MYCOLOGY

CHAPTER 29 Dermatophytosis
Dermatophytosis(or) Dermatomycosis
NAIP

Superficial infection of keratinized layers of the skin,

hair, feathers and horns of farm, domestic and wild
animals, birds and man.
Ring-shaped lesions- called as ringworm.
Dermatophytes classified into 3 genera, having
specific target tissue which it infects.
Microsporum infects hair & skin.
Trichophyton infects hair, skin & nail
Epidermophyton infects skin & nail
History

Sabouraud (1910) detailed work on systematic

-

and scientific studies of the dermatophytes.

In 1959 the sexual state of some of the

dermatophytes was identified- Class Ascomycetes.

Gentles (1958) published the first report on the oral

administration of griseofulvin
-Treatment for experimental dermatophytosis in a
guinea pig.
Natural habitat

Soil is rich with dermatophytes

Obligate parasites of animals.
In general, the more chronic the infection and
the more adapted the parasite is to the host, the
less severe the inflammatory response will be.
M. gypseum -
natural soil inhabitant that is a
common cause of dermatophytosis, while most
of the other common animal pathogens are
normally found only on animals.
Morphology NAIF

In non-parasitic phase, including culture, dermatophytes

produce septate, branching hyphae -mycelium.
Conidia aerial mycelium (either macroconidia or
-

microconidia).
Shape, size, structure, arrangement, and abundance of
conidia are diagnostic criteria.
Most species in the genus Microsporum produce
predominately macroconidia
Most Trichophytorn species produce predominately
microconidia with few or no macroconidia.

Trichophyton Microsporum Epidermophyton

Morphology NAIF

Hyphal peculiarities spirals, nodules, "rackets,"

"chandeliers," and chlamydoconidia
Pigmentation useful in dermatophyte differentiation.
In tissue sections, arthroconidia can often be identified.
This "parasitic" phase arthrospore can remain infectious
for years.
Arthroconidia are indistinguishable from species to species.
Chlamydoconidia are also commonly seen in some
dermatophytes in vitro and their presence may be of use
diagnostically in the absence of other conidia.
Sexual spores (ascospores) are absent in the parasitic
phase
Growth characteristics NA

Dermatophyte test medium (DTM) or Sabouraud's dextrose agar,

Acidity (pH 5.6) renders it mildly bacteriostatic and selective.
Selectivity enhanced by addition of cycloheximide
(500ug/ml), which inhibits other fungi, and gentamicin and
tetracycline (100 ug/ml of each), or chloramphenicol (50 ug/ml).
Aerobes-and nonfermenters.
Attack proteins and deaminate amino acids.
Grow optimally at 25°C to 30°C and require several days to
weeks of incubation.
Dermatophytes in skin and hair (but not in culture) produce
a green fluorescence due to a tryptophan metabolite that is visible
under a Wood's light.
Of animal dermatophytes, only Microsporum canis produces this
reaction
Virulence factor

Proteolytic enzymes (elastase, collagenase, keratinase)

determine virulence, particularly in severe inflammatory
disease.
Highly specialized for utilizing keratin as food source.
Localization in keratinized epidermis attributed to the
-

lack of sufficient available iron elsewhere.

Highly species-specific, have a keratinase, only
hydrolyze keratin froma particular animal species.
Broad acting keratinases can invade the skin of
many different species (e.g. T. mentagrophytes).
Pathogenesis NAIP

Conidia enters the skin through an abrasion, germinate, and hyphae

begin to grow in the stratum corneum.
Portions of mycelium differentiate into arthroconidia. This growth
pattern in the hairless skin predominates with some dermatophytes
(M. nanum, T. rubrum).
Hair invasion- begins with germination of a spore near a follicular
orifice.
Hyphae invade the hair follicle and enter the cortex of the hair by
dissolving the keratin.
Hyphae and conidia are carried to the surface by the growing hair,
which often breaks off.
Hair invasion may be
a. endothrix
(arthroconidia develop within the hair shaft only and the cuticle
remains intact) or
b. ectothrix
(arthroconidia develop outside the hair shaft and hyphae are within the
hair shaft; the cuticle is destroyed).
Pathogenicity
More commonly in very young, old, or sick animals and
most often in stabled rather than pastured animals.
Peak incidence occurs in the winter.
Characteristic lesion - hyperkeratosis with septate hyphae,
and arthroconidia in the stratum corneum.
Hair shaft to become weak and break, resulting in circular,
scaly areas of alopecia with or without crust formation.
Arthroconidia within or outside the hair shaft - arthrospores.
"id" reactions Due to fungi or their products disseminating
in the body and to hypersensitivity reaction (delayed-type,

or immediate).
M. canis - "kerions"- vesicles in and around the lesion.
Lesions NAIP

Similar in different animal

species.
Most common areas of
infection
Dogs and cats- head and
extremities.
Horses and sheep neck
and girth
Cattle and sheep head
and neck.
Fowl (fowl favus or white
comb) white moldy
crusts develop on the
Comb and wattle.
 Diagnosis
Based on Direct Examination

Fluorescence of hairs is useful for identification of hairs that

may be infected with dermatophytes.

