, abdominal pains,
diarrhea, nausea and vomiting, bloody diarrhea .
>90 percent even if treated
Cutaneous
Sores developing from early itchy skin bumps,
blisters, ulcerating with black centers .
>20 percent untreated
< 1 percent treated
DIAGNOSTIC PROCEDURES
Cutaneous
■■ Culture of vesicular fluid, exudates, eschar, Gram stain.
■■ Blood culture if systemic systems present.
■■ Biopsy for immunochemistry.
Inhalational
■■ Chest x-ray for widened mediastinum, pleural effusions, infiltrates,
pulmonary congestion.
■■ Affected tissue biopsy for immunohistochemistry.
■■ Any available sterile site fluid for Gram stain, PCR, culture.
■■ Pleural fluid cell block for immune histochemistry.
Gastrointestinal Blood cultures, oropharingeal swab collection
LABORATORY CRITERIA FOR IDENTIFICATION OF BACILLUS ANTHRACIS
From clinical samples
Encapsulated Gram-positive rods.
From growth on sheep blood agar
Large Gram-positive -rods, non-motile, non-hemolytic.
From clinical and culture samples
PCR.
Confirmatory criteria
Capsule production, lysis by gamma phage, direct fluorescent
antibody assay.
weakness, swelling of neck, chest, abdomen and
genitals as well as bloody diarrhea. Death occurs
within about three days.
In pigs, dogs and cats, there may be acute
septicemia and sudden death, or there may be
oropharyngitis with rapid swelling of the throat
sometimes leading to suffocation. Chronically,
pigs may show symptoms and eventually recover.
In man, there are three forms of the disease,
inhalational, gastrointestinal and cutaneous.
The gamma phage assay is used to identify
cultures of Bacillus anthracis utilizing the affinity
of gamma phage for this bacillus, entering it
and destroying it. In a lawn of the bacilli the
bacteriophage will produce round zones of lysis
about 1 centimeter in size. In mass screenings,
the PCR test is useful in detecting anthrax antigen
in questionable samples. To prepare such samples
or biopsy material, they must be heat-inactivated
before use.
The direct fluorescent antibody assay (DFA)
Because the clinical symptoms alone do not allow incorporates two antibody types:
a clear cut diagnosis, laboratory confirmation
■■ Monoclonal IgM against cell wall
is necessary. The CDC suggests the following
polysaccharide antigen.
diagnostic procedures and laboratory criteria for
■■ Monoclonal IgG against capsule antigen.
identification of Bacillus anthracis:
For the test itself, 45 μl of sample is mixed
Blood and lymphatic tissue are the material of
with 5 μl of one of these antisera, incubated at
choice for identification of the pathogen. Bacterial 37 degrees C for 30 minutes, diluted in 1ml of
culture of the agent is not complicated: routine
phosphate -buffered saline-tween 20, centrifuged
culture media, such as 5 percent sheep blood
(14,000 x g for 3 minutes) and re-washed
agar at 35 degrees C, in ambient air will produce
and re-centrifuged. The cell pellet of the final
colonies within a day. The bacterium stains
wash is then examined under UV microscope.
Gram-positive but not necessarily all the time as it Bright green fluorescence of the whole body is
may vary or even appear Gram-negative at times.
considered positive, and the sample is considered
However, both the presence of spores and Grampositive when both the cell wall and the capsule
positivity tend confirm the diagnosis.
antibody are positive.
Penicillin will inhibit growth. Sporulation of the
cultured organism is facilitated by growing it in
tryptose agar at 37 degrees C for 48 hours and
then at 23 degrees C for two weeks. Sporulation
can be confirmed microscopically using the
Schaeffer-Fulton stain (vegetative cells reddish,
spores green). In egg yolk agar it will produce a
wide zone of lecithinase.
The effectiveness of a vaccine against anthrax
requires the measurement of the immune
responses induced by it, antibody being one
of them. Antibody neutralizing lethal-factor
toxicity in a mouse macrophage-like cell line
can be measured by determining the number
of surviving cells by their uptake of indicator
chromophores and measuring the cell lysate
Elite
spectrophotometrically or by identifying the
cells carrying the chromophore by means
of fluorescence microcopy. The indirect
hemagglutination test employing anthrax antigencoated tanned sheep erythrocytes is another test
used to measure antibody. ELISA and Western
blotting can be used as well.
Brucellosis
Brucellosis: Brucella abortus, Brucella suis,
Brucella melitensis and Brucella canis are
Gram-negative, non-motile, non-spore-forming
rods causing serious infections in animals and
man. Early signs are abortion, stillborn, and
weak calves; retention of placenta; and a drop in
milk yield. There may be orchitis, infection of
accessory sex glands, and abscesses in testicles.
Some cattle develop swollen arthritic joints.
The ELISA test includes Brucella melitensis
as antigen using the standard procedure of
adding serum dilutions to antigen-coated wells,
incubating at room temperature for one hour,
washing the reaction wells diligently (three
times), adding enzyme-linked conjugate,
incubating for 30 minutes, rinsing again three
times, adding substrate, i.e., chromogen, and after
another 10 minutes, adding stopping solution and
reading absorbency at 415-620 nm.
Antibody titers are useful for monitoring treatment
success. Complement fixation tests can be used,
but they may be too complicated for the field.
Leptospirosis
Leptospirosis: Leptospira are free-living long,
thin, motile spirochetes surviving for extended
periods and remaining infective in surface
Infection occurs through mucous membranes
waters, swamps, rivers, riverbanks, soil and mud.
and spreads through the lymphatic system,
Floods tend to increase the number and extent of
occasionally producing abscesses in lymph nodes, outbreaks. There are about 17 species and more
and spleen and inflammation of mammaries, It
than 200 serologically different variants.
enters the uterus and placenta and interferes with
Infection causes ill-defined symptoms of fever,
fetus nutrition while producing toxins at the same
icterus, hemoglobinuria, infertility, abortion,
time, weakening or killing the fetus.
renal failure, and death. Acute leptospirosis in
The Brucella milk ring test (BRT) is done once a calves is usually more severe than in older cattle,
year in the U.S., and all young stock is vaccinated with a fever (40.5-41 degrees C), loss of appetite,
to reduce chances of transmission.
breathing difficulties and pulmonary congestion,
anemia, hemoglobinuria, and icterus. However,
In man, the early symptoms include undulating
the normal course runs about a week or a little
fever (Malta fever), muscular pain and
more with full recovery.
migratory arthralgia, and sweating. There
always is septicemia (melitococcemia), anemia
and leukopenia, and possibly liver damage as
suggested by increased alanine aminotranferease
(ALT) and aspartate aminotransferase (AST), and
frequently orchitis.
In older cattle, there is an abrupt reduction in
milk production, down by 10 to 75 percent; the
milk is yellowish and thick, contaminated with
blood, with thick clots and high cell counts, and
the udder is flabby and soft. Recovery is quick,
maybe two weeks, to full milk production in most
In chronic cases, it affects bones and joints,
cows. A few cows may not regain their initial
causing back pain. The vertebral column is often
affected with brucellar spondylitis and spinal cord production level for that season.
compression (X-ray diagnosis). Liver biopsies
In beef cattle, the disease is less remarkable. Still,
may show granulomatous hepatitis.
there may be abortions within six to 12 weeks
after infection and more commonly in the last
To confirm the diagnosis of brucellosis, the
trimester, stillbirths, premature births, and weakly
organism can be cultured in tryptose broth from
calves. A number of abortions occurring at about
the blood during melitococcemia, the bone
marrow, and from infected tissues (liver, spleen). the same time may be the first sign of the disease,
with younger animals more likely to abort.
It may take up to two months to grow.
The fluorescence in situ hybridization (FISH)
test allows visualization of specific bacteria
without preceding cultivation or amplification.
This procedure identifies the presence of a
brucella-specific 16S rRNA gene sequence by
using a fluorescent-labeled oligonucleotide probe
with a matching sequence and visualizing it by
fluorescence microscopy.
Antibody develops within about three weeks
after onset of the disease. However, the finding
of antibody in endemic areas is of questionable
value, because it would be around anyway. For
antibody detection, the most often used procedure
would be the standard tube agglutination test
(STA): two-fold dilutions of the serum are mixed
with Brucella abortus antigen, shaken, incubated
at 37 degrees C overnight, and the highest dilution
still agglutinating the antigen is considered the
antibody titer of that particular sample.
Elite
Colostral antibody in calves from previously
infected cows may provide protection for half a
year. Because the organism tends to localize in
the uterus and oviducts, infertility may become
a problem.
In man infected by contact with infected tissues
or surface water contaminated with the urine
of infected animals, the symptoms are flu-like
but rapid-onset, with fever and chills, headache,
malaise, dry cough, nausea, vomiting and
diarrhea, myalgia, and ocular pains.
Confirmatory laboratory tests include
demonstration of the pathogen in urine and
affected tissues employing PCR and fluorescent
antibody techniques. The culture of leptospira is
slow, with a doubling time of eight to 16 hours,
and requirements are critical and not the same for
different serotypes.
For antibody measurement, the microscopic
agglutination test can be performed with live or
killed leptospira. Because of the great number
of potential serovariants, a screen of all samples
versus all available antigens is performed at one
single serum dilution, perhaps 1/25. The positive
samples are then diluted to obtain a measure of
the antibody level in the patient.
Listeriosis
Listeriosis: Listeria monocytogenes is a small,
motile, Gram-positive, non-sporeforming coccoid
bacterium with long filaments. There are several
serotypes, variants based on somatic and flagellar
antigens. This organism is extremely resistant.
It is widely distributed in nasal secretions, feces
and soil and causes occasionally life-threatening
disease in domestic fowl and parrots.
Infection occurs by inhalation or wound
contamination. It has been found in obviously
healthy birds that might serve as carriers or
reservoirs for the agent. The disease picture
ranges from depression and listlessness to
encephalitis, torticollis, paresis and paralysis,
stupor and sudden death.
It is common in wild and domesticated animals,
sheep and cattle, and can cause encephalitis,
circling, facial paralysis and mastitis with
relatively high fatality rates. It can produce
abortions, miscarriages, stillbirths and septicemia
in the newborn.
In man, a person may be infected without
evidence of disease or will notice fever, myalgia,
gastrointestinal affliction with nausea, vomiting
and diarrhea. Immunocompromised individuals
are, as always, more likely to be severely
affected. Pregnant women also tend to be more
affected and infection may lead to premature
delivery, miscarriage or stillbirth and infection of
the newborn.
Occasionally, the central nervous system is
implicated as well, producing symptoms such as
stiff neck, headache, loss of balance, confusion
and convulsions.
To confirm a diagnosis, culture the pathogenic
organism on blood and tryptose agar or brainheart infusion. Samples of cerebrospinal
fluid of animals with central nervous system
involvement, aborted placenta, and the fetus
are all likely to contain the organism. The
cerebrospinal fluid is expected to have greatly
increased protein concentration and infiltration of
large mononuclear cells. If all else fails, ground
brain tissue should be cultured and recultured at
39 degrees C for several weeks.
Immunofluorescence allows identification of
organisms by employing specific fluoresceinisocyanate-labeled antibody to the antigens of
that organism. ELISA has been used for the same
purpose after a preliminary enrichment period in
broth culture.
