Showing posts with label denticles. Show all posts
Showing posts with label denticles. Show all posts

Sunday, 10 May 2015

Sporothrix schenckii Complex ("Revisited")



Sporothrix schenckii Complex –Hyphomycetes  (Dimorphic Fungus)

Note1: I first added a post entitled Sporothrix schenckii back in November of 2008 while bedbound, recovering from a serious injury.  I had just discovering ‘blogging’ and toyed with the idea of posting a few film photos that I had tucked away in a drawer – just for the fun of it.  The poor quality photo demanded it be upgraded once I decided to keep up my blog ‘Fun with Microbiology’.  Finally, here is the upgraded blog post, perhaps more accurately entitled Sporothrix schenckii complex Revisited.

Note2:  In recent years, gene sequencing studies have revealed that the species previously known as Sporothrix schenckii is actually composed of several species.  The currently accepted species names of the species which comprise the complex are as follows:  S.albicans (formerly Sporothrix pallida), S.brasiliensis, S.globosa, S.luriei, S.mexicana, and S.schenckii , ‘sensu strictu’.  As many clinical laboratories may not have routine access to molecular technology for specific speciation, this blog simply describes Sporothrix schenckii complex with macroscopic, microscopic and physiological features found in most current textbooks.

Ecology:
Sporothrix is a cosmopolitan (found just about everywhere) fungus which is commonly isolated from soil and decomposing plant matter.  Peat moss is a particularly well known source of Sporothrix.  It may also be found on living plants such as rose bushes leading to what has been termed ‘rose handler’s disease’ where the fungus gains entry to the host through thorny pricks.


Pathogenicity:
Sporothrix schenckii is the agent responsible for sporotrichosis, a chronic infection that most frequently begins as a skin puncture with introduction of the fungus into the subcutaneous tissues.  Eventually it will involve the lymph nodes and lymphatic channels that drain the infected area.  Implantation of the fungus is usually by puncture by items contaminated with plant material harbouring the fungus such as wood splinters, sphagnum moss, hay or thorns as previously mentioned.  Pulmonary (respiratory) infection may also develop in predisposed individuals after inhaling fungal spores.  Rare cases of disseminated Sporothrix infection with a fatal outcome have been reported.
Laboratory acquired infections have also been reported.

Sporothrix schenckii is a thermally dimorphic fungus, meaning it can take on one of two forms depending on the temperature it finds itself in.

·         At 25ᵒC exhibits the mould (filamentous) form, with a glabrous, moist texture.  Initially white or cream coloured, the colony may acquire a black colour with aging
·         At 37ᵒC exhibits a yeast-like form, with a creamy texture, cream to beige in colour.

Macroscopic Morphology:
The mould or filamentous phase in greater detail:
Growth is described by most sources s moderately rapid to rapid, with the colony becoming mature within 7 days.
The filamentous phase grown on SAB or PDA is generally cream coloured with some sources describing orange to orange-grey colouration.  As the fungus matures, a salt & peppery brown or black colour develops with the colony retaining a narrow whitish border.  Isolates may vary in their colour, some being dark/black from initial growth.  Stock cultures kept for long periods may lose their dark colour completely.
The reverse of darkly pigmented colonies is usually dark in the center with a progressively lighter periphery.
Sporothrix initially has a moist appearance but becomes wrinkled and leather to velvety in texture as it ages.

Sporothrix schenckii - SAB, 30ᵒC, 3 weeks incubation (Nikon)

The Yeast Phase:
The yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).   At 37ᵒC, Sporothrix exhibits a yeast-like form, with a creamy texture, cream to beige in colour which also may darken with aging.
(Yeast Phase Photo further below)

 

Microscopic Morphology:
Fillamentous Mould Phase:
Sporothrix produces narrow (1 – 2 µm dia.) hyaline, septate and branching hyphae.
Sporothrix produces two types of conidia:
·         Slender, tapering conidiophores arise at right angles from undifferentiated hyphae.   Hyaline conidia are produced at a small swelling at the conidiophores apex by sympodial growth resulting in a “rosette-like” appearance.  These conidia (2 – 3 X 3 – 6 µm) are tear-drop shaped to round in appearance and unless disturbed, remain attached to the conidiophore in young cultures via thread-like denticles. (Rosette)
·         Single thick-walled brown to black (dematiaceous) sessile conidia (2 – 4 µm dia.) can also be present, arising directly from the hyphae.