Hairs and skin scales infected with M. canis or M. audouinii

emit a bright greenish fluorescence under UV light
(Wood's lamp)
Diagnosis NAF

Based on Microscopic examination

>Skin scrapings & hair - presence of hyphae and arthroconidia.
scraping9 material from the margins of any lesion and the full
thickness of the keratinized epidermis.
Hair is plucked, so as to include the intrafollicular portion.
Sample is placed on a slide, flooded with 10% to 20% KOH or NaOH
with a cover slip, and heated gently.
Individual, spherical arthroconidia are recognizable at higher
magnification
Branching hyphae and chains of arthroconidia -
hairless skin.

Based on culture
DTM medium will turn red as the dermatophyte is growing, and the
fungus itself will usually be hyaline and fluffy.
ldentification confirmed by microscopy.
Very long, narrow hyphae with distinctive shapes and micro or
macroconidia dermatophyte infection.
Differential diagnosis

Differentiate fromn
Insect bites
Urticaria
Bacterial infections
seborrheaic dermatitis
Interdigital dermatitis
Treatment NAIP

Small animals - griseofulvin - orally

Drug is incorporated into the keratin of the tissue
and renders the skin resistant to infection.
Requires prolonged therapy (given for at least a
month, or 2 weeks), particularly if the infection is in
the nail. Some toxicity may occur.
Ketoconazole and fluconazole is also effective
Topical treatments salicylic and benzoic acids,
iodine, natamycin-s, and imidazole derivatives used
for large animals for skin infections, but not for nail
infections.
CHAPTER 30 Candidiasis
Candidiasis
Host (s) Disease (s)
|Chicken, turkey, pigeon, Crop mycosis or Avian moniliasis
ducklings
Swine Stomach ulcers and cutaneous
candidiasis
Puppies, kittens, calves|Mycotic stomatitis and enteritis
and foals
Mares and bitches Genital tract infection

C.albicans, C.tropicalis, C.pseudotropicalis, C.parapsilosis,

C.krusei &C.rugosa bovine mastitis
-

C.parapsilosis - bovine abortion

C.rugosa - pyometra in mare.
Candida albicans infections moniliasis, candidiasis&
Candidosis.
Candidiasis of the alimentary tract thrush.
 Natural habitat NAIP

All Candida occur saprophytically.

Candida species commonly present in the crop
of birds.
C.albicans worldwide in distribution
C.albicans Commensal of the oral, gastro
intestinal and genital tract of many species of
animals and humans.
Pathogenesis NAIP

Infections are endogenous in origin

Predisposing factors - immunosuppression, prolonged
antibiotic therapy & malnutrition.
Disseminated candidiasis (or) systemic candidiasis -
immunosuppressed animals.
Candida possesses adhesions consisting of fibrillar peptide.
Mannans - affinity for the fibronectin on the surface of cells
Yeast forms - responsible for tissue damage.
Inhibition of yeast cell division results in hyphal elements that
invade tissues.
Virulence factors cell wall glycoprotein, proteases,
neuraminidase, chitin, mannoprotein & lipids.
Cell-wall glycoproteins endotoxin like activity.
Granulomatous lesions are rare.
Symptomns
Poultry unsatisfactory growth, stunted
appearance, roughness of feathers, listlessness,
loss of appetite, shrunken appearance of chest &
tendency to stand around with head drowned
on shoulder.
Acute: High mortality in young birds
Cattle :Pneumonic & enteric symptoms, mastitis
may be mild and self-limiting, spontaneous
recovery may occur within a week.
Vomiting, diarrhea & emaciation pigs.
Diagnosis

Specimens
Scrapings from lesions, centrifuged milk samples, biopsy or
tissue samples in 10% formalin for histopathology.

Based on morphology
C.albicans grows as oval, budding yeast cell on agar
cultures & in animal tissues.
Pseudohyphae are also produced in animal tissue byy
elongation of yeast cells that fails to separate.
In Gram stained smears - purple-blue yeast cell.
In PAS-haematoxylin (or) methaneamine silver stains of
tissue sectionsthin walled oval, budding yeast cells
and/or in the form of pseudohyphae.
Diagnosis NAIF

2. Based on isolation and identification

On Blood agar or SDA without inhibitors, incubated at 37C,

aerobically, for up to 5 days.
white to cream, shiny, high convex colonies, pleasant beery smell.
Grams or lactophenol cotton blue or methylene blue stain reveal thin
walled budding yeast cell and pseudohyphae.

On BiGGy agar (Bismuth-sulphite-glucose- glycine- yeast agar)

Most bacteria inhibited by the Bismuth sulphite.
C.albicans and C.tropicalis strongly reduce the Bismuth sulphite to
Bismuth sulphide.
C.albicans smooth, circular brownish colonoies and no color
diffusion into the surrounding medium.
Colonies of C.tropicalis are similar but there is diffuse blackening of
the medium after 72 hrs.
Diagnosis

3. Germ tube or serum tube test

A small inoculum from an isolated colony is
suspended 0.5 ml of sheep, bovine, rabbit or
in
human serum and incubated at 370C for 2-3 hrs.

A drop of the preparation is examined under

phase contrast or high objective of the light
microscope.