A highly sophisticated approach to the
identification of different species of Listeria,
utilizing the “rapid microarray-based assay for
the reliable detection and discrimination of six
Page 17
species of the Listeria genus,” i.e., the “onetube multiplex PCR amplification of a long
list of virulence factor genes, the synthesis of
fluorescently labeled single-stranded DNA,
and hybridization to the multiple individual
oligonucleotide probes specific for each Listeria
species,” is beyond the scope of this review and
will be reviewed elsewhere. (For additional detail
and references see:”Volokhov Dmitriy, Avraham
Rasooly, Konstantin Chumakov and Vladimir
Chizhikov Identification of Listeria Species by
Microarray-Based Assay, Journal of Clinical
Microbiology, 2002; 40(12): 4720-4728.”)
Lyme disease
Lyme disease: Borrelia burgdorferi is a spiral
shaped, two-membrane bacterium with two
flagellae. Borreliosis exists worldwide; the
pathogen is tickborne (Ixodes pacificus, Ixodes
scapularis) and depends on the tick for its survival.
Blacklegged ticks live for two years and have
three feeding stages: larvae, nymph and adult.
Tick eggs are laid in the spring and hatch as
larvae in the summer. Larvae feed on mice, birds,
and other small animals in the summer and early
fall. When a young tick feeds on an infected
animal, the tick takes bacteria into its body along
with the blood meal, and it remains infected for
the rest of its life.
After this initial feeding, the larvae become
inactive as they grow into nymphs. The following
spring, nymphs seek blood meals to fuel their
growth into adults. When the tick feeds again,
it can transmit the bacterium to its new host.
Usually the new host is another small rodent, but
sometimes the new host is a human.
Most cases of human illness occur in the late
spring and summer when the tiny nymphs are
most active and human outdoor activity is greatest.
Adult ticks feed on large animals and sometimes
on humans. In the spring, adult female ticks lay
their eggs on the ground, completing the life cycle.
Although adult ticks often feed on deer, these
animals do not become infected. Deer are
nevertheless important in transporting ticks and
maintaining tick populations.
Early signs of the infection are of neurological,
cardiac and renal nature, including fever, loss of
appetite, lymphadenopathy, lameness (swollen,
painful joints), and lethargy.
Renal borrelosis in dogs is expressed by uremia,
hyperphosphatemia, nephropathy and generalized
edema. It is usually fatal. The cardiac symptoms
show disorder of the electrical conducting
system and bradycardia while the neural disorder
includes seizures and facial paralysis. To confirm a
presumptive diagnosis based on these symptoms,
the pathogen should isolated and identified.
Culture is technically difficult and slow, and
different media have been tested. The one of
choice seems to be BSK-H medium, complete,
with 6 percent rabbit serum, sterile-filtered,
Borrelia burgdorferi-tested. PCR has been used
on isolates from joints, periarticular tissue and
blood to identify the pathogenic organism.
Page 18
The presence of antibody to Borrelia burgdorferi
is useful as a corroborating factor, however, by
itself, it cannot identify the disease-producing
agent. ELISA, IFA, and Western Blot have been
used to measure antibody levels.
Nocardiosis
Nocardiosis: Nocardia are weakly staining
Gram-positive, weakly acid-fast, filamentous
rod-shaped bacteria. They are saprophytic, exist
worldwide in organic matter and soil, and are
part of the normal oral microflora. Nocardiosis is
considered an opportunistic infection, secondary
to a weakened condition, immunosuppressive
treatment in transplant recipients, HIV or other
diseases affecting the immune system.
Overall, it can involve the pulmonary,
neurological, cardiac and lymphatic system. It
can produce keratitis and it can be disseminated
throughout the body with serious prognosis.
The extent of symptoms and severity of the
disease is a direct function of the state of
immunocompetency of the patient.
The pulmonary form consists of progressive
pneumonia, fever, night sweats, chest pain and
cough. When the nervous system is affected, it
exhibits signs of meningitis, lethargy, headache,
neurological deficit, seizures, confusion. The
cardiac affectation is endocarditis and damage to
heart valves, while the lymphocutaneous disease
produces erysipela-like cellulitis, inflammation
of the lymphatic system and the formation of
nodules along its pathways.
Nocardia may grow aerobically on 5 percent
sheep blood or chocolate agar within two to five
days or require up to four weeks. Antibiotics
treatment of the patient from which the sample
was obtained may slow down culture of the
organism. Faster growing contaminants may
cover its presence. Different strains of Nocardia
can be distinguished by their growth and
hydrolysis patterns and varied resistance to
different kinds of antibiotics.
The very nature of the disease, requiring
individuals of lowered immune-responsiveness,
suggests the inadequate development of
antibody. The complement fixation test was
able to detect antibody to Nocardia. So were
the passive hemagglutination test, indirect
immunofluorescence (IFA), ELISA, Western Blot.
Q fever
Q fever is a zoonosis caused by Coxiella burnetii
with cattle, sheep and goats as primary reservoir.
The infected animal sheds the pathogen with its
milk, urine, feces, amniotic fluid and plazenta.
Coxiella burnetii has been found in ticks, and
ticks have been shown to play a minor role in
transmission. Being very resistant to heat, drying,
and many desinfectants, Coxiella burnetii will
survive for many months in nature.
The infected domestic ruminant may have
no symptoms or loss of appetite and late
abortion. Infertility, necrotizing placentitis and
sporadic abortions are indicative but do need
confirmation. Cats may have transient fever,
anorexia and drowsiness lasting for a few days.
Immunofluorescence antibody tests on samples
taken two weeks apart are used to determine a
recent infection.
Dry, flying barnyard dust is the main source of
infection for man. Early clinical signs – and only
about 50 percent of those infected will show any
symptoms – come on all of a sudden: high fever,
chills, sweats, sore throat, non-productive cough,
severe headache and chest pain, confusion,
malaise, myalgia, as well as abdominal pain,
nausea, vomiting and diarrhea. Fever usually
subsides after a couple of weeks, but weight loss
may continue for some time.
About one-third to one half of diseased patients
will develop pneumonia, while most of those
with abnormal liver function tests will develop
hepatitis. One to two of 100 acutely diseased
individuals may die, while most will fully recover
within a few months even without treatment.
Still, there are few who will not fully recover
within six months and develop the chronic form
with more serious complications, including
endocarditis and valvular heart disease. More
than half of them may die.
Because the symptoms of Q fever are not very
distinctive, laboratory tests are necessary to
confirm a diagnosis. Confluent cultures of human
embryonic fibroblast cells are inoculated with
samples of infected tissue (blood, bone marrow
aspirate, placenta, etc.) and incubated at 37
degrees C for one to two weeks under 5 percent
CO². The causative agent may be identified in
smears or sections of infected tissue samples
using PCR or immunohistochemical staining.
Coxiella burnetti has two important antigens:
antigen phase II stimulates antibody earlier,
about two weeks post-infection, and to higher
levels than antigen phase I, which induces its
antibody to appear later, toward the more chronic
stage of the disease. These relationships are
important because they will help to specify the
stage of disease. However, both types antibody
may persist for months if not years post-initial
infection.
A better indicator for the acuteness of the
disease would be the determination of IgM
titers or IgM/IgG ratios of two samples taken a
week apart. Other antibody testing procedures
that have been used are complement fixation,
microimmunofluorescence and ELISA.
Shigellosis
Shigellosis: Shigella is a Gram-negative, nonspore-forming, rod-shaped bacterium causing
dysentery in its host. Its only hosts are primates:
man and ape. Transmission occurs through
fecal-oral route, through food from fecally
contaminated water and careless, unsanitary
food handlers. The symptoms of shigellosis are
sudden fever, nausea and vomiting, abdominal
pain, cramping, watery diarrhea containing blood,
mucus, pus, rectal pain and tenesmus.
The disease runs its course within four to eight
days without treatment other than oral reElite
hydration; in severe cases, it may last up to six
weeks. Although the symptoms are very much
distinctive and descriptive for shigellosis, proof
of diagnosis must be provided. The culture of
Shigella is straightforward, because the organism
is not very demanding in its growth conditions
and nutritional requirements. Serological
responses can be determined by passive
hemagglutination, ELISA, EIA, and so on.
Tularemia
Tularemia is a serious infectious disease caused
by Francisella tularensis (fastidious, Gramnegative, nonmotile, pleomorph coccoid rod)
in rabbits and hares as well as man. It is passed
on through ticks, deer flies and other biting flies
and mosquitoes. Its symptoms include headache,
fever, sweating and chills, myalgia and arthralgia,
ulcerating sores, and weight loss. There are five
types of the disease:
Form of
tularemia
Symptoms
Ulceroglandular/ Local lesion at bite site,
glandular
ulceration, local lymph
nodes swell, become tender,
produce thick pus like
matter.
Oculoglandular
Conjunctivitis, ulcerating
sores, local lymph nodes,
very painful.
Oropharyngeal/
gastrointestinal
Ulcerating sores in mouth,
throat, intestinal tract,
nausea, vomiting, bloody
diarrhea.
Pulmonary
Infection by inhalation,
severe pneumonia.
Typhoidal
Generalized, no skin or
lymph node involvement,
headache, confusion, shock.
If untreated, about 5 percent of the diseased will
die. Treatment may reduce this to 1 percent.
The diagnosis of tularemia must be firmed up,
especially when dealing with the pulmonary and
typhoidal mode, by blood culture, serology, chest
x-ray, and by PCR. The culture medium to isolate
and identify Francisella tularensis must contain
buffered charcoal, yeast extract, cystein and
maintain aerobic conditions. Colonies develop
within 72 hours. Culturing the organism requires
extreme, i.e., biohazard, safety measures and
should be carried out only under those conditions.
The Gram stain will show small, Gram-negative
rods, pleomorph, poorly staining, and the cells
appear coccoidal with bipolar staining. Both
conventional PCR and real-time PCR have
been used to identify the organism quickly and
efficiently.
For antibody measurements, both the tube- and
micro-agglutination can be used with almost
equal results, although the tube agglutination
test is slighter more sensitive to the higherend dilutions of the sample. The tube test uses
test tubes (13mm x 100mm) with dilutions of
Elite
serum sample in 0.85 percent saline, addition of
antigen, overnight incubation at 37 degrees C
and reading for presence of agglutination. The
micro-agglutination replaces the test tubes with
U-bottomed microtiter plates and uses much
smaller portion of reagent, incubation and reading
remaining the same. The ELISA test and Western
blot have been used as well.
However, in the best of cases, antibody will
have to develop before it can be measured,
which may take two to three weeks if not
longer in immunosuppressed individuals,
who would be the ones most likely to benefit
from early treatment. Thus, tests with quicker
turnaround times are sought. Historically, the
delayed-type hypersensitivity reaction following
intracutaneous injection with Francisella antigen
was used. It produced a response within four days
post-infection. There are no commercial skin test
antigens available today.
Eliassen and Olcen et al., in 2008 reported on a
lymphocyte stimulation test that would provide
an earlier response. Not only did the immune
response appear earlier, but it was also more
sensitive. It was measurable before humoral
antibody was detected.
The measurement of lymphocyte stimulation
has been greatly improved by the arrival of
flow-cytometry, capable of measuring thousands
of paticles ranging in size from 0.2 μm to 150
μm, and the ability to measure stimulated
lymphocytes in whole blood. FASCIA, or
flow-cytometric assay of specific cell-mediated
immune response in activated whole blood,
requires test tubes, formalin-killed vaccine
strain material as antigen, peripheral sample
blood collected into heparin-containing vacuum
tubes and diluted one in 10 in RPMI 1640 and
glutamine and 10 μg/ml of gentamycin, positive
and negative control samples.
