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Application of Immunological

Presented by

Mahmoud El-Hariri M.V.Sc (2005)

For Ph.D. degree, in Veterinary Medicine, Microbiology (Bacteriology, Immunology and Mycology)

Application of immunological

and molecular genetic methods

for diagnosis of fungi

Under Supervision of:

•Prof. Dr. Mohamed Kamal Refai

•Prof. Dr. Heidy Mohamed Shawky

• It causes cryptococcosis infections

commonly starts following

inhalation of the organism

• The invasive fungus has a

predilection for respiratory &

nervous systems of human &

animals

Cryptococcus neoformans

Human Cryptococcosis

Bovine Cryptococcosis

Canine Cryptococcosis

Feline Cryptococcosis

Pulmonary forms

• Acute

• Chronic

Disseminated forms

• CNS affection

• Cutaneous

• Mastitis (Cattle)

• Nasal infection

Cryptococcosis

• Candida albicans has predilection for

skin & mucous membrane of human

& animals.

• It may invade blood & tissue causing

systemic infection.

Candida albicans

• Opportunistic fungi of mucous

membrane of all birds and mammals

(dog, foals , cows,…..).

• It causes candidosis infections

commonly starts under stress,

antibiotic , steroids or direct contact.

Candida albicans

• Is a zoophilic dermatophyte.

• Cats & dogs are the main reservoir.

• It known as ringworm infection in

animals & tinea infection in human.

Microsporum canis

Cryptococcosis

Candidosis

Ringworm

Aim of the work

Establish effective methods for rapid

identification & differentiation of

three fungal species through:

1. Determination of molecular

genetic markers for a broad group of

fungi by using universal primers.

2. Utilizing the differences in the

lengths of ITS regions PCR

amplicon to achieve specific

identification of pathogenic fungi.

Aim of the work

3. Using a RFLP-PCR for ITS

region of rDNA as a valuable

molecular typing markers of

different fungal species.

Aim of the work

4. Using of ISSR-RAPD for the

comparative identification of closely

related fungal species.

5. Phylogenetic analysing of genetic

polymorphism intraspecies by ISSR

analysis of fungal isolates from

culture colonies.

Aim of the work

Molecular genetic markers

Tandem Repeat

Anonymous Markers

1–Minisatellites

(VNTR)

2–Microsatellites

(STR or SSR)

1-AFLPs & RFLPs.

2-RAPDs.

3-ISSRs

Experimental design

2- Conventional identification

3- Molecular typing & identification

1- Isolation of fungal elements

b. SDA with chloramphenicol .

c. SDA with with cycloheximide .

d. SD broth .

Isolation of fungal elements

a. Samples

Experimental design

2- Conventional identification

3- Molecular typing & identification

1- Isolation of fungal elements

Conventional methods

1. Indian ink stain.

2. Lactophenol cotton blue.

1. Modified tobacco agar medium.

2. Eucalyptus agar medium.

3. Pal's medium.

C. BCE on differential media :

A. Macroscopic features.

B. Microscopic features.

Conventional methods

D. Biochemical test:

1. Urea agar test.

2. Sugar fermentation.

3. Sugar assimilation.

E. Germ tube & Growth on rice agar

Experimental design

2- Conventional identification

3- Molecular typing & identification

1- Isolation of fungal elements

Molecular methods

From Cr. neoformans (Botes, 2007).

From C. albicans (Xiang et al., 2006).

From M. canis (Makimura et al., 2001).

A. DNA extraction:

B. Rapid mini-preparation DNA extraction for all fungal isolates.

Molecular methods

1. PCR amplification of ITS region.

C. Molecular typing & identification:

2. RFLP-PCR for amplicon of ITS1-ITS4

3. Microsatelliate & ISSR markers

4. Phylogenetic analysis

PCR amplification of ITS regions

•Using three universal primers

ITS1, ITS3 and ITS4.

•Analysis of fungal ITS-regions PCR

amplicons size.

What is ITS ?

Internal transcribed spacers are the

regions between the 18S gene and the 28S

gene of ribosomal DNA (rDNA).

Eukaryotic organisms have two ITS; ITS1

is located between the 18S & the 5.8S

genes, and ITS2 is located between the

5.8S & the 28S genes

NT

S

ETS

18S 5.8S 28S ITS1 ITS2

NTS: Non- Transcribed Spacer ETS: External Transcribed Spacer 18S: 18S gene of Ribosomal DNA ITS1: Internal Transcribed Spacer 1 5.8S: 5.8S gene of Ribosomal DNA ITS2: Internal Transcribed Spacer 2 28S: 28S gene of Ribosomal DNA

5́ 3́

ITS1-Pr.