Small, thin walled tubes will be seen projecting

from some of the yeast cells. This is characteristic
of C.albicans.
Diagnosis

4. Demonstration of Chlamydospore (Dalmu's technique)

Subsurface inoculation is made on corn meal- tween 80 or

chlamydospore agar and the plates are incubated at 30°C
for 2-4 days
A thin coverslip is placed on the surface of the agar and

examined under high power microscope to demonstrate

thick walled chlamydospores borne on the tips of
pseudohyphae.
Clusters of smaller blastospores may also be present
 Candida albicans

Chlamydospores of Candida albicans

 Diagnosis

5. Based on biochemical tests

Characters C.albicans C. tropicalisC.pseudotropicalisC. parapsilosis

Ability to utilize Acid (A) and A &G A&G A&G

1. Glucose Gas (G)

2.Sucrose A A&G A&G

3.Maltose A&G A&G
4.Lactose A&G
5. Chlamydospore
on corn meal agar

6. Animal inoculation
Rabbits and mice iv and ilp.
Abscesses develop in the kidney
Prevention and control
Nystatin (Mycostatin)
in feed candidiasis in chickens, turkeys, swine,
dogs and cats.
in mammary gland treat mastitis in cattle.
-

Amphotericin B - most effective drug systemic

-

candidiasis.

Ketoconazole and clotrimazole mucocutaneous

candidiasis.
CHAPTER 31 Cryptococcosis
Cryptococcosis

Cryptococcosis (European blastomycosis or Torulosis)

-encapsulated yeast Cryptococcus neoformans
-

Of the 19 species of Cryptococcus, only C.neoformansis

pathogenic for animals and humans.
subacute or chronic mycotic infections of man and various
species of animals involving the CNS, the respiratory
system and eye.

Host (s) Disease (s)

Dogs and cats Subcutaneous and nasal granulomas,
Meningitis, Blindnesss
Horses |Nasal granuloma
Cattle CryptocOccal mastitis
|Human Cryptococcal meningitis
 Morphology 4AIP

There are two varieties

Cryptococcus neoformans var neoformans (serotypes A and D)
Cryptococcus neoformans var gati (Serotypes B and C).
C. neoformans- member of the fungi imperfecti.
Filobasidiella neoformans teleopmorphic (sexual stage) state of
serotypes A and D
Filobasidiella bacillispora teleomorphic state of serotypes B and C.
-

C. neoformans spherical to oval, thin walled, budding yeast

mucoid polysaccharide capsule varies in thickness
in animal tissues very large, the width ofthe capsule exceeding the
d.m. of the parent cell.
Daughter cells single, bud from the parent cell by a thin neck.
Gram positive.
Lactophenol cotton blue or nigrosin stains demonstrate the spherical
budding yeast surrounded by a capsule.
 NAIP

C. neoformans is a spherical to oval, thin walled, budding yeast that

varies greatly in d.m, surrounded by a mucoid polysaccharide capsule
Natural habitat

Present in skin, mucous membrane and intestinal tract of

normal animals and birds and in dust.
Faeces of birds (particularly pigeons) and soil
contaminated by avian excreta - reservoir of types A and D.
Pigeon -not infected
concentrated in pigeon faeces -high content of creatinine.
creatinine inhibits many other micro organisms but can be
utilized by C. neoformans.
survive in pigeon droppings for more than a year.
C. neoformans has a worldwide distribution
Cultural characters NAIP

Grow well on blood agar or on SDA (without cycloheximide).

&Streaked as for bacteria, incubate aerobically at 37C for two weeks.
Chocolate agar under 5% Co2 at 37 C- demonstrate capsule
C. neoformans can grow at temp. up to 40 C.
Colonies smooth, moist, shiny, white and become wrinkled, cream
to brownish granular colonies on further Incubation
Bird seed agar (which contain Guizotia abyssinica seeds)
selective medium for C. neoformans.
Contains di and polyphenolic compounds.
Incubated at 37 C for at least a week.
Cryptococcus species use creatinine and produce melanin-pigmented
(brown) colonies.
C. neoformans is highly urease positive.
Pathogenesis

Infections are exogenous and are usually acquired by

inhalation
Animal to animal transmission is not known to occur.
Infection is usually localized in the nasal cavity or paranasal
sinuses and later extension to the brain and meninges.
Virulence is largely associated with the antiphagocytic
and immunosuppressive capsule and a unique enzyme
diphenol oxidase.
*Lesions resemble myxomatous neoplasm; infection of the
meninges resemble tubercular meningitis.
Infections extend to the optic nerve resulting in blindness.
Subcutaneous granuloma in cervical or pedal regions.
Symptoms
In cows-cryptococcal mastitis
In mild infections no clinical signs except swelling of the
affected glands.
In severe infections- fever, swelling and firmness of the udder.
Milk secretion gradually diminishes.
Milk will appear grey, white, highly viscid and mucoid.

In dogs
Itaffects the CNS, causing incoordination, hyperaesthesia and
nasal discharge.
Subcutaneous granulomos seen around the ear, face and feet.