The BSA was added to cover attachment sites on
the plastic bottom not covered by the antigen and
block them from adsorbing other components of
the test.
For use, the plates were rinsed again, and test
serum dilutions were added and incubated at
room temperature for 3-5 hours. After another
rinse of alkaline-phosphatase-conjugated rabbitto-human, IgG and IgM was added and incubated
overnight again at room temperature. The plates
were rinsed again, phosphatase substrate was
added, and absorbance was read at 405 nm.
Summary overview of diagnostic
procedures
Initial observations: When communicating
with the owner of a diseased animal, a number
of questions should come to mind and should
be answered.
Diagnostic considerations
Type of animal
■■ Race, sex, size and
weight, age.
Central
nervous system
■■ Behavior: normal,
obtunded, stuporous or
comatose.
■■ Attentiveness, bright
and alert, posture, gait,
balance.
■■ Pupils constricted,
dilated, equal size,
responsive to light.
■■ Breathing pattern,
seizures, convulsions,
ataxia, circling.
■■ Response to pain
stimulus.
Circulatory
■■ Color: mucous
membranes, gums,
conjunctiva, capillary
refill time.
■■ Pulse strength and rate,
heart rate and regularity,
skin turgor.
■■ Temperature of
extremities, decreased
urine production.
■■ Agility, weakness, signs
of blood loss.
■■ Electrocardiogram,
tachycardia,
hypovolemia,
bradycardia.
Respiratory
distress
■■ Coughing, shortness of
breath, stance of animal.
■■ Open mouth, flaring
nostrils, chest wall
motion.
■■ Cyanosis, hypoxemia.
Gastrointestinal
■■ Nausea, vomiting,
diarrhea, loss of
appetite, weight loss.
■■ Stool: appearance (firm,
watery), frequency.
■■ Distended abdomen.
For the test, antigen was placed into the test
tubes, the diluted blood sample was added and
the test was incubated in humidified atmosphere
at 37 degrees C under 5 percent CO² in air.
After one, two, and three days, the tubes were
gently mixed and samples were removed
and centrifuged (300xg for five minutes).
Supernatants were kept at -80 degrees C for
cytokine (IFN-γ) analysis. Flow cytometry was
used to measure lymphocyte proliferation.
The researchers compared ELISA and the
tube agglutination test with their lymphocyte
stimulation test, in which they measured cytokine
release from stimulated lymphocytes. The
lymphocyte stimulation test produced an immune
response two to four days before humoral
antibody appeared.
Their ELISA test produced results for both IgG
and IgM antibody. The ELISA test procedure
was performed in flat-bottomed microtiter plates.
Antigen was added to the plates and allowed to
adsorb overnight at room temperature, rinsed
to remove excess antigen and stored in the
refrigerator under 0.5 percent bovine serum
albumin in phosphate-buffered saline until use.
Page 19
Renal
Systemic
Present
medication
■■ Urination.
■■ Frequency.
■■ Color.
■■ Fever, loss of appetite,
weakness, debilitation,
depression.
■■ Bleeding from orifices,
blood in stool, urine.
■■ Yellow mucosae: icterus
(liver), hemolysis.
■■ Pale/white: blood loss,
anemia, shock.
■■ Brick red: sepsis,
polycythemia,
hyperthermia.
■■ Blue: hypoxia.
■■ Poison, abnormal
intake, chemicals
around house.
Once the general symptomatology of a disease
has been determined, laboratory tests are
necessary to confirm a presumptive diagnosis,
to identify the pathogen causing it and to decide
on a course of action. Laboratory confirmation,
generally, involves isolating the agent, identifying
it and determining its effect on the diseased body,
which includes the symptoms observed and the
body’s defense response.
Isolation
Collect
sample
■■ From blood, stool, urine,
cerebrospinal fluid, suspicious
tissue (biopsy), pharyngeal
swabs, tracheal lavage,
amniotic fluid, aborted fetus.
Inoculate
culture
■■ Culture plate, liquid medium,
indicator animals (embryonated
eggs, mice, hamsters etc), cell
cultures;
■■ Substrates, growth conditions
and procedures dependent on
suspected pathogen.
Harvest
product
■■ For identification: morphology
of pathogen, antigenicity, DNA
content, infectivity for other
indicator substrates.
Store
harvest
■■ Aliquots: unadulterated (-70ºC),
prepared for eventual use.
Sample collection and manipulation must be
carried out under conditions that preclude
contamination from other sources: hands,
equipment, chemicals involved in sample
collection and preservation, airborn contaminants,
dust, or from hair or orifices of technician.
The microscopic recognition of a specific
pathogenic organism is not always easy or clearcut, although there are pictorial representations
to be found everywhere on the Web (the Google
image gallery, Wikipedia or similar sites and,
especially, the image library of the CDC at
http://www.dpd.cdc.gov/DPDx/html/Image_
Library.htm).
Page 20
AGENT
Plasmodium
Giardia
Toxoplasma
Cryptosporidium
Trychomonas
Leishmania
Trypanosoma
Anthrax
Brucella
Leptospira
Listeria
Borrelia
Nocardia
Coxiella
Shigella
Francisella
Identification
SOURCE/CULTURE
IDENTIFICATION
Red blood cells in RPMI 1460.
Microscopy: in red blood cells.
(Giemsa, H&E).
Enteroscopy, string test,
Microscopy: trophozoites, cysts
ova, parasites, antigen in stool.
(tri-chrome stain).
Tissues, body fluids (CSF, intra Microsopy: In tissues, body fluids
ocular humor).
(Giemsa, immunofluorescence), PCR
In mice; cell culture (human fibroblast)
Presence of IgA, IgM, IgG; Live tachyzoites
take up alkaline methylene blue.
Stool.
Microscopy: Giemsa, H&E,
Fluorescent microscopy with auramine
DFA, IFA, ELISA; PCR.
Vagina; Diamond’s TYM
Microscopy: Giemsa, pap stain.
medium.
Spleen, bone marrow, lymphatic Microscopy: Giemsa, H&E, Leishman;
system, Skin lesions; buffy coat fluorescent dye-tagged antibody
or cultured in biphasic Novy(fluorescein isothiocyanate-conjugate,
MacNeal-Nicolle (NNN) culture rhodamide B isothiocyanate-conjugate).
medium.
Liver infusion tryptose medium Microscopy: Giemsa, Wright-Giemsa.
with fetal bovine serum or 3
percent human urine; Liquid
medium L4NHS.
5 percent sheep blood agar;
Microscopy: Gram-positive; should show
bicarbonate agar is used to
spores to confirm.
induce capsule formation.
Slow growing blood culture on
Microscopy: dense clumps Gram-negative
Castenada medium.
coccobacilli.
From blood, serum, fresh urine
Dark field microscopy: spirochete, motile,
and possibly fresh kidney
Gram-negative, 2 flagellae spiral-shaped
biopsy grow in Ellinghausenbacteria that are 6-20 μm long and 0.1 μm.
McCullough-Johnson-Harris
(EMJH) medium, with 0.2-1
percent rabbit serum.
Listeria selective agar: Peptone Microscopy: Gram-positive, nonspore23.0; starch 1.0; sodium chloride forming, catalase-positive rod, motile via
5.0; agar-agar 13.0 (Columbia
flagella at 30 degrees C and below but not
agar); esculin 1.0; ammonium
at 37 degrees C, facultatively anaerobic,
iron (III) citrate 0.5; lithium
oxidase negative, and hemolytic.
chloride 15.0.
Seems to grow better in solid
Microscopy: Gram-negative, motile, twoBSK-based medium in anaerobic membrane, flat-waved spirochete
conditions at above 35ºC.
microaerophillic and slow-growing.
Routine bacterial, fungal, and
Gram-positive, strictly aerobic,
mycobacterial media, slow
filamentous, branching, weakly acid-fast
growing: distinctive appearance bacilli.
and odor.
Animal inoculation, chickembry, Antibody response; indirect
BHK-21 cell line, Vero cells;
immunofluorescence assay (IFA) 1 percent ACCM-agarose. immunohistochemical staining DNA: trans PCR.
Stool, white blood cells,
Gram-negative, non-spore forming rodmucous in stool, containing 2a
shaped bacteria; serotype 2a-specific
lipopolysaccharide (LPS)
monoclonal antibodies coupled to gold
N4-Agar (high level of yeast
particles “dipstick”.
extract).
Cysteine heart agar with
Gram-negative, facultative intracellular,
chocolatized 9 percent sheep
aerobic pleiomorphic coccobacillus, nonblood, supplemented with
motile, non-spore-forming; real time PCR;
antibiotics (CHAB-A).
direct immunofluorescence.
Elite
Sometimes it is still not easy to locate the pathogenic agent among all the debris
and other organisms on the field being examined much less to distinguish it
from similar looking alternatives. In this case, pathogen-specific fluorescent
dye-tagged antibody (fluorescein isothiocyanate-conjugate, rhodamide B
isothiocyanate-conjugate) can be employed to specifically attach to and identify
the agent by means of fluorescent microscopy. These antibody conjugates are
readily available commercially. If a specific antibody to a particular pathogen
is not available in the fluorescent dye conjugated form, fluoresein-labeled
antibody to the serum or antibody type of the species of animal that had
produced the pathogen-specific antibody could be used to advantage (IFA).
Instead of linking the antibody with a fluorochrome, the enzyme immune
assay, (EIA), also called enzyme linked immuno-sorbent assay (ELISA),
achieves similar effects by conjugating the antibody with an enzyme, such
as horseradish peroxidase, that will act upon chromogenic substrates such
as TMB (3,3’,5,5’-tetramethylbenzidine), DAB (3,3’-diaminobenzidine)
or ABTS (2,2’-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid) to
visualize the amount of antigen that retains the specific antibody by the
intensity of coloration. Varieties of these reagents are commercially
available singly or in kit form.
6
Enzyme-labeled
specific AB
Diluents
8
Rinse repeatedly to remove unattached
matter.
Add, allow to react according to
instructions.
Rinse repeatedly to remove unattached
matter.
Incubate for limited time, stop
enzyme action, read signals in
spectrophotometer.
Chromogenic
substrate
Sandwich enzyme-linked immuno-sorbent assay (ELISA)
STEP
MATERIAL
PROCESS
1
Capture AB for AG
Coat surfaces of flat-bottomed
that is sought
polystyrene microtiter plate with AB.
2
Incubator
Allow attachment to surface (several
hours, overnight).
3
Diluent with BSA
Rinse, remove unattached AB; BSA to
block uncovered plastic area.
4
AG sample matter
Add to AB-coated surface, incubate as
instructed.
5
Diluent
Rinse repeatedly to remove unattached
matter.
6
AB from different host Add, allow to react according to
animal
instructions.
7
Diluents
Rinse repeatedly to remove unattached
AB.
8
Enzyme-labeled AB to Add, allow to react according to
host animal sandwich instructions to measure specific AB
AB
retained by pathogen; not retained if
negative.
9
Diluents
Rinse repeatedly to remove unattached
AB.
10
Chromogenic
Incubate for limited time, stop
substrate
enzyme action, read signals in
spectrophotometer.