 Sporothrix schenckii -Initial look at the growth attached to a cover slip from a slide culture.
(100X, LPCB, DMD-108)

Sporothrix schenckii - A closer look where detail begins to emerge.
(400X, LPCB, DMD-108)

Sporothrix schenckii - ditto
(400X, LPCB, DMD-108)

Sporothrix schenckii - At yet a higher magnification, fine structures emerge. Conidia are easily seen at the tips of conidiophores.  At the center of this photo one such structure is seen in a 'rosette' arrangement, typical for this fungus.
(1000X, LPCB, DMD-108)

Sporothrix schenckii - hyphae bearing conidiophores with conidia being produced sympodially at the apex.  (1000X, LPCB, DMD-108)

Sporothrix schenckii - appearing somewhat in 3-D, the hypha is seen running across the photo with conidiophores extending at right angles from the hypha.  Conidia formation is is seen at different stages with the conidiophore at the right having accumulated numerous conidia.
(1000X, LPCB. DMD-108)

Sporothrix schenckii - again in "3-D" perspective, you can see a number of hyphae below, and out of the plane of focus of the camera, bearing conidiophores which appear to be rising upwards, towards the camera/viewer.  Conidia are present at the apex.
(1000X, LPCB, DMD-108)

Sporothrix schenckii -septate hyphae are show and two well defined conidiophores are shown with a 'rosette' of conidia at the apex.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -another look at the conidiophores with conidia accumulated at the apex (tips).  Fine thread-like denticles can be seen attaching the ellipsoid or tear drop shaped conidia to the conidiophore.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -yet another view, as above.
(1000+10X. LPCB, DMD-108)

Sporothrix schenckii -what is meant as a "rosette" configuration of conidia.  This is typical of Sporothrix schenckii and assists in its identification.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - I particularly like this photo.  Like a shower of delicate flowers.  What is seen are the conidia rosettes at the tips of unseen conidiophores and hyphae, below the plane of focus.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -a single hypha running through the photo, bearing conidia in typical rosette pattern.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -single, sessile and begining to show dark pigment (dematiaceous), are seen along the hypae.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -ditto.  Black pigment much more pronounced in this photo.
(1000X, LPCB, DMD-108)

Sporothrix schenckii - again, as above.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - just another photo...
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -Sessile (attached directly to hypha) with many developing a dark pigmentation (dematiaceous) which gives the macroscopic colony its distinctive colour.
(1000X, LPCB, DMD-108)

Sporothrix schenckii - dematiaceous cells are more abundantly produced as the colony ages.
(1000X, LPCB, DMD-108)

Sporothrix schenckii -sessile dematiaceous conidia are seen lining a hypha running through the photo, as well as some conidiophores bearing the tear-drop shaped conidia.
(1000X, LPCB, DMD-108)

Sporothrix schenckii -another view of the sessile, dematiaceous conidia lining the hypha with a few conidiophores extending from the hypha showing the delicate rosette arrangement at the tips.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -and just another photo showing what was described in the last few photos.
(1000X, LPCB, DMD-108)

Sporothrix schenckii -a nice rosette formation in the center of the photo.
(1000+10X, LPCB, DMD-108)

The Yeast Phase:

As previously mentioned, the yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).  Yeast cells are round to ovoid of varying size (1 – 3 X 3 – 10 µm), producing single or multiple buds often resembling rabbit ears or ‘Mickey Mouse’ ears extending from the primary yeast cell.