In cats
chronic nasal and ocular discharge, granulomas and blindness
Diagnosis NAIP

Specimens
CSF, lesions or exudates, mastitic milk, biopsies and tissues
1. Direct microscopy
Demonstration of budding yeast with a large capsule by India ink,
Nigrosin and LPCB staining methods.
2. Histological sections stained by the PAS-haemotoxylin stain- stain
outline the yeast cell but not the capsule- appear as a clear area
surrounding the celI.
3.Based on isolation and identification: production of brown pigment on
birdseed agar.
4. Based on urease production
5. By Animal inoculation
Mice intra cerebral or intra peritoneal die within 4 days to two
weeks.
Gelatinous lesions in abdominal cavity and lungs.
Budding encapsulated yeast demonstrated from the lesions.
-

6. By serology Slide LAT,tube agglutination, CFT, ELISA & IFAT

Treatment

Amphotericin B isthe drug of choice.

ketoconazole and fluconazole
CHAPTER 32 Classical histoplasmosis
Classical histoplasmosis NA

Chronic, granulomatous disease

-Histoplamsa capsulatum var capsulatum
Synonyms Small form histoplasmosis, Daling's disease,
Reticuloendothelial cytomycosis.
Habitat
Heavily concentrated in the feces of black birds, sea gulls, starlings and
pigeons
Bats are also important vectors of this disease.
soil in bat caves, bird roosts, chicken houses and silos
Morphology
Facultative, intracellular parasites of macrophages.
small, oval yeast cells with orwithout buds.
Daughter cells are attached to mother cells by a narroW attachment point.
Yeast cells are relatively small (2-4 um).
>A clear halo is seen around darker-staining central material.
Cultural characters
Sabouraud dextrose agar with chloramphenicol but
without cycloheximide at 25° C
Brain heart infusion agar with blood at 37 C
At 25° C, the mycelial phase has two types of conidia
small, round microconidia, large thick-walled,
macroconidia with knob-like projections.
Yeast phase grows at 37° C on blood agar.
Pathogenesis
NAIP

Inhalation and ingestion of spores. Infections subclinical or benign.

Dogs are particularly susceptible
Also in cattle, cats, swine, horses, sheep, and wild animals.
Not reported in birds.
Not contagious.
Following inhalation of spores, macrophages phagocytize the organisms
and an inflammatory response ensues.
Fungus is either killed, or local granulomas form with calcification.
Host immunity and the number of spores inhaled determine which form
the disease manifests itself as.
Macrophages may carry the organisms to various body sites and actually
help to disseminate it - disease of the reticuloendothelial system.
Enlargement of the liver and spleen, and nodules on the tongue, ocular
involvement, and abortion
Clinical signs
Basically pulmonary.
Asymptomatic or benign and self limiting.
Fever, night sweats, weight loss and hemoptysis.
In disseminated cases hepatomegaly and splenomegaly
develop, with anemia and leucopenia.
Chronic pulmonary infections associated with cough,
dyspnea, chest pain, hemoptysis and weight loss.
Cavities may develop in the apex or subapical regions of
the lungs.
Lesions
Epithelioid and giant cell granulomas of the
lung are charactersitic.
Invasion of cells of the reticuloendothelial
system in the adrenal glands, bone marrow,
gastro intestinal tract, liver, lymphnode and
spleen.
Lesions tend to become calcified.
Diagnosis

Specimens: CSF, biopsies, bone marrow, lymph nodes, or

buffy coat - Gomori methanamin or Periodic acid-schiff
stains -organisms within macrophages.
Yeast cells are relatively small. A clear halo is seen
around darker-staining central material.
Skin test.
Complement fixation test and immuno diffusion test
- confirmatory test
A latex agglutination test and fluorescent antibody test
screening tests.
Treatment

Amphotericin B is the drug of choice

Ketoconazole
Sulfonamides
Ethyl vallinate.
CHAPTER 33 Epizootic lymphangitis
Epizootic lymphangitis
Histoplosma capsulalum and H. farciminosum

*Cord-like appearance of the subcutaneous lymphatic vessels,

especially of the limbs, neck and chest
Development of a series of pyogranulomas, discharge from
which contains yeast-like cells. Rarely, pneumonia and
conjunctivitis.
H. farciminosum Rivolta (1873)- first demonstrated in pus
-

Tokishiga (1896)- first pure cultures

Yeast double- contoured oval or ovoid body.
Saprophytic stage is mycelia form
Cultural characteristics NA

Grows slowly when the yeast phase is grown on media rich in

protein and in an atmosphere enriched with CO2.
Sabouraud's dextrose agar enriched with 2.5% glycerol
*Brain heart infusion agar enriched with 10% horse blood
Nutrient agar supplemented with 2% dextrose
Growth is very slow and appears after four to eight weeks of
incubation at 25°C.
Colonies of the mycelial form yellowish/light brown to deep
brown, convoluted, waxy and cauliflower-like.
Conversion of the mycelial form to the yeast form achieved by
incubating at 35°C to 37C.
Complete conversion to the yeast form is achieved only after four to
five repeated serial transfers onto fresh media every eight days.
Biochemical characteristics

Catalase positive
Urease positive
Assimilation of ammonium sulphate as the sole
sOurce of nitrogen.
Not ferment sugars
Liquefy gelatin
Reduction of nitrate occurs
Resistance