The Western blotting technique, also called enzyme-linked
electroimmunotransfer blotting (EITB), involves the separation and
concentration of the likely antigen sample from its background matter by
using SDS-PAGE electrophoresis, transferring the electrophoresis product
onto a nitrocellulose membrane and proceeding as in indirect enzymelinked immunesorbent assay.
Western Blot (EITB)
STEP
PROCESS
2
With separation and concentration
product (AG) from SDS-PAGE.
Rinse in distilled water.
3
Block membrane
4
Rinse
5
AG specific antibody
6
Incubate
3 percent gelatin in tris-buffered saline
(TBS), overnight at 4 degrees C.
Rinse copiously with TBS-Tween (3-5
times).
Human AB specific for AG expected in
concentration product (Step 1).
2 hours at room temperature.
7
Rinse
8
9
MATERIAL
Nitrocellulose
membrane
Destain
1
Direct enzyme-linked immuno-sorbent assay (ELISA)
STEP
MATERIAL
PROCESS
1
Capture AB for AG
Coat surfaces of flat-bottomed
that is sought
polystyrene microtiter plate with AB.
2
Incubator
Allow attachment to surface (several
hours, overnight).
3
Diluent with BSA
Rinse, remove unattached AB; BSA to
block uncovered plastic area.
4
AG sample matter
Add to AB-coated surface, incubate as
instructed.
Elite
Diluent
7
Direct fluorescent assay (DFA)
STEP
MATERIAL
PROCESS
1
Infected matter
Fix on microscopic slide or flat
bottomed microtiter plate.
2
Diluent containing
Rinse repeatedly; BSA to block
BSA
uncovered plastic area.
3
Pathogen-specific AB Add to infected matter, incubate for
fluorescent dye labeled required time and temperature.
4
Test diluent
Rinse repeatedly: remove unattached
antibody.
5
UV microscope
Read; along with positive and negative
control samples.
Indirect fluorescent assay (IFA)
STEP
MATERIAL
PROCESS
1
Infected matter
Fix on microscopic slide or flatbottomed microtiter plate.
2
Diluent containing
Rinse repeatedly; BSA to block
BSA
uncovered plastic area.
Add to infected matter, incubate for
3
Pathogen-specific
required time and temperature.
Antibody from given
animal species
4
Test diluent
Rinse repeatedly: remove unattached
antibody.
5
“given animal-species” Add and incubate for required time and
specific AB fluorescent temperature.
dye labeled
6
Test diluent
Rinse repeatedly: remove unattached
antibody.
7
UV microscope
Read; along with positive and negative
control samples.
5
Rinse copiously with TBS-Tween (3-5
times).
Labeled AB to specific Horseradish peroxidase-conjugated antiAB
human γ globulin.
Incubate
2 hours at room temperature.
Page 21
green, like ethidium bromide, is a nucleic acid stain that binds to the doublestranded DNA product of polymerization, producing a bright green but not to
the single stranded DNA. Isolation and identification of the pathogenic agent
are crucial for early treatment and prognosis of the disease it caused.
Rinse copiously with TBS-Tween (3-5
times).
Enzyme substrate
DAB (3,3’- diaminobenzidine) or
11
similar to visualize result.
The above assay systems base pathogen identification on morphology
and antigenic coat components of the pathogenic agent. The polymerase
chain reaction is based on the duplication of a selected short section of
DNA and re-duplication from its product exponentially to arrive at billions
of identical copies within a very short period of time. This selective
amplification makes it a very efficient and highly sensitive procedure for
the identification of minute traces of DNA.
10
Rinse
However, the development of an effective host immune response and its
persistence in the long term must be verified to measure effectiveness
of such treatment and protection from reinfection. The above mentioned
immunoassays applied to the identification of pathogens can, of course,
just as easily be modified to permit antibody assays and to measure that
response. Most antigens and antibody are commercially available.
Direct fluorescent antibody assay (DFA)
STEP
MATERIAL
PROCESS
1
Specific antigen
Attach to well bottoms of microtiter
plates.
2
Diluent containing
Rinse repeatedly; BSA blocks nonBSA
specific attachment to free plastic area.
3
Test AB dilutions
Add to wells; incubate for required time
and temperature.
4
Diluent
Rinse repeatedly: remove unattached
antibody.
5
Fluorescein-conjugated Fluorescein-conjugated AB will attach
AB to test AB donor
to test AB retained by Ag lawn in
animal
bottom of well; incubate for required
time and temperature.
6
Diluent
Rinse repeatedly: remove unattached
antibody.
7
UV microscope
Read; along with positive and negative
control samples.
Enzyme-linked immuno-sorbent assay (Elisa)
STEP
MATERIAL
PROCESS
1
Specific antigen
Coat bottoms of polystyrene microtiter
plate with AG.
2
Incubator
Allow attachment to surface (several
hours, overnight).
3
Diluent with BSA
Rinse, Remove unattached AG; BSA
to block uncovered plastic area.
4
Test AB dilutions
Add to AG coated surface, incubate as
instructed.
5
Diluent
Rinse repeatedly to remove unattached
AB.
6
Enzyme-labeled AB to
Add, allow to react according to
test AB
instructions.
7
Diluents
Rinse repeatedly to remove unattached
AB.
8
Chromogenic substrate Incubate for limited time, stop
enzyme action, read signals in
spectrophotometer.
It is used on DNA traces left at crime scenes, a hair, a drop of blood and, of
course, of traces of pathogenic matter found in the course of an infectious
disease. It is found in stool, urine, blood and other body fluids, infected
tissue, lymph nodes, skin ulcerations and so forth. The test itself requires:
■■ A starter DNA sequence (“target”: template containing the target
region).
■■ An excess of deoxynucleoside triphosphates (dNTPs: DNA building
blocks).
■■ An excess of primers, complementary to the target DNA.
■■ Taq DNA polymerase (temperature optimum of ~70 degrees C).
■■ Buffer solution including the divalentcation Mg² and monovalent cation
potassium.
As the primer is locating and connecting with its complementary segment
on the target DNA, the polymerase will commence synthesizing that
section of DNA with the dNTPs provided. Messenger RNA (mRNA) can
be processed as well by using reverse transcriptase to convert mRNA into
complementary DNA (cDNA) and then proceeding as with DNA.
The PCR has become a standard procedure in many laboratories, and
equipment and essential reagents have become commercially available.
Polymerase chain reaction (PCR)
STEP
PROCESS
MATERIALS AND PROCEDURES
1
Initialization step Target/template DNA found in stool, infected
tissue etc, dNTP’s, primers, taq DNA
polymerase, buffer + Mg²; DNA thermal
cycler; Combine reagents in test tube (0.2 to
0.5 ml size).
Place into thermal cycler, heat to 94 to 96
degrees C for 5 (1 to 9) minutes.
2
Denaturation step 94 to 98 degrees C for 20 to 30 seconds: melt
hydrogen bonds between double strands of
DNA produce 2 single stranded molecules.
3
Annealing step
50 to 65 degrees C for 20 to 40 seconds to
allow annealing of primers to the singlestranded DNA target; temperature and time
are critical to limit non-specific annealing
(background noise); polymerase cum
primer-template hybrid will commence DNA
synthesis.
4
Elongation step
72 degrees C DNA polymerase doubles all
existing DNA in system.
5
Repeat steps 2, 3 Recycle steps 2, 3 and 4 about 30 to 40 times.
and 4
6
Final elongation
70 to 74 degrees C for 5 to 15 minutes after
last cycle to allow full extension of singlestranded DNA molecules.
7
Holding until use In the refrigerator.
8
Agarose gel
Separate PCR products by size; read using
electrophoresis
ethidium bromide stain.
Additionally, antibody titers can be determined by agglutination and
complement fixation tests. Drops of a suspension of the suspected organism
are placed on microscopic slides and mixed with a battery of known
antisera and identified by its agglutination with the specific antibody to this
organism. This same test has been performed in tubes and microtiter plates.
Similarly, agglutination tests have been found early on to be a practical
procedure to measure antibody that might agglutinate antigen-coated red
blood cells or antigen-coated latex particles. Also, antibody levels could
be determined by their blocking of antigen-induced agglutination of red
blood cells.
The complement fixation test can identify and measure antigen or antibody.
Real-time PCR is an improvement because it permits real-time observation of
The presence of both in a sample will bind and use up the defined amount
the process of polymerization. All that is required is a real-time machine tied
of complement in the test and thus compete with the indicator system
into a computer with real-time software able to read nucleic acid stains. Cybre (sheep red blood cells and anti-sheep red blood cell antibody) necessary to
Page 22
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visualize the presence of complement. If either
antigen or antibody is absent from the test tube,
the complement is available to activate sheep red
blood cell lysis and make it visible.
References and suggested reading
1. Addis, D. G., J. P. David, J. M. Roberts, and E. E. Mast.
Epidemiology of giardiasis in Wisconsin: increasing
incidence of reported cases and unexplained season trends.
Am. J. Trop. Med. Hyg. 1992; 47:13-19
2. Adone, R., P. Pasquali, G. La Rosa, C. Marianelli, M.
Muscillo, A. Fasanella, M. Francia, and F. Ciuchini.
Sequence analysis of the genes encoding for the major
virulence factors of Bacillus anthracis vaccine strain
“Carbosap.” J. Appl. Microbiol. 2002; 93: 117-121
3. Alm E W, Oerther D B, Larsen N, Stahl D A, Raskin
L. The oligonucleotide probe database. Appl Environ
Microbiol. 1996; 62:299–306.
4. Al-Olayan EM, Williams GT, Hurd H: Apoptosis in
the malaria protozoan, Plasmodium berghei: a possible
mechanism for limiting intensity of infection in the
mosquito. Int J Parasitol 2002; 32:1133-1143.
5. Amann R I, Binder B J, Olson R J, Chrisholm S W,
Devereux R, Stahl D A. Combination of 16S rRNAtargeted oligonucleotide probes with flow cytometry for
analyzing mixed microbial populations. Appl Environ
Microbiol. 1990; 56:1919–1925
6. Amann R I, Ludwig W, Schleifer K H. Phylogenetic
identification and in situ detection of individual microbial
cells without cultivation. Microbiol Rev. 1995; 59:
143–1694.
7. Anderson MR, et al. Evaluation of vaginal complaints.
JAMA 2004; 291(11): 1368-1379.
8. Angeles AM, Sugar AM. Identification of a common
immunodominant protein in culture filtrates of three
Nocardia species and use in etiologic diagnosis of
mycetoma. J Clin Microbiol. 1987 Dec;25(12):2278–2280.
9. Angeles AM, Sugar AM. Rapid diagnosis of nocardiosis
with an enzyme immunoassay. J Infect Dis. 1987
Feb;155(2):292–296
10. Ariza J et al. Treatment of human brucellosis with
doxycycline plus rifampin or doxycycline plus
streptomycin. Ann Intern Med 1992; 117: 25–30
11. Arumugaswamy R and LF Gibson: Listeria in Zoo
Animals and Rivers. Australian Veterinary Journal. 1999;
77(12): 819-820
12. Atherton, F., C. P. Newman, and D. P. Casemore. An
outbreak of waterborne cryptosporidiosis associated with
a public water supply in the UK. Epidemiol. Infect.1995;
115: 123-131
13. Avrameas S, Uriel J. Méthode de marquage d’antigènes
et d’anticorps avec des enzymes et son application en
immunodiffusion. C R Acad Sci Hebd Seances Acad Sci D
1966; 262: 2543-2545.
