Ascaris lumbricoides

Ascaris lumbricoides (Intestinal Nematode)  “Roundworm”

Geographic Distribution:

Ascaris lumbricoides can be found throughout the world but is more commonly found in moist temperate and tropical regions.  Ascaris is increasingly prevalent where poor sanitation exists.  In cultures where human excrement (night soil) is used as fertilizer, infection may again be greater.

Pathogenicity:

Infection occurs with the ingestion of fully embryonated eggs (ova).  Infection with Ascaris lumbricoides is termed Ascariasis and it affects more of the world’s population than any other parasitic disease.   Ingestion of small numbers of eggs may be asymptomatic for the host however larger numbers may cause Ascaris pneumonitis[i] or Loeffler’s[ii] syndrome.  Those infected with Ascaris lumbricoides may develop asthmatic attacks, shortness of breath, wheezing or a persistent cough.  A heavy infection in the small intestine may result in a bowel obstruction, especially in children.  Symptoms may include fever and generalized malaise possibly with abdominal distension, associated tenderness and possible vomiting.

Life Cycle:

Ingested embryonated eggs travel to the duodenum (small intestine) where they hatch and begin an obligatory migration throughout the body before they return to the duodenum to mature into adulthood.  Hatched larvae start the migration by penetrating the duodenum wall to enter the blood or lymphatic system which eventually carries the larvae to the liver and the heart to eventually enter pulmonary circulation.  In the lungs, the larvae break free of the capillaries to enter the aveoli where they continue to grow.  After about 3 weeks, they begin to migrate through respiratory passages to reach the esophagus where they are swallowed and once again reach the small intestine.

At about two to three months post ingestion of the eggs, the now mature worms begin to lay their own eggs.  It has been estimated that mature female worms may produce an average of 200,000 eggs daily.  Ascaris lumbricoides females are oviparous[iii].  When passed, the eggs require about 2 to 3 weeks outside of the host to develop into the infective embryonated stage.  While the eggs are susceptible to excessive heat and drying, they can remain viable in moist soils for long periods of time.

Ascaris Worm:

The adult Ascaris lumbricoides worms area creamy-white in colour, occasionally with a pinkish cast.  Female worms range from 20 to 35 cm in length.  Males are generally shorter and usually do not exceed 30 cm.  Female worms are also generally thicker (3-6 mm) than the more slender males (2-4 mm).  Males can be distinguished from females by their incurved tail.  Females have a straight tail.  Adult worms are believed to live up to a year.  Ascaris worms do not attach themselves to the intestinal wall but rather maintain their position through constant movement.

 Ascaris lumbricoides: Adult male worm show with its distinctive curved tail.  This particular worm was about 24 cm or 10 in in length, (Nikon-Macroscopic)

Ascaris Eggs (Ova):

Diagnosis of ascariasis is by the demonstration of the characteristic eggs in the feces.

Fertile eggs are broadly oval in shape and typically yellow-brown in colour, stained by the bile in freshly passed stools.  They measure 45 to 75 µm in length by 35 to 50 µm in breadth.  The outer albuminoid coat is coarsely mammillated covers a smooth shell which is difficult to distinguish in laboratory preparations.  Some confusion in diagnosis may occur if the Ascaris egg has lost its mammillated coat (decorticated) as it may somewhat resemble Hookworm or Trichostrongylus species ova.   Ascaris eggs contain only one cell when passed in the feces.

Infertile eggs are elongate, about 85 – 95 µm by 43 – 47 µm in size.  Their mammillated layer may vary from being coarsely irregular to a relatively smooth layer, almost devoid of mammillations. The internal contents of infertile eggs appear as a mass of disorganized, refractile granules.

Note:  All the following photos are take from the re-suspended sediment after fecal concentration.

 Ascaris lumbricoides egg seen in the center of the photograph.

(Nikon, 250X)

  Ascaris lumbricoides egg  has a distinctive appearance.  This egg shows the rough looking surface due to the mammillated albuminoid coat.

(DMD-108, 400X)

 Ascaris lumbricoides: another photo of the egg with its rough mammillated coat.

(Nikon, 400X)

 Ascaris lumbricoides: a photo of the egg with an alternate focus inserted for comparison.  As the egg is three-dimensional, adjusting the focus can aid in visualizing the egg's structure.

(Nikon, 400X)

 Ascaris lumbricoides: as above.

(DMD-108, 400X)

 Ascaris lumbricoides: Two A.lumbricoides eggs seen in the same field.

(Nikon, 400X)

 Ascaris lumbricoides: Typical appearance of the Ascaris egg.  The presence of these eggs in a fecal specimen is diagnostic for an Ascaris infection.

(DMD-108, 400+10X)

 Ascaris lumbricoides: The thick albuminoid mammillated layer is quite evident on this fertile Ascaris egg.  (DMD-