Resistant to physical and chemical agents

survive for at least a month in the dust of stables.
Viable and virulent after desiccation in the laboratory for 25
months.
Survive for up to ten weeks in non-sterile water at 26°C.
 Pathogenesis
Horses, mules and donkeys
Horses under six years of age are most susceptible.
Mode of transmission direct and indirect contacts
Incubation several weeks to six months.
Initial invasion of the skin- thro' lymphatic vessels to the
regional lymph nodes- internal organs.
Skin nodular and chronic suppurating lesions in overlying
lymph vessels and nodes.
Mucosal lesions confined to the upper respiratory tract and
eyes.
Nasal infection with mucopurulent discharge containing large
numbers of the fungus.
Clinical signs

Cutaneous form
30pen granulomatous wound along the course of a lymphatic
vessel, ulcerate, or undergo alternating periods of discharge
and closure for some weeks before healing with residual scar
formation.
esions -
most common in forelimbs, chest wall and neck. In
severe cases, skin over the entire body may be affected.
Lesions begin as indolent, chancre-like papules, becoming
larger over the course of weeks, and eventually form irregular
pyogranulomatous nodules, which frequently ulcerate.
Mortality - 10% to 15%.
Clinical signs contd..

Ophthalmic form
Conjunctivitis or a naso-lachrymal infection.
Rarely becomes generalized.
watery discharge from one or both eyes, swelling of the
eyelids, development of papules, ulcerating button-like
growths on the conjunctiva and/or on the nictitating
membrane
Clinical signs contd..
Respiratory form
Lesions mostly confined to upper respiratory tract.
On the nasal mucosa, the lesions begin as yellowish
papules or nodules and these soon form crater-like
granulating ulcers that bleed easily.
Lesions are usually found near the external nares and in
lungs.

Asymptomatic carriers
dentified clinically by the identification of fibrocalcific skin
lesions at previous sites of infection.
Positive result to an intradermal sensitivity test
Positive reactions to serological tests.
Lesionss
Pyogranulomas
Purulent discharge of thickened superficial lymphatic
vessels
Enlargement of regional lymph nodes.
Histopathologically a typical granulomatous tissue
reaction occurs with a predominance of the large
macrophages, many of which contain oval organisms in
the cytoplasm.
Tissues stained by Gram stain -ovoid double-contoured
yeast-like cells.
Periodic-acid Schtff or Gomori's methanamine silver stains
very useful to demonstrate the presence of the
organisms.
Typical nodules of liquefied foci in the pleura, spleen, liver
-

and bone marrow.

Diagnosis
Direct smear examination and culture technique
Serological tests
Electron microscopic examination
Animal inoculation
Intra dermaltest

Differential diagnosis
Glanders
Strangles
Ulcerative lymphangitis
Sporotrichosis
Treatment
10% Sodium iodide, 100ml, ilv, repeated weekly for four
weeks.
Amphotericin B @ 0.2 mg/kg body weight,iv, three
times on alternate days.
Remove the scabs cleaned daily with an iodine solution
for seven days.
Griseofulvin combined with iodides and local surgical
treatment.
Surgical treatment opening the nodules and packing
-

with gauze soaked in 7% tincture of iodine.

CHAPTER 34 Coccidioidomycosis
Coccidioidomycosis
Benign, inapparent, or mildly severe upper respiratory
infection that resolves naturally. On occasion, the disease
may become an acute or chronic, disseminating, fatal
mycosis.

Eoccidioides immitis- most virulent fungal pathogen.

Dust storms increase the incidence of disease.

Dogs most frequently affected

Cats, swine, sheep, cattle, human and nonhuman
primates, and some 30 species of non-domestic mammals.
Morphology NAIP

In the soil
C. Immitis slender septate hyphae that
-
give rise, on
thicker secondary branches, to chains of infectious
arthroconidia.
Arthroconidia bulging, thick-walled cells, separated by
empty cells, through which breaks OCCur when
arthroconidia are dispersed.
In tissue
Arthroconidia spherical sporangia with bifringent
walls, "spherules", which by internal cleavage produce
several hundred "endospores"
walls disintegrate, allowing dissemination of endospores,each
of which may repeat the cycle or, on a nonliving substrate,
give rise to mycelial growth.
Endospores can experimentally initiate disease.
Sexual spores are not known.
Cultural Characteristics NAIP

Sabouraud's or blood agar at 25° C or 37° C moist

white colony develops that later is covered with a fluffy
mycelium.
Haemolysis on Bovine blood agar
Arthroconidia produced in 5 to 7 days.
-

LPCB staining Thick-walled, barrel-shaped arthroconidia

alternating with empty disjunctor cells is characteristic
lsolate can be reconverted to the sporangial phase by
animal inoculation or cultivation in a spherule medium.
Sporangial phase produced at 40°Cin media
containing casein hydrolysate, glucose, biotin, glutathione,
and a salt mixture.
Resistance