14. Avrameas S. Coupling of enzymes to proteins with
glutaraldehyde. Immunochemistry 1969; 6:43-52
15. Barbour A G. Isolation and cultivation of Lyme disease
spirochetes. Yale J Biol Med. 1984;57:521–525
16. Barbour A G, Tessier S L, Todd W J. Lyme disease
spirochetes and ixodid tick spirochetes share a common
surface antigenic determinant defined by a monoclonal
antibody. Infect Immun. 1983;41:795–804
17. Bauwens, L., F. Vercammen, and A. Hertsens. Detection
of pathogenic Listeria spp. in zoo animal faeces: use of
immunomagnetic separation and a chromogenic isolation
medium. Vet. Microbiol. 2003; 91:115-123
18. Beer HH, Jallerat E, Pflanz K, Klewitz TM. Quantification
of cellulose nitrate membranes for lateral-flow assays. IVD
Technol 2002; 8:35-42
19. Berri M, Nathalie Arricau-Bouvery and Annie Rodolakis.
PCR-Based Detection of Coxiella burnetii from Clinical
Samples Methods in Molecular Biology, 2003; 216 (2),
153-161
20. Beyer, W., P. Glockner, J. Otto, and R. Bohm. A nested
PCR method for the detection of Bacillus anthracis in
environmental samples collected from former tannery sites.
Microbiol. Res. 1995; 150:179-186
21. Beyer, W., S. Pocivalsek, and R. Bohm. Polymerase chain
reaction-ELISA to detect Bacillus anthracis from soil
samples—limitations of present published primers. J. Appl.
Microbiol. 1999; 87: 229-236
22. Bharti AR, Nally JE, Ricaldi JN, et al. Leptospirosis: a
zoonotic disease of global importance. Lancet Infect Dis.
2003;3(12):757-771
23. Bhattarai NR, Van der Auwera G, Khanal B, De Doncker
S, Rijal S, Das ML, et al. PCR and direct agglutination as
Leishmania infection markers among healthy Nepalese
Elite
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
subjects living in areas endemic for kala-azar. Trop Med
Int Health. 2009; 14:404–411.
Bingol A, Yucemen N, Meco O. Medically treated
intraspinal brucella granuloma. Surg Neurol 1999; 52:
570–576.
Black, S. F., D. R. Gray, D. R. Fenlon, and R. G. Kroll.
Rapid RAPD analysis to distinguish Listeria species and
Listeria monocytogenes serotypes using a capillary air
thermal cycler. Lett. Appl. Microbiol.1995; 20:188-190
Blessmann J, Tannich E. Treatment of asymptomatic
intestinal Entamoeba histolytica infection. N. Engl. J. Med.
2002; 347 (17): 1384
Blumer Sharon O, Kaufman Leo. Microimmunodiffusion
Test for Nocardiosis. J Clin Microbiol. 1979
Sep;10(3):308–312.
Boiron P, Provost F. Use of partially purified 54-kilodalton
antigen for diagnosis of nocardiosis by Western
blot (immunoblot) assay. J Clin Microbiol. 1990
Feb;28(2):328–331
Boone, J. H., T. D. Wilkins, T. E. Nash, J. E. Brandon, E.
A. Macias, R. C. Jerris, and D. M. Lyerly. TechLab and
Alexon Giardia enzyme-linked immunosorbent assay kits
detect cyst wall protein 1. J. Clin. Microbiol.1999; 37:
611-614
Bouharoun-Tayoun H, P Attanath, A Sabchareon, T
Chongsuphajaisiddhi, and P Druilhe. Antibodies that
Protect Humans against Plasmodium Falciparum Blood
Stages do not on their own Inhibit Parasite Growth and
Invasion In Vitro, but Act in Cooperation with Monocytes.
Journal of Experimental Medicine 1990; 172(6): 16331641
Bruce-Chwatt LJ. DNA probes for malaria diagnosis.
Lancet 1984; 1:795
Bubert, A., I. Hein, M. Rauch, A. Lehner, B. Yoon, W.
Goebel, and M. Wagner. Detection and differentiation of
Listeria spp. by a single reaction based on multiplex PCR.
Appl. Environ. Microbiol. 1999; 65: 4688-4692
Burgdorfer W, Barbour AG, Hayes SF, Benach JL,
Grunwaldt E, Davis JP. “Lyme disease-a tick-borne
spirochetosis?”. Science 1982; 216 (4552): 1317–9.
Carter EH, Suhrbier A, Beckers PJ, Sinden RE: The in
vitro cultivation of P. falciparum ookinetes, and their
enrichment on Nycodenz density gradients. Parasitology
1987; 95 (1):25-30.
Casemore, D.P., Garder, C.A., and O’Mahony, C.
“Cryptosporidial infection, with special reference to
nosocomial transmission of Cryptosporidium parvum: a
review.” Folia Parasitol, 1994; 41 (1): 17-21.
CDC. Tularemia associated with a hamster bite-Colorado, 2004. MMWR Morb Mortal Wkly Rep. 2005;
53(51):1202-1203
Centers for Disease Control and Prevention. Interim
guidelines for investigation of and response to Bacillus
anthracis exposures. Morb. Mortal. Wkly. Rec. 2001;
50:987-990.
Centers for Disease Control and Prevention.
Recommendations for test performance and interpretation
from the Second National Conference on Serologic
Diagnosis of Lyme Disease. Morb Mortal Wkly Rep.
1995;44:590–591
Chan, R., J. Chen, M. K. York, N. Setijono, R. L. Kaplan,
F. Graham, and H. B. Tanowitz. 2000. Evaluation of a
combination rapid immunoassay for detection of Giardia
and Cryptosporidium antigens. J. Clin. Microbiol. 38:393394.
Cheun, H. I., S. I. Makino, T. Shirahata, I. Uchida,
K. Takeshi. A simple and sensitive detection system
for Bacillus anthracis in meat and tissue. J. Appl.
Microbiol.2001; 91:421-426
Christopher G, Cieslak T, Pavlin A, Eitzen E. Biological
warfare. A historical perspective. JAMA 1997; 278:412–7.
Chuang YY, Huang YC, Lin SY. Outbreak of Shigella
sonnei gastroenteritis in Northeastern Taiwan. Pediatr
Infect Dis J. 2006; 25(1):92-94
Cox F, Hughes WT. Contagious and other aspects of
nocardiosis in the compromised host. Pediatrics. 1975
Jan;55(1):135–138.
Current, W. L., and L. S. Garcia. Cryptosporidiosis. Clin.
Microbiol. Rev. 1991; 3:325-358
Curry WA. Human nocardiosis. A clinical review with
selected case reports. Arch Intern Med. 1980;140(6):818–
826
Damude DF, Jones CJ, Myers DM. A study of leptospirosis
among animals in Barbados W.I. Trans R Soc Trop Med
Hyg. 1979; 73(2):161-168.
Dearsly AL, Nicholas J, Sinden RE: Sexual development
in Plasmodium berghei: the use of mitomycin C to separate
48.
49.
50.
51.
52.
53.
54.
infective gametocytes in vivo and ookinetes in vitro. Int J
Parasitol 1987; 17:1307-1312
Deborggraeve S, Boelaert M, Rijal S, De Doncker S,
Dujardin JC, Herdewijn P, et al. Diagnostic accuracy of
new Leishmania PCR for clinical visceral leishmaniasis
in Nepal and its role in diagnosis of disease. Trop Med Int
Health. 2008; 13:1378–1383
Deng, M. Q., D. O. Cliver, and T. W. Mariam.
Immunomagnetic capture PCR to detect viable
Cryptosporidium parvum oocysts from environmental
samples. Appl. Environ. Microbiol. 1997; 63:3134-3138
Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia
as a biological weapon: medical and public health
management. JAMA. 2001; 285(21):2763-2773
Desjeux P. Leishmaniasis. Public health aspects and
control. Clin Dermatol. 1996; 14:417–23.
Devine, P., Doyle, C., and Lambkin, G. Combined
determination of Coxiella burnetii-specific
immunoglobulin M (IgM) and IgA improves specificity
in the diagnosis of acute Q fever.Clinical and Diagnostic
Laboratory Immunology1997; 4:384-386.
Divo AA, Geary TG, Davis NL, Jensen JB. Nutritional
requirements of Plasmodium falciparum in culture. I.
Exogenously supplied dialyzable components necessary
for continuous growth. J Protozool. 1985; 32(1):59-64
Doing, K. M., J. L. Hamm, J. A. Jellison, J. A. Marquis,
and C. Kingsbury. False-positive results obtained with the
Alexon ProSpecT Cryptosporidium enzyme immunoassay.
J. Clin. Microbiol. 1999; 37:1582-1583.
55.
56. Doyle, P.S., Crabb, J., and Petersen, C. “AntiCryptosporidium parvum antibodies inhibit infectivity in
vitro and in vivo.” Infect Immun, 1993; 6(10): 4079-84.
57. DuPont, H. L., C. L. Chappell, C. R. Sterling, P. C.
Okhuysen, J. B. Rose, and W. Jakubowski. The infectivity
of Cryptosporidium parvum in healthy volunteers. N. Engl.
J. Med. 1995; 332: 855-859
58. Dupont, H.T., Thirion, X., and Raoult, D.
Q fever serology: cutoff determination for
microimmunofluorescence. Clinical and Diagnostic
Laboratory Immunology, 1994; 1:189-196.
59. Eddlestone SM: Visceral leishmaniasis in a dog from
Maryland. J Am Vet Med Assoc 2000; 217:1686-1688
60. Eliasson H, Broman T, Forsman M. Tularemia: current
epidemiology and disease management. Infect Dis Clin
North Am. 2006; 20(2):289-311,
61. Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin
Microbiol Rev. 2002;15(4):631-46
62. el-Zaatari FA, Reiss E, Yakrus MA, Bragg SL, Kaufman
L. Monoclonal antibodies against isoelectrically focused
Nocardia asteroides proteins characterized by the
enzyme-linked immunoelectro-transfer blot method. Diagn
Immunol. 1986;4(2):97–106.
63. Elschner MC et al. Burkholderia mallei infection in a horse
imported from Brazil. Equine Veterinary Education 2009;
21:147-150
64. Enderlin G, Morales L, Jacobs RF, Cross JT Streptomycin
and alternative agents for the treatment of tularemia:
review of the literature. Clin. Infect. Dis. 1994; 19 (1):
42–47
65. Engvall E, Perlmann P. Enzyme-linked immunosorbent
assay (ELISA). Quantitative assay of immunoglobulin G.
Immunochemistry 1971; 8: 871-874
66. Engvall E, Perlmann P. Enzyme-linked immunosorbent
assay, Elisa. 3. Quantitation of specific antibodies by
enzyme-labeled anti-immunoglobulin in antigen-coated
tubes. J Immunol. 1972; 109(1):129–135.
67. Everard CO, Fraser-Chanpong GM, James AC, et al.
Serological studies on leptospirosis in livestock and
chickens from Grenada and Trinidad. Trans R Soc Trop
Med Hyg. 1985; 79(6):859-864
68. Fasanella, A., S. Losito, T. Trotta, R. Adone, S. Massa, F.
Ciuchini, and D. Chiocco. Detection of anthrax vaccine
virulence factors by polymerase chain reaction. Vaccine
2001; 19:4214-4218
69. Fayer, R., J. M. Trout, and M. C. Jenkins. Infectivity
of Cryptosporidium parvum oocysts stored in water at
environmental temperatures. J. Parasitol. 1998; 84:11651169
70. Flanigan, T.P. and Soave, R. “Cryptosporidiosis.” Prog
Clin Parasitol, 1993; 1-20
71. Fournier, P.E., Marrie, T.J., and Raoult, D. Diagnosis of Q
fever. Journal of Clinical Microbiology, 1998; 36:18231834.