 Sporothrix schenckii -the very same organism added to the nutritionally rich Brain-Heart Infusion (BHI) agar and incubated at 37ᵒC for ten days.  A typical pasty, yeast form develops which is why this organism is 'dimorphic'. (Nikon)

Sporothrix schenckii -yeast cells seen in a suspension in Lactophenol Cotton Blue.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - Sporothrix in the yeast phase may show single yeas like cells with some of the population budding with typical "rabbit ears" or "Mickey Mouse" ears.  They appear as two elongated cells projecting from the same surface of the parent cell.  One can be seen here, one-third in from the center left edge of the photo.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -"Mickey Mouse' ear configuration in the cell at the upper center.
(1000X, LPCB, Nikon)

Sporothrix schenckii -again, yeast cells with two attached daughter cells on the same side giving the apearance of "rabbit ears" or "Mickey Mouse" ears.
(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -ditto (as above) -inset -floppy 'rabbit ears"
(1000X, LPCB, DMD-108)

Sporothrix schenckii - that's it, I'm done!
(1000+10X, LPCB, DMD-108)


Physiology:
Sporothrix is resistant to cycloheximide; however growth is inhibited at temperatures of 39 - 40ᵒC.

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Monday, 23 February 2015

Ochroconis species



Ochroconis species (Hyphomycete)  Fungus

Ecology:
Ochroconis species are primarily soil saprobes (live on decaying vegetative matter), found in the soil worldwide.   As of 2014, there are thirteen recognized species of Ochroconis.

Pathology:
Ochroconis species have been recovered from central nervous system (CNS) infections as well as pulmonary (lung) infections, from both immunocompromised and immuocompetent hosts.  In particular, Ochroconis gallopava is considered to be a neurotropic opportunist and proposals have been made to place this fungus into a new genus, Verruconis. 
Ochroconis species are considered to be mesophilic (preferring moderate temperatures) however they can cause disease in several species of cold-blooded animals, particularly fish such as coho salmon and rainbow trout.  Ochroconis species are known to cause encephalitis in chickens, turkeys and other fowl.

Macroscopic Morphology:
The rate of growth is rather slow growing as measured by the expanding colony but will mature to produce conidia usually within 5 days.
The texture is described as velvety to felt-like or floccose.
The colony colour is usually a reddish-brown to chocolate brown to a dark olive-grey.  The reverse is a dark brown to black.
A red to brown pigment may diffuse into the medium.

 Ochroconis on Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 3 weeks. (Nikon)

Ochroconis- same organism as above but with different background and lighting to show variations in texture and pigment.  SAB, 30˚C, 3 weeks. (Nikon)



Ochroconis on SAB - colony center rises off and above the agar surface resembling and inverted shallow bowl.  Looked like a small hollow mountain! 
Below, right - shows the Reverse of the Ochroconis presented here.  The lighter section which appears in the center of the larger colony is the area that has "cupped" and lifted off of the surface of the agar.  (Nikon

Microscopic Morphology:
Ochroconis produces septate hyphae which are hyaline (clear) to pale brown in colour.
Conidiophores are also hyaline to pale brown. They arise erect and unbranched from the hyphae and usually have a knobby or bent appearance.  The conidiophores have apical denticles in a sympodial arrangement from which the conidia have formed.  Conidia (2.5 – 4.5 µm X 11 – 18 µm) are usually 2 to 4 celled, depending on the species.  Conidia are cylindrical to club shaped and after detachment from the conidiophore (denticle), an inconspicuous frill may remain on both the denticle and the conidium base.

Ochroconis species - edge of growth of slide culture as initially viewed at low magnification.  Hyphae radiating out from point of inoculation after 1 week of incubation.
(LPCB, 250X, DMD-108)

Ochroconis species - Conidia extending from hyphae now become evident at this higher magnification. (LPCB, 250X, Nikon)

Ochroconis species - as we once more increase magnification individual conidia attached to phialides can be seen in more detail.  (LPCB, 400X, Nikon)

Ochroconis species - another view with conidia attached to their phialides. Brown pigmentation seen in lower right of photo.  (LPCB, 400X, Nikon)

Ochroconis species - one more view.  Remember, the fungus grows in three dimensions and even here, within the space between a microscope slide and a cover slip, hyphae and phialides extend forward, into the photo and some backwards, out of the photo.  It is for this reason that the photos often appear to be out of focus as only those features that lie relatively flat along the focal plane appear clearly in the picture. (LPCB, 500X, Nikon)

Ochroconis species - a massive ammount of conidia with the most mature to the right where the brown pigment is most evident. (LPCB, 500X, Nikon)