Arthroconidia resist drying and tolerate heat and

saliniity.
In summer heat, C. immitis survives in soil layers
nearer the surface than its competitors.
When conditions favor growth again after rains,
C. immitis repopulates the superficial soil layers
first, ensuring its widespread dispersal.
Pathogenesis
Inhalation of dust with arthrospores.
proteases, T suppressor cell activator, and leukotactic
agents
Leukocytes in-vitro encourage arthroconidial
metamorphosis to spherules.
Serine protease liberated during the growth ofthe fungus
in-vivo (digests elastin, collagen, and immunoglobulins).
Cell-mediated immune responses arrest the process at this
stage following stimulation of TH -1, lymphocytes that
activate macrophages.
With inadequate cell-mediated immunity, dissemination
can occur to bones, skin, abdominal viscera, heart, genital
tract, and eye (and rarely to brain and meninges).
Symptoms
Young Boxer dogs and Doberman pinschers - highly
susceptible
Male dogs - Highest prevalence - 4 to 7 years of age.
Pulmonary disease asymptomatic, symptomatic of
variable degree, benign and chronic, or progressive.
Dissemination dog and human, depends on host
resistance & level of exposure.
cough, fever, lameness due to bone involvement, or
discharging sinuses from deep lesions.
Cattle, sheep, and swine - asymptomatic, limited to lungs
and regional lymphnodes and undiagnosed until slaughter.
 Lesions
White granulomas varying from miliary nodules to irregular
masses.
Peritoneal, pleural, and pericardial effusions
SystemicC disease meninges, bones, joints, and
subcutaneous and cutaneous tissues.
Lesions lung, brain, liver, spleen, and kidney.
Acute cases - burrowing abscesses are common.
Chronic -
focal and suppurative granulomatous lesions are
common without caseation or calcification.
Severe, disseminated disease only in dogs and humans.
Cattle and swine - disease restricted to a few tuberculous-like
lesions in the lymph nodes and sometimes the lung.
Diagnosis
Based on Direct Examination of Specimens
Animal fluids and tissues are examined for spherules by wet
mount in saline containing 10% KOH.
Endospores -hematoxylin and eosin & Gomori methanamine
silver stain

Spherules

Based on Culture
Mycelial growth evident within a week presence of
-

arthroconidia in a lactophenol cotton blue wet mount

Based on serological test
Coccidioidin skin test, immunodiffusion test, complement
fixation test, latex agglutination test, and tube precipitin test.
Treatment

Amphotericin B is the drug of choice.

Nystatin and Ketoconazole are also effective.
CHAPTER 35 Aspergillosis
Aspergillosis
N

Primarilya disease of the respiratory system

characterized by inflammatory, granulamatous,
necrotising lesions.
Haematogenous spread of the organism leads to
lesions in eye, skin, meninges and respiratory tract.
Mainly caused by A.fumigatus, A.flavus and A.nige.

Host (s) Disease (s)

Avian species Acute Aspergillosis
Chicken, turkey, ducks, (Brooder's pneumonia)
pigeon, quails Chronic Aspergillosis
Bovine Abortion, pneumonia and mastitis
Ovine Pneumonia, and abortion
Horse Abortion and diarrhoea
Dog Ear and nasal infection
Cat Fatal pulmonary aspergillosis
Natural habitat

Play an important role in decomposition process in soil and

elsewhere.
Saprophytic
Prolific spore producers
Airborne dissemination of their spores contributes to their
ubiquity.
common contaminant of laboratory.
Pathogenic form recovered from the skin and URT of
healthy animals and also from air, dust, straw, hay, soil,
litter, faeces, old nesting materials and dirty incubators etc.
Occurrence and spread is more in high humidity and
moderate temperature.
Cultivation
Grows very well in ordinary Sabourauds Dextrose Agar with
chloramphenicol.
Cycloheximide should never been incorporated in the
media.

Characters A.fumigatus A.niger A.flavus

Macroscopic Velvetty or Wooly. At first Velvetty, yellow to
morphology of powdery. white to yellow green or brown
the colony At first white then and then turning Reverse: Red brown
turning to dark to dark brown to
bluish green. black.
Reverse of the Reverse: white to
colony will be yellow
white to tan
|Morphology of Single, usually Double, cover Single and double,
conidiospore one on upper half entire vesicle cover entire vesicle,
and sterigmata of the vesicle, form radiate point out in all direction
parellel to axis of head.
stalk.
Aspergillosis: Fungal growth
NAIP

On SDA, Growth of Aspergillus parasiticus On Czapek dox agar, colonies are granular, flat,
Velvetty or powdery at first white then often with radial grooves, yellow at first but
turning to dark bluish green. quickly becoming bright to dark yellow-green with
age
Aspergillosis
NAIP

Conidiophore of A. flavus with Conidial head of A. flavus

conidium-bearing cell.
Pathogenesis

Predisposing factors - prolonged antibiotic treatment and

steroidal treatment.
Inhalation and ingestion of considerable numbers of spore
result in infection.
Ability to penetrate the egg and infect the embryo
(embryopathy in incubation).
Causes pneumomycosis in newly hatched chicks
weak chicks and die few days after hatch.
Very short incubation period.
Avian aspergillosis

Acute Aspergillosis (brooder pneumonia)

High morbidity and mortality in very young chicks.
loss of appetite, high temperature, listlessness,
foetid diarrhoea, convulsion and die with in 24-48hrs
of the onset of symptoms.