72. Franks AH, Hermie J. M. Harmsen,* Gerwin C.
Raangs, Gijsbert J. Jansen, Frits Schut,† and Gjalt W.
WellingVariations of Bacterial Populations in Human
Page 23
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
Feces Measured by Fluorescent In Situ Hybridization
with Group-Specific 16S rRNA-Targeted Oligonucleotide
Probes Appl Environ Microbiol. 1998 ; 64(9): 3336–3345
Fraser CM, Casjens S, Huang WM, et al. (December
1997). “Genomic sequence of a Lyme disease spirochaete,
Borrelia burgdorferi”. Nature 390 (6660): 580–6
Garcia, L. S., A. C. Shum, and D. A. Bruckner. 1992.
Evaluation of a new monoclonal antibody combination
reagent for direct fluorescent detection of Giardia cysts
and Cryptosporidium oocysts in human fecal specimens. J.
Clin. Microbiol.2000; 30: 3255-3257
Garcia, L. S., R. Y. Shimizu, and C. N. Bernard. . Detection
of Giardia lamblia, Entamoeba histolytica/Entamoeba
dispar, and Cryptosporidium parvum antigens in human
fecal specimens using the Triage Parasite Panel enzyme
immunoassay. J. Clin. Microbiol. 38:3337-3340
Gay F, Traore B, Zanoni J, Danis M, Fribourg-Blanc A.
Direct acridine orange fluorescence examination of blood
slides compared to current technique for malaria diagnosis.
Transactions of Royal Society of Tropical Medicine and
Hygiene 1996; 90:516-518
George LW: 2002. Listeriosis. In Smith, Bradford P.
large Animal Internal Medicine. 3rd edition, Mosby,
Philadelphia, :A 946-949
Giovannoni S J, DeLong E F, Olsen G J, Pace N R.
Phylogenetic group-specific oligodeoxynucleotide probes
for identification of single microbial cells. J Bacteriol.
1988; 170:720–726
Godoy D, Randle G, Simpson AJ, et al. “Multilocus
Sequence Typing and Evolutionary Relationships among
the Causative Agents of Melioidosis and Glanders,
Burkholderia pseudomallei and Burkholderia mallei”. J
Clin Microbiol. 2003; 41: 2068–2079.
Goodgame, R.W. “Understanding intestinal spore-forming
protozoa: cryptosporidia, microsporidia, isospora, and
cyclospora.” Ann Intern Med, 1996; 15; 124 (4): 429-41.
Goodman J L, Bradley J F, Ross A E, Goellner P, Lagus A,
Vitale B, Berger B W, Luger S, Johnson R C. Bloodstream
invasion in early Lyme disease: results from a prospective,
controlled, blinded study using the polymerase chain
reaction. Am J Med. 1995; 99:6–12.
Grosjean NL, Vrable RA, Murphy AJ, Mansfield LS:
Seroprevalence of antibodies against Leishmania spp
among dogs in the United States. J Am Vet Med Assoc
2003; 222:603-606
Guerin PJ, Olliaro P, Sundar S, Boelaert M, Croft SL,
Desjeux P, et al. Visceral leishmaniasis: current status
of control, diagnosis, and treatment, and a proposed
research and development agenda. Lancet Infect Dis. 2002;
2:494–501
Guidoboni M, Ferreri AJ, Ponzoni M, Doglioni C, Dolcetti
R (January ). “Infectious agents in mucosa-associated
lymphoid tissue-type lymphomas: pathogenic role and
therapeutic perspectives”. Clinical Lymphoma & Myeloma
2006; 6(4): 289–300
Gupta A, Polyak CS, Bishop RD, Sobel J, Mintz ED.
Laboratory-confirmed shigellosis in the United States,
1989--2002: epidemiologic trends and patterns. Clin Infect
Dis 2004; 38:1372-1377
Haase A, Janzen J, Barrett S, Currie B (July ). “Toxin
production by Burkholderia pseudomallei strains and
correlation with severity of melioidosis”. Journal of
medical microbiology 1997; 46 (7): 557–63.
Hanscheid T, Grobusch MP: How useful is PCR in the
diagnosis of malaria? Trends Parasitol 2002; 18: 395-398
Haque R, Ali IKM, Akther S, Petri WA Jr. Comparison
of PCR, isoenzyme analysis, and antigen detection for
diagnosis of Entamoeba histolytica infection. J Clin
Microbiol 1998; 36: 449-452.
Harman M et al. Brucellar spondylodiscitis MRI diagnosis.
Clin Imaging 2001; 25: 421–427
Havlícek J, Gasová ZG, Smith AP, Zvára K, Flegr J.
“Decrease of psychomotor performance in subjects with
latent ‘asymptomatic’ toxoplasmosis”. Parasitology 2001;
122 (5): 515–520
Hawker JI et al. A large outbreak of Q fever in the West
Midlands: windborne spread into a metropolitan area?
Communicable disease and public health (England) 1998;
1(3): 180-187.
92.
93. Heidrich HG, John E. K. Mrema, David L. Vander
Jagt, Philip Reyes and Karl H. Rieckmann.Isolation of
Intracellular Parasites (Plasmodium falciparum) from
Culture Using Free-Flow Electrophoresis: Separation of
the Free Parasites According to Stages. The Journal of
Parasitology, 1982; 68(3):443-450
94. Herder S, Simo G, Nkinin S, Njiokou F. Identification of
Page 24
trypanosomes in wild animals from southern Cameroon
using the polymerase chain reaction (PCR). Parasite. 2002;
9(4):345-349
95. Heyworth, M.F. “Immunology of Giardia and
Cryptosporidium infections.” J Infect Dis, 1992; 166 (3):
465-72.
96. Hook EW Trichomonas vaginalis – no longer a minor
STD. Sexually transmitted diseases 1999; 26 (7): 388–389
97. Ho-Yen DO, Joss AW, Balfour AH, Smyth ET, Baird D,
Chatterton JM “Use Of The Polymerase Chain Reaction To
Detect Toxoplasma Gondii In Human Blood Samples”. J.
Clin. Pathol. 1992; 45 (10): 910–913.
98. http://www.cdc.gov/toxoplasmosis/factsheet.html
99. Humphreys DW, Crowder JG, White A. Serological
reactions to Nocardia antigens. Am J Med Sci.
1975;269(3):323–326
100.Hung CC, Deng HY, Hsiao WH, Hsieh SM, Hsiao CF,
Chen MY, Chang SC, Su KE. Invasive amebiasis as
an emerging parasitic disease in patients with human
immunodeficiency virus type 1 infection in Taiwan. Arch
Intern Med. 2005; 165 (4): 409–415.
101.Huppert JS, et al. (). Use of an immunochromatographic
assay for rapid detection of Trichomonas vaginalis in
vaginal specimens. Journal of Clinical Microbiology,2005;
43(2): 684-687
102.Ikaheimo I, Syrjala H, Karhukorpi J, et al. In vitro
antibiotic susceptibility of Francisella tularensis isolated
from humans and animals. J Antimicrob Chemother. 2000;
46(2):287-290
103.Inglesby T, Henderson D, Bartlett J, Ascher M, Eitzen
E, Friedlander A, et al. Anthrax as a biological weapon.
JAMA 1999; 281: 1735–45.
104.Ivins, B. E., J. W. J. Ezzell, J. Jemski, K. W. Hedlund, J.
D. Ristroph, and S. H. Leppla. Immunization studies with
attenuated strains of Bacillus anthracis. Infect. Immun.
1986; 52:454-458
105.Janse CJ, Mons B, Rouwenhorst RJ, Van der Klooster
PF, Overdulve JP, Van der Kaay HJ: In vitro formation of
ookinetes and functional maturity of Plasmodium berghei
gametocytes. Parasitology 1985; 91(1): 19-29.
106.Jernigan, J. A., D. S. Stephens, D. A. Ashford, C.
Omenaca, M. S. Topiel, M. Galbraith, M. Tapper, T. L.
Fisk, S. Zaki, T. Popovic, R. F. Meyer, C. P. Quinn, S.
A. Harper, S. K. Fridkin, J. J. Sejvar, C. W. Shepard,
M. McConnell, J. Guarner, W. J. Shieh, J. M. Malecki,
J. L. Gerberding, J. M. Hughes, and B. A. Perkins.
Bioterrorism-related inhalational anthrax: the first 10 cases
reported in the United States. Emerg. Infect. Dis. 2001;
7:933-944
107.Johnson, D.W., Pieniazek, N.J., Griffin, D.W., Misener,
L., and Rose, J.B. “Development of a PCR protocol for
sensitive detection of Cryptosporidium oocysts in water
samples.” Appl Environ Microbiol, 1995; 61 (11): 384955.
108.Jones KD. Troubleshooting protein binding in
nitrocellulose membranes. Part 1: principles. IVD Technol
1999; 5:32-41.
109.Jones KD. Troubleshooting protein binding in
nitrocellulose membranes. Part 2: common problems. IVD
Technol 1999; 5:26-35.
110.Jones JL, Kruszon-Moran D, Wilson M, McQuillan G,
Navin T, McAuley JB (). “Toxoplasma gondii infection in
the United States: seroprevalence and risk factors”. Am J
Epidemiol 2001; 154 (4): 357–365.
111.Jones JL, Kruszon-Moran D, Sanders-Lewis K, Wilson M.
“Toxoplasma gondii infection in the United States, 19992004, decline from the prior decade”. Am J Trop Med Hyg
2007; 77 (3): 405–10.
112.Juranek, D.D. “Cryptosporidiosis: sources of infection and
guidelines for prevention.” Clin Infect Dis, 1995; 21 (1):
S57-61.
113.Jurca T, Ruzic-Sabljic E, Lotric-Furlan S, Maraspin
V, Cimperman J, Picken R N, Strle F. Comparison of
peripheral and central biopsy sites for the isolation of
Borrelia burgdorferi sensu lato from erythema migrans
skin lesions. Clin Infect Dis. 1998; 27:636–638
114.Kain KC, Keystone JS: Malaria in travelers. Epidemiology,
disease, and prevention. Infect Dis Clin North Am 1998;
12:267-284
115.Kain KC, MacPherson DW, Kelton T, Keystone JS,
Mendelson J, MacLean JD: Malaria deaths in visitors to
Canada and in Canadian travellers: a case series. Canadian
Medical Association Journal 2001; 164: 654-659
116.Karlsson M, Hovind-Hougen K, Svenungsson B,
Stiernstedt G. Cultivation and characterization of
spirochetes from cerebrospinal fluid of patients with Lyme
borreliosis. J Clin Microbiol. 1990;28:473–479
117.Kehl, K. C., H. Cicirello, and P. L. Havens. Comparison
of four different methods for the detection of
Cryptosporidium species. J. Clin. Microbiol.1995; 33:416418
118.Keusch, G.T., Hamer, D., Joe, A., Kelley, M., Griffiths, J.,
and Ward, H. “Cryptosporidia--who is at risk?” Schweiz
Med Wochenschr, 1995; 125 (18): 899-908.