ITS4-Pr.

ITS3-Pr.

ITS-Pr.1: Internal Transcribed Spacer Primer1 ITS-Pr.3: Internal Transcribed Spacer Primer3 ITS-Pr.4: Internal Transcribed Spacer Primer4

Internal Transcribed Spacer (ITS)

NT

S

ETS

18S 5.8S 28S ITS1 ITS2

5́ 3́

ITS1-Pr.

ITS4-Pr.

ITS3-Pr.

ITS1-Pr. (5-TCCGTAGGTGAACCTGCGG-3) Leaw et al. 2006

ITS3-Pr. (5-GCATCGATGAAGAACGCAGC-3) Leaw et al. 2006

ITS4-Pr. (5-GCATATCAATAAGCGGAGGA-3) Leaw et al. 2006

Internal Transcribed Spacer (ITS)

ITS2

NT

S

ETS

18S 28S ITS1

5́ 3́ 5.8S

ITS4-Pr.

ITS3-Pr.

Cr.neoformans 354 -369

C. albicans 334-346

M. canis 314 - 325

PCR amplification by ITS3-ITS4

5.8S ITS1 ITS2

NT

S

ETS

18S 28S 5́ 3́

ITS1-Pr.

ITS4-Pr.

M. canis 728- 766

Cr. neoformans 540 - 569

C. albicans 500 - 530

PCR amplification by ITS1-ITS4

Amplicon

Size Range

Amplicon

Size Range

C. albicans 334 – 346 500 - 530

Cr. neoformans 354 – 369 540 - 569

M. canis 314 - 325 728 - 766

ITS regions amplicon size

Molecular methods

1. PCR amplification of ITS region.

C. Molecular typing & identification:

2. RFLP-PCR for amplicon of ITS1-ITS4

3. Microsatelliate & ISSR Marker

4. Phylogenetic analysis

Differntiation of fungal species

(Cr. neoformans & C. albicans) by

RFLP-PCR amplicon of ITS1-ITS4

primers by:

Hae lll

Bam Hl

RFLP-PCR for amplicon of ITS1-ITS4 primer

Organism

C. (R.)

C. (I.)

Cr. n. (R.)

Cr. n. (I.)

ITS Amplicon

500 - 530

ITS Amplicon 540 – 569

Bam Hl digest

fragments

296 296 537

-----

557

----- 225 228

Hae lll digest

fragments

365 360 330 325

117 117 238 235

Why ITS is a Good Marker ?

The level of variation in this region

makes it suitable as a MARKER

for detecting genetic variation among

genera, species and within species

Molecular methods

1. PCR amplification of ITS region.

C. Molecular typing & identification:

2. RFLP-PCR for amplicon of ITS1-ITS4

3. Microsatelliate & ISSR Marker

4. Phylogenetic analysis

• It is a Simple Sequence Repeat or small

arrays of tandem repeats of 2 to 4bp in

length: di- (CA)n, tri- (CAG)n, tetra-

(GACA)n nucleotide repeats.

• Evenly distributed through genome

mostly located in intron and some in

coding region.

Microsatellite Marker

1-Produced higher levels of species

polymorphism with lower error rate

than RAPDs (Dominant Marker)

2-Universal primer no prior sequence

knowledge is required.

3-Have proven useful in plants, birds

and invertebrates.

ISSR Markers

Why we use GACA4 for ISSR ?

Molecular methods

1. PCR amplification of ITS region.

C. Molecular typing & identification:

2. RFLP-PCR for amplicon of ITS1-ITS4

3. Microsatelliate & ISSR Marker

4. Phylogenetic analysis

Depending on result of GACA4 ISSR

banding profile the similarity and

polymorphism degree between three

fungal species were used to establish

the correlative dendrogram between

fungal species

Phylogenetic analysis

1-Application of universal primers

can be used for amplification of

PCR products specific for more

than one fungal species

Conclusion

2-Simple differntiation of fungal

species by PCR-RFLP of ITS

region amplicon.

Conclusion

3-Comparitive differentiation of

more than one fungal species is

possible by universal ISSR

primer.

Conclusion

4-Serotyping of Cr. neoformans

isolates can be done by molecular

biology methods

Conclusion

5-Phylogenetic analysis of closely

related fungal species is easily

achieved by ISSR polymorphism.

Conclusion

6-It is advisable to use more than

one molecular genetic markers

for confirmation of molecular

identification of fungi.

Conclusion

Thank You