Chronic Aspergillosis
Individual adult birds orfew birds in a flock.
Mild Symptoms: anaemia, yellowing of faeces and
the presence of respiratory rattle.
Aspergillosis

Aspergillus infection in
a young duck

A. fumigatus growing in air sacs of a hen

Brooder pneumonia JP

Granulamatous
nodule in lung
Aspergillosis in cattle
NAl

High humidity and temperature encourage the growth

of molds when hay and straw are stored and this
constitutes the source of infection for cattle.
A. fumigatus is the primary cause of mycotic abortion, but
other Aspergillus species A.flavus, A.nidulans, A.niger,
A.terreus and A.versicolor also are found to be associated
with abortion.
Infections by inhalation into lungs or by ingestion
Carried to the placenta in the blood stream from lesions in
the respiratory tract or ulcers, mycotic ruminits or other
lesions of the digestive tract.
Slowly developing fungal placentitis (one to two months)
and interfere with the nutrition of the foetus, resulting in
foetal death and abortion.
Chronic form lead to purulent vaginitis, cervicitis and
endometritis, results in infertility.
Aspergillosis in cattle
NA

Abortion- 6-7 months of gestation.

Aborted foetus -
discrete, raised ringworm type lesions
on the skin of head and neck.
Thickened, haemorrhagic, odematous and necrotic placenta.
Grey cotyledons, inter cotyledonary area will be leathery, grey
and tan in color.
Lung and airsac grayish or yellowish gaseous exudates with
mycelia are commonly seen.
Organism colonise the bronchi, forming a compact spherical
Colony fungus ball.
Fungal balls produced mostly by A.niger than A.fumigatus.
-

Characteristic nodular lesions in alimentary canal, kidneys

and ovaries
Diagnosis
Direct microscopic examination.
Wet mount preparation of sputum, nasal discharge, milk,
uterine discharge, fetal stomach contents.
Confirmed by isolation of pathogens from the stomach
contents of the aboted fetuses and placenta.
Demonstration of fungal hyphae in the foetal tissue (By
using 10% KOH or LPCB)
Demonstration of pattern of condiospore and sterigmata by
slide culture method.
Animal pathogenicity test
Allergic test.
For confirmatory diagnosis
1. Repeated isolation
2. Absence of any other pathogen
3. Recovery from unexposed tissue and demonstration of
hyphae.
Prevention and Treatment
Aspergillosis can be prevented by
Reducing spore exposure to the animals
Removal of potential source of spore contamination
Maintain stress free environment
Prophylactic use of 5-fluorocytosine inhibits inhaled spore
germination.

Treatment
Amphotericin-B, 5-fluorocytosine -fungistatic,
administerd orally inhibit spore germination.
-

Ketoconazole effective against cutaneous and gastro

intestinal Aspergillosis.
CHAPTER 36 Mycotoxin and mycotoxicoses
Mycotoxin and Mycotoxicoses NAIF

Fungal toxins - mycotoxins

Diseases produced by fungal toxins - mycotoxicosis

History
Epidemics of ergot poisoning - Claviceps purpura recorded
-

in middle ages- ergotism identified in 1875.

During 1942 1947, the disease alimentary toxic aleukia
(ATA)Outbreak - consumption of mold cereal grains.
First animal mycotoxicosis in East Anglia, England in 1960-
more than 1,00,000 turkey poults died of an unknown disease
(Turkey X disease).
Groundnut meal revealed the presence of Aspergillus flavus
mycelia and the toxic metabolites by thin layer
chromatography (TLC) called aflatoxins.
-
General features of mycotoxin formation
P
Over 100 species of toxigenic fungi elaborating mycotoxins.
Same mycotoxin can be produced by different fungi and the same fungus
can produce different mycotoxins.
Toxin production moisture, temperature, suitability of substrate and
appropriate oxygen tension.
optimum conditions for toxin production are relatively specific for each
fungus.
e.g. Fusarium - at freezing temperature,
A. flavus at 25°C.
susceptibility of different crops to mould infection is governed by the
presence of suitable substrates.
*Damage to the seed coat by insects, mechanical harvesting, severe frost or
other factors may predispose crops to fungal attack. Insects may also serve
as carriers of fungal spores.
*Field fungi invade the grains before harvest and required greaterwater
activity for growth e.g. Fusarium, Helminthosporium and Cladosporium
Storage fungi invade the grains after harvest during drying and in storage
e.g. Aspergillus, Penicillium
Mycotoxins

Species Toxins
A.flavus and Aflatoxins
A.parasiticus
A. ocheraceus Ochratoxin
Fusraium roseumTrichothecane (t-2) toxin
Pencillium citrinum Citrinin
A.nidulans and Sterigmatocyosin
A.versicolor
 Characteristics of mycotoxins
Greek word 'mykes' meaning 'fungus' and the Latin word
toxicum' meaning 'poison'.
Group of compounds produced by some strains of certain fungi
that cause illness or death when ingested by man or
animals.
low molecular weight, non-antigenic, heat stable secondary
fungal metabolites.
activate at low concentrations.
wide spectrum of toxic effects, like carcinogenic, mutagenic,
teratogenic and immunosuppressive.
Acquired immunity does not occur following exposure.
Each toxin affects specific target organs or tissues
Target organs/ tissues Toxins
Vascular system Aflatoxins
Digestive system Aflatoxins
Mucous membrane Trichothecane (t-2) toxin
Urinary system Ochratoxin
Reproductive system Zearalenone (Fusarium toxin)
Cutaneous system Sporidesmin
Mycotoxicosis NAIP

Determined by several factors

Species of toxigenic fungus
Concentration of mycotoxin in the food
Age, sex and health status of the exposed animal
*Target organs or tissue affected
Duration of exposure to contaminated feed.