119.Kevube RA, Wardlaw SC, Patton CL. Detection of
haemoparasites using quantitative buffy coat analysis
tubes. Parasitology Today 1989; 5:34.
120.Kliemann W, Ruoff K. Bioterrorism: implications for the
clinical microbiologist. Clin Microbiol Rev 2001; 14:
364–81.
121.Knight A, Sinden RE: The purification of gametocytes
of Plasmodium falciparum and P. yoelii nigeriensis by
colloidal silica (Percoll) gradient centrifugation. Trans R
Soc Trop Med Hyg 1982; 76:503-509.
122.Korich, D.G., Mead, J.R., Madore, M.S., Sinclair, N.A.,
and Sterling, C.R. “Effects of ozone, chlorine dioxide,
chlorine, and monochlorine on Cryptosporidium parvum
oocyst viability.” Appl Envion Microbiol, 1990; 56 (5):
1423-1428.
123.Krawczyk M. Serological evidence of leptospirosis in
animals in northern Poland. Vet Rec. 2005; 156(3):88-89.
124.Krieger JN and Alderete JF. Trichomonas vaginalis and
trichomoniasis. In: K. Holmes, P. Markh, P. Sparling et al
(eds). Sexually Transmitted Diseases, 3rd Edition. New
York: McGraw-Hill, 1999, 587-604.
125.Lang GH. Q fever: an emerging public health concern in
Canada. Can J Vet Res 1989; 53: 1-6.
126.Lang G, Waltner-Toews D, Menzies P. The seroprevalence
of coxiellosis (Q fever) in Ontario sheep flocks. Can J Vet
Res 1991; 55: 139-142.
127.Lee SH, Kara UA, Koay E, Lee MA, Lam S, Teo D. New
strategies for the diagnosis and screening of malaria. Int J
Hematol 2002; 76(1): 291-293.
128.Lee, M. A., G. Brightwell, D. Leslie, H. Bird, and A.
Hamilton. Fluorescent detection techniques for real time
multiplex strand specific detection of Bacillus anthracis
using rapid PCR. J. Appl. Microbiol.1999; 87:218-223. 15.
129.Levett PN, Whittington CU, Camus E. Serological survey
of leptospirosis in livestock animals in the Lesser Antilles.
Ann N Y Acad Sci. 1996; 791:369-377.
130.Levine LS, Long GW, Oberst R et al. Rapid diagnosis
of malaria by acridine orange staining of centrifuged
parasites. Lancet 1989; 1:68-71.
131.Lifeso RM, Harder E, Mc Corkell. Spinal brucellosis. J
Bone Joint Surg 1985; 67: 345–351.
132.Lioyd RB, Naiper LE. The blood meal of sand-flies
investigated by means of precipitin antisera. Indian J Med
Res. 1930; 18:347–359.
133.Long GW, Jones TR, Rickman LS, Fries L, Egan J,
Wellde B et al. Acridine orange diagnosis of Plasmodium
falciparum; evaluation after experimental infection. The
American Society of Tropical Medicine and Hygiene 1994;
51(5): 613-616
134.Mackenzie, W. R., N. J. Hoxie, and M. E. Proctor. A
massive outbreak in Milwaukee of Cryptosporidium
infection transmitted through the public water supply. N.
Engl. J. Med. 1994; 331:161-167
135.Makino, S. I., H. I. Cheun, M. Watarai, I. Uchida, and K.
Takeshi. Detection of anthrax spores from the air by realtime PCR. Lett. Appl. Microbiol. 2001; 33: 237-240
136.Marshall, M. M., D. Naumovitz, Y. Ortega, and C. R.
Sterling. Waterborne protozoan pathogens. Clin. Microbiol.
Rev.1997; 10: 67-85
137.Martínez-Subiela S, Tecles F, Eckersall PD, Cerón JJ:
Serum concentrations of acute phase proteins in dogs with
leishmaniasis. Vet Rec 2002; 150:241-244
138.Maurin, M., and Raoult, D. Q fever. Clinical Microbiology
Reviews, 1999; 12:518-553
139.McConkey SE, López A, Shaw D, Calder J: Leishmanial
polyarthritis in a dog. Canine Vet J 2002; 43:607-609
140.Merien F, Amouriaux P, Perolat P, et al. Polymerase
chain reaction for detection of Leptospira spp. in clinical
samples. J Clin Microbiol. 1992; 30(9):2219-2224
141.Miles LEM, Hales CN. Labelled antibodies and
immunological assay systems. Nature 1968; 219:186-189
142.Mörner T . The ecology of tularaemia. Rev. Sci.
Tech. 1992; 11(4): 1123–30
143.Montoya JG, et al. Diagnosis and management of
toxoplasmosis. Clinics in Perinatology. 2005;32:705
144.Moody AH, Chiodini PL. Methods for the detection of
blood parasites. Clin Lab Haematol 2000; 22:189-201.
145.Moody A. Rapid diagnostic tests for malaria parasites. Clin
Microbiol Rev 2002; 15:66-78.
146.Morgan-Ryan UM, Fall A, Ward LA, Hijjawi N, Sulaiman
Elite
I, Fayer R, et al. Cryptosporidium hominis n. sp.
(Apicomplexa: Cryptosporidiidae) from Homo sapiens. J
Eukaryot Microbiol 2002; 49: 433-440.
147.Morlais I, Grebaut P, Bodo JM, Djoha S, Cuny G, Herder
S. Detection and identification of trypanosomes by
polymerase chain reaction in wild tsetse flies in Cameroon.
Acta Trop. 1998;70(1):109-117.
148.Morlais I, Grebaut P, Bodo JM, Djoha S, Cuny G.
Characterization of trypanosome infections by polymerase
chain reaction (PCR) amplification in wild tsetse flies in
Cameroon. Parasitology. 1998; 116 (6):547-554
149.Mousa AM et al. Neurological complications of brucella
spondylitis. Acta Neurol Scand 1990; 81: 16–23.
150.Mühlberger N, Jelinek T, Behrens RH, Gjørup I, Coulaud
JP, Clerinx J, Puente S, Burchard G, Gascon J, Grobusch
MP, Weitzel T, Zoller T, Kollaritsch H, Beran J, Iversen
J, Hatz C, Schmid ML, Björkman A, Fleischer K, Bisoffi
Z, Guggemos W, Knobloch J, Matteelli A, Schulze MH,
Laferl H, Kapaun A, McWhinney P, Lopez-Velez R,
Fätkenheuer G, Kern P, Zieger BW, Kotlowski A, Fry G,
Cuadros J, Myrvang B: Age as a risk factor for severe
manifestations and fatal outcome of falciparum malaria
in European patients: observations from TropNetEurop
and SIMPID Surveillance Data. Clin Infect Dis 2003; 36
:990-995
151.Munderloh UG, Kurtti TJ: The infectivity and purification
of cultured Plasmodium berghei ookinetes. J Parasitol
1987; 73:919-923.
152.Nadelman R B, Nowakowski J, Forseter G, Bittker S,
Cooper D, Goldberg N, McKenna D, Wormser G P. Failure
to isolate Borrelia burgdorferi after antimicrobial therapy
in culture-documented Lyme borreliosis associated with
erythema migrans: report of a prospective study. Am J
Med. 1993;94:583–588
153.Nas K et al. Management of spinal brucellosis and
outcome of rehabilitation. Spinal Cord 2001; 39: 223–227
154.Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ.
Intraleucocytic malaria pigment and prognosis in severe
malaria. Trans R Soc Trop Med Hyg 1995; 89:200-204.
155.Nigrovic LE, Wingerter SL. Tularemia. Infect Dis Clin
North Am. 2008; 22(3):489-504,
156.Njiokou F, Simo G, Nkinin SW, Laveissière C, Herder
S. Infection rate of Trypanosoma brucei s.l., T. vivax,
T. congolense “forest type”, and T. simiae in small wild
vertebrates in south Cameroon. Acta Trop. 2004;92(2):139146
157.Njiokou F, Laveissière C, Simo G, Nkinin S, Grébaut
P, Cuny G, Herder S. Wild fauna as a probable animal
reservoir for Trypanosoma brucei gambiense in Cameroon.
Infect Genet Evol. 2006; 6(2):147-153
158.Njiru ZK, Constantine CC, Guya S, Crowther J, Kiragu
JM, Thompson RC, Dávila AM. The use of ITS1 rDNA
PCR in detecting pathogenic African trypanosomes.
Parasitol Res. 2005; 95(3):186-192
159.Nocton J J, Dressler F, Rutledge B J, Rys P N, Persing D
H, Steere A C. Detection of Borrelia burgdorferi DNA by
polymerase chain reaction in synovial fluid from patients
with Lyme arthritis. N Engl J Med. 1994;330:229–234
160.Noppadon Tangpukdee, Chatnapa Duangdee, Polrat
Wilairatana, Srivicha Krudsood. Malaria Diagnosis: A
Brief Review Korean J Parasitol. 2009; 47(2): 93-102.
161.Ono T, Ohnishi Y, Nagamune K, Kano M:
Gametocytogenesis induction by Berenil in cultured
Plasmodium falciparum. Exp Parasitol 1993; 77:74-78.
162.Ozates M et al. Lumbar epidural brucellar abscess causing
nerve root compression. Spinal Cord 1999; 37: 448–449.
163.Paul M . “Immunoglobulin G Avidity In Diagnosis
Of Toxoplasmic Lymphadenopathy And Ocular
Toxoplasmosis”. Clin. Diagn. Lab. Immunol. 1999; 6 (4):
514–518.
164.Penchenier L, Simo G, Grébaut P, Nkinin S, Laveissière
C, Herder S. Diagnosis of human trypanosomiasis, due to
Trypanosoma brucei gambiense in central Africa, by the
polymerase chain reaction. Trans R Soc Trop Med Hyg.
2000;94(4):392-394
165.Penn RL, Kinasewitz GT. Factors associated
with a poor outcome in tularemia. Arch Intern
Med. Feb 1987;147(2):265-268. 166.Perez-Castrillon JL, Bachiller-Luque P, Martin-Luquero M,
et al. Tularemia epidemic in northwestern Spain: clinical
description and therapeutic response. Clin Infect Dis. 2001;
33(4):573-576. 167.Perlmann P, Berzins K, Perlmann H, Troye-Blomberg M,
Wahlgren M, Wahlin B. Malaria vaccines: immunogen
selection and epitope mapping [Review]. Vaccine 1998;
6:183-187
168.Picken M M, Picken R N, Han D, Cheng Y, Ruzic-
Elite
Sabljic E, Cimperman J, Strle F. A two year prospective
study to compare culture and polymerase chain reaction
amplification for the detection and diagnosis of Lyme
borreliosis. Mol Pathol. 1997;50:186–193.
169.Pieniazek, N. J., F. J. Bornay-Llinares, S. B. Slemenda,
A. J. da Silva, I. N. S. Moura, M. J. Arrowood, O. Ditrich,
and D. G. Addis. New Cryptosporidium genotypes in HIVinfected persons. Emerg. Infect. Dis.1999; 5: 444-449
170.Pieroni P, Caroline Dawn Mills, Colin Ohrt, Mary
Anne Harrington, Kevin C. Kain. Comparison of the
ParaSight™-F test and the ICT Malaria Pf™ test with the
polymerase chain reaction for the diagnosis of Plasmodium
falciparum malaria in travelers. Trans R Soc Trop Med
Hyg. 1998; 92(2): 166-169
171.Pile J, Malone J, Eitzen E, Friedlander A. Anthrax as
a potential biological warfare agent. Arch Intern Med
1998:158:429–34.