Ochroconis species - edge of  slide culture (as previous)
(LPCB, 500X, Nikon)

Ochroconis species - a hypha weaves its way from top center to bottom center of this photograph.  Along its length you can see phialides with attached conidia.  (LPCB, 500X, Nikon)

Ochroconis species -a two-celled conidium is seen attached to a phialide that extends from the hypha.  The conidium shows a slight constriction near the center.
(LPCB, 1000X, Nikon)

Ochroconis species -Numerous phialides with attached conidia shown here.  Phialides seen bearing multiple conidia.  Dark pigmentation is also evident as the colony ages.  Most conidia are two-celled, some showing a slight constriction near their center, others (center) showing the furthest end of the conidium being larger than that nearest the phialide from which it originated.  The arrow points to a phialide which has lost it's conidium -a slight scare remains.  
(LPCB, 1000X, DMD-108)

Ochroconis species -a mass of darkly pigmented, septate hyphae as well as a dark blue conidium seen near the left of the photo (LPCB, 1000X, DMD-108)

Ochroconis species -Hyphae with phialides bearing conidia.  Arrows point to phialies with multiple (two) conidia, the one on the left showing the scar remaining after the conidium has detatched.
(LPCB, 1000X, DMD-108)

Ochroconis species -a three-celled conidium appears to be present along with the more numerous two-celled conidia.  (LPCB, 1000+10X, DMD-108)

Ochroconis species -center of photo, conidium attached to a long phialide.
(LPCB, 1000X, DMD-108)

Ochroconis species -Nice photo of a phialide bearing two conidia attached to the parent hypha.
(LPCB, 100X, DMD-108)

Ochroconis species -yet another photo showing much the same.  Phialides bearing multiple conidia with the arrows showing the ragged attachment points which remain after the conida have detatched.
(LPCB, 1000+10X, DMD-108)

Ochroconis species -septation within the hyphae are clearly visible as is the developing brown pigmentation.  Tree Conidia still attached to their brownish pigmented phialides which extend from the hyphae.  Near center, one phialide is seen with a detached conidium nearby.
(LPCB, 1000X, DMD-108)

Ochroconis species -Aging phialides & conidia.  Brown phialide in center of photo has a thinner denticle at it's apex to which the conidium is attatched.
(LPCB, 1000X, DMD-108)

Ochroconis species -again, septations in hyphae are clearly evident.  Conidium near center of photo appears to be supported by a bent denticle.  (LPCB, 1000+10X, DMD-108)

Ochroconis species -conidium at apex attached to phialide by a short denticle.
(LPCB, 1000X, DMD-108)

Ochroconis species - Seen more clearly here, the two-celled conidium is attached to the phialide via a somewhat wavy denticle.  The phialide, in turn is attached to the hypha from which it originated.
(LPCB, 1000+10X, DMD-108)

Ochroconis species -  Here we are looking at the conidium 'head-on' so it appears spherical.  The conidium is attatched to the hypha by this darkly pigment, and apparently degenerating, phialide-denticle structure. (LPCB, 1000+10X, DMD-108)

Ochroconis species - Almost done here.  A phialide bearing two conidia.
(LPCB, 1000+10X, DMD-108)

Ochroconis species
(LPCB, 1000+10X, DMD-108) 

Ochroconis species - Phialide bearing multiple conidia.
(LPCB, 1000+10X, DMD-108) 

Physiology:
Growth is inhibited by cycloheximide.
I'm uncertain as to which specific species I have pictured in this blog as the conidia occasionally show more than 2 cells and can be constricted in the center.  The majority of the conidia from the isolate presented here are two-celled and rather ellipsoidal or cylindrical in shape


Differentiation of the more common species:
Conidia usually 4-celled = Ochroconis tshawytschae
Conidia usually 2-celled = Ochroconis gallopava
Conidia distinctly clavate (club-shaped), with the upper cell wider than the basal cell = Ochroconis humicola
Conidia broadly ellipsoidal and constricted at the septum = Ochroconis constricta

Ochroconis constricta is not known to be pathogenic
 
A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava.

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