Mainly by ingestion, inhalation or direct skin contact. Mycotoxicosis occurs in

three forms.
1. Acute primary mycotoxicosis
Due to high to moderate amounts of mycotoxins are consumed.
Marked signs of disease or death.
2. Chronic primary mycotoxicosis
Due to moderate to low levels of mycotoxin intake.
Reduced productivity, reproductivity and inferior market quality
3. Secondary mycotic diseases
Due to intake of very low concentration of specific mycotoxins
impairment of immune system results in predispose to infectious
diseases Salmonellosis, candidiasis and coccidiosis.
Clinical features of mycotoxicoses NAIE

Not transmissible to incontact animals.

Outbreaks are often seasonal and sporadic, associated with
certain batches of stored food or particular types of
pasture.
decreased growth rate or immunosuppression
antibiotics are usually ineffective.
Recovery depends on the type and amount of mycotoxin
ingested and the duration of the exposure to contaminated
feed.
laboratory demonstration of mycotoxins in suspected food,
or in the tissues, secretions or excretions of affected
animals.
Characteristic lesions in target organs of affected animals
are important supporting diagnostic evidence.
Aflatoxicosis
Name aflatoxin derives from Aspergillus flavus toxin.
Toxic compounds produced by A. flavus and A,parasiticus during growth
on a variety of cereal grains and food stuffs such as maize, cotton seed
and groudnuts.
High humidity and high temperature during pre-harvesting, harvesting
transportation and storage as well as damage to feed crops by
insects, drought and mechanical injury during harvesting favours the
growth and toxin production of Aspergillus flavus.
moisture content of the substrate greater than 15%, temp.25°C and
adequate aeratio favours toxin production.
-

They are bisfuranocumasin compounds with toxic, carcinogenic,

teratogenic and mutagenic activity.
Four majoraflatoxins are B1, B2, G1 and G2.
B1and B2 produce blue colour and G1, G2 - green fluorescence TLC.
M1, M2 are hydroxylated metabolites of B1 and B2 that are excreted
in the milk of lactating animals such as dairy cows.
Biological effects of aflatoxin

Acute toxicity
Hepatic injury and nervous signs ataxia and
convulsions.
Death may occur suddenly.

Chronic toxicity
Reduction in efficiency of food conversion,
depressed daily weight gain, decreased milk
production in dairy cattle
Susceptibility to inter-current infections due to
immunosuppression.
Symptoms
NAI

Young pigs, calves, turkey poults and ducklings highly

susceptible.
B1 most hepatogenic, carcinogenic, teratogenic and embryotoxic
effects
Signs in calves blindness, circling. grinding of teeth, diarrhoea,
tenesmus and convulsions.
Sheep are highly resistant.
In dairy cattle M1 and M2 are excreted in the milk.
In pigs - drowsiness, inappetence, jaundice, weight loss and yellow
urine.
Ducklings -most susceptible avian species to aflatoxins.
Signs include anorexia, poor growth rate, ataxia and opisthotonus
followed by death.
In birds over three weeks of age, subcutaneous haemorrhages of
legs and feet.
Lesions NAIP

Principle target organ is liver.

Depending on the severity of intoxication,
hepatomegaly with necrosis, marked bile duct
hyperplasia will occur.
Acute hepatic failure and massive haemorrhage
due to impaired blood clotting, increased capillary
fragility leading to death may occur with higher
doses
In chronic toxicity, in addition to liver damage,
degenerative changes in the kidney, thymus
cortical aplasia leading to decreased cell mediated
immune response.
Diagnosis NAIP

Chemical identification in food samples and biological assays

for toxicity- confirmatory.
Demonstration A. flavus and A. fumigatus and of potentially toxic
levels of mycotoxins in the food, tissues, secretions helpful for
diagnosis.
Concentration of aflatoxin B1 in excess of 100ug kg of feed are
considered toxic for cattle.
Thin layer chromatography and HPLC more sensitive for
determine aflatoxins levels in the food.
Radio immuno assay and ELISA.

Biological assays
Ducklings. Bile duct proliferation in one-day-old ducklings and
chick embryo bioassay are highly useful.
Control and prevention

Prevention of contamination at all stages of food

production, storage and use preferred
method of preventing aflatoxicosis.
Physical removal and chemical treatment of
aflatoxin contaminated feeds such as acids,
alkalies and aldehydes,
Degrading aflatoxins with oxidizing agents of
selected gases ammonia
High affinity inorganic compounds benzoic
and propionic acid widely used as
preservatives for stored agricultural products.