172.Pinto MJ, Rodrigues SR, Desouza R, Verenkar MP.
Usefulness of quantitative buffy coat blood parasite
detection system in diagnosis of malaria. Indian J Med
Microbiol 2001; 19:219-221.
173.Priest, J. W., J. P. Kwon, D. M. Moss, J. M. Roberts, M.
J. Arrowood, M. S. Dworkin, D. D. Juranek, and P. J.
Lammie. Detection of enzyme immunoassay of serum
immunoglobulin G antibodies that recognize specific
Cryptosporidium parvum antigens. J. Clin. Microbiol.
1999; 37: 1385-1392
174.Prina E, Roux E, Mattei D, Milon G. Leishmania DNA is
rapidly degraded following parasite death: an analysis by
microscopy and real-time PCR. Microbes Infect. 2007;
9:1307–1315
175.Qi, Y., G. Patra, X. Liang, L. E. Williams, S. Rose, R. J.
Redkar, and V. G. Del Vecchio. Utilization of the rpoB
gene as a specific chromosomal marker for real-time PCR
detection of Bacillus anthracis. Appl. Environ. Microbiol.
2001; 67: 3720-3727
176.Ramisse, V., G. Patra, H. Garrigue, J. L. Guesdon, and
M. Mock. Identification and characterization of Bacillus
anthracis by multiplex PCR analysis of sequences on
plasmids pX01 and pX02 and chromosomal DNA. FEMS
Microbiol. Lett. 1996; 145:9-16
177.Raoult, D., Houpikian, P., Tissot-Dupont, H.,
Riss, J.M., Arditi-Djiane, J., and Brouqui, P.
Treatment of Q fever endocarditis: comparison of 2
regemins containing doxycycline and ofloxacin or
hydroxychloroquine. Archives of Internal Medicine,
1999; 159:167-173.
178.Read, T. D., S. L. Salzberg, M. Pop, M. Shumway, L.
Umayam, L. Jiang, E. Holtzapple, J. D. Busch, K. L.
Smith, J. M. Schupp, D. Solomon, P. Keim, and C. M.
Fraser. Comparative genome sequencing for discovery of
novel polymorphism in Bacillus anthracis. Science 2002;
296: 2028-2033
179.Rodriguez MC, Margos G, Compton H, Ku M, Lanz H,
Rodriguez MH, Sinden RE: Plasmodium berghei: routine
production of pure gametocytes, extracellular gametes,
zygotes, and ookinetes. Exp Parasitol 2002; 101:73-76
180.Rosenblatt, J. E., L. M. Sloan, and S. K. Schneider.
Evaluation of an enzyme-linked immunosorbent assay for
the detection of Giardia lamblia in stool specimens. Diagn.
Microbiol. Infect. Dis.1993; 16:337-341
181.Rosenblatt, J. E., and L. M. Sloan. Evaluation of an
enzyme-linked immunosorbent assay for detection
of Cryptosporidium spp. in stool specimens. J. Clin.
Microbiol. 1993; 31:1468-1471
182.Rosoff, J. D., C. A. Sanders, S. S. Sonnad, P. R. De
Lay, W. K. Hadley, F. F. Vincenzi, D. M. Yajko, and
P. D. O’Hanley. Stool diagnosis of giardiasis using a
commercially available enzyme immunoassay to detect
Giardia-specific antigen 65 (GSA 65). J. Clin. Microbiol.
1989; 27:1997-2002
183.Russell KL, Montiel Gonzalez MA, Watts DM, et al. An
outbreak of leptospirosis among Peruvian military recruits.
Am J Trop Med Hyg. 2003; 69(1):53-57
184.Sachs J, Malaney P: The economic and social burden of
malaria. Nature 2002 ; 415: 680-685.
185.Sankaran-Kutty M, Marwah S, Kutty MK. The skeletal
manifestations of brucellosis. Int Orthop 1991; 15: 17–19
186.Schell MA, Ricky L, Ulrich et al. “Type VI secretion is a
major virulence determinant in Burkholderia mallei”. Mol
Microbiol 2007; 64 (6): 1466–1485.
187.Schmid GP, Kornblatt AN, Connors CA, et al. Clinically
mild tularemia associated with tick-borne Francisella
tularensis. J Infect Dis. Jul 1983; 148(1):63-67. 188.Schwartz I, Wormser G P, Schwartz J J, Cooper D,
Weissensee P, Gazumyan A, Zimmermann E, Goldberg N
S, Bittker S, Campbell G L, et al. Diagnosis of early Lyme
disease by polymerase chain reaction amplification and
culture of skin biopsies from erythema migrans lesions. J
Clin Microbiol. 1992;30:3082–3088
189.Schwebke, JR, Hook EW III (). High rates of trichomonas
vaginalis among men attending a sexually transmitted
diseases clinic: Implications for screening and urethritis
management. Journal of Infectious Diseases,2003; 188:
465-468.
190.Scott, G.H., and Williams, J.C. Susceptibility of Coxiella
burnetii to chemical disinfectants. Annals of the New York
Academy of Sciences, 1990; 590:291-296.
191.Shane A, Crump J, Tucker N, Painter J, Mintz E. Sharing
Shigella: risk factors and costs of a multi-community
outbreak of shigellosis. Archives Pediatrics and Adolescent
Med 2003; 157:601-603.
192.Sharif HS et al. Brucellar and tuberculosis spondylitis:
comparative imaging features. Radiology 1989; 171:
419–425
193.Siden-Kiamos I, Vlachou D, Margos G, Beetsma A, Waters
AP, Sinden RE, Louis C: Distinct roles for pbs21 and
pbs25 in the in vitro ookinete to oocyst transformation of
Plasmodium berghei. J Cell Sci 2000; 113(19): 3419-3426.
194.Sobel J, Cameron DN, Ismail J, et al. A prolonged outbreak
of Shigella sonnei infections in traditionally observant
Jewish communities in North America caused by a
molecularly distinct bacterial subtype. J Infect Dis 1998;
177:1405-1408.
195.Soper D. American Journal Of Obstetrics And Gynecology
2004; 190 (1): 281–290.
196.Sprague LD et al. P Trichomoniasis: under control or
undercontrolled? revalence-dependent use of serological
tests for diagnosing glanders in horses. BMC Veterinary
Research 2009; 5:32
197.Stabler A, Reiser MF. Imaging of spinal infection. Radiol
Clin North Am 2001; 39: 115–135
198.Stanley SL “Amoebiasis”. Lancet 2003; 361 (9362):
1025–1034
199.Stephenson J. RAMP: a quantitative immunoassay
platform takes shape. IVD Technol 1998; 4: 51-56.
200.Sugar AM, Schoolnik GK, Stevens DA. Antibody
response in human nocardiosis: identification of two
immunodominant culture-filtrate antigens derived from
Nocardia asteroides. J Infect Dis. 1985 May;151(5):895–
901.
201.Tasova Y, Saltoglu N, Sahin G, Aksu HSZ. Osteoarticular
involvement of brucellosis in Turkey. Clin Rheumatol
1999; 18: 214–219
202.Trampuz A, Matjaz Jereb, Igor Muzlovic, Rajesh M
Prabhu. Clinical review: Severe malaria. Critical Care
2003; 7:315-323
203.Uhl, J. R., C. A. Bell, L. M. Sloan, M. J. Espy, T. F. Smith,
J. E. Rosenblatt, and F. R. Cockerill III. Application of
rapid-cycle real-time polymerase chain reaction for the
detection of microbial pathogens: the Mayo-Roche Rapid
Anthrax Test. Mayo Clin. Proc. 2002; 77: 673-680
204.Vitale, G., R. Pellizzari, C. Recchi, G. Napolitani, M.
Mock and C. Montecucco. Anthrax lethal factor cleaves the
N-terminus of MAPKKs and induces tyrosine/threonine
phosphorylation of MAPKs in cultured macrophages.
Biochem. Biophys. Res. Commun. 1998; 248:706-711
205.Volokhov Dmitriy, Avraham Rasooly, Konstantin
Chumakov and Vladimir Chizhikov Identification of
Listeria Species by Microarray-Based Assay, Journal of
Clinical Microbiology, 2002; 40(12): 4720-4728
206.Wagner-Wiening, C., and Kimmig, P. “Detection of viable
Cryptosporidium parvum oocysts by PCR.” Appl Environ
Microbiol, 1995; 61 (12): 4514-6.
207.Weinstock H, Berman S, Cates W. Sexually transmitted
disease among American youth: Incidence and prevalence
estimates, 2000. Perspectives on Sexual and Reproductive
Health 2004; 36: 6-10
208.Weiss MM, Vanderberg JP: Studies on Plasmodium
ookinetes: II. In vitro formation of Plasmodium berghei
ookinetes. J Parasitol 1977; 63: 932-934
209.Wolfe, M. S. Giardiasis. Clin. Microbiol. Rev. 1992; 5:
93-100
210.Zamora A, Bojalil Lf, Bastarrachea F. Immunologically
Active Polysaccharides From Nocardia Asteroides And
Nocardia Brasiliensis. J Bacteriol. 1963; 85:549–555.
211.Zimmerman, S. K., and C. A. Needham. Comparison of
conventional stool concentration and preserved-smear
methods with Merifluor Cryptosporidium/Giardia direct
immunofluorescence assay and ProSpecT Giardia EZ
microplate assay for detection of Giardia lamblia. J. Clin.
Microbiol. 1995; 33:1942-1943
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INFECTIOUS DISEASES
DIAGNOSTICs (a)
prokaryotes and eukaryotes
(unicellular agents)
10. Toxoplasmosis is the third leading cause
of death due to food-borne illnesses in the
world.
Choose True or False for questions 1-15 then
complete your test online at
www.elitecme.com.
11. Antibody tests cannot be used to diagnose
toxoplasma.
TrueFalse
Final Examination Questions
1. About 25 percent of emerging human
diseases are zoonoses.
TrueFalse
TrueFalse
12. To identify toxoplasma, a culture source
should come from body fluids.
2. In the ideal parasitism, both host and parasite
benefit and help each other to survive.
TrueFalse
TrueFalse
13. Blood and lymphatic tissue are the material of
choice for identification of Bacillus anthracis.
3. Prokaryotes make up the most abundant
biomass on earth.
TrueFalse
TrueFalse
14. In older cattle, an abrupt reduction in milk
production, down by 10 to 75 percent, milk
that is yellowish thick, contaminated with
blood, with thick clots and high cell counts,
and a flabby and soft udder all point to
infection with Leptospira.
4. A cat stillbirth or weak, debilitated newborns
suggest Toxoplasma gondii.
TrueFalse
5. Congenital toxoplasmosis may have
serious implications for children, including
premature birth, damage to the central
nervous system and the eyes, skin and ears.
TrueFalse
15. Culturing for Francisella tularensis requires
extreme, ie., biohazard, safety measures.
TrueFalse
TrueFalse
6. Bacillus anthracis can survive infective in the
soil for only a few weeks.
TrueFalse
7. Leptospira survive for a long time in
contaminated surface waters, which seem to
be the major source of reinfestation.
TrueFalse
8. Nocardia infects horses frequently.
TrueFalse
9. The absence of parasitized erythrocytes
in a single blood smear is a definitive
demonstration that there is no malaria.
TrueFalse
Page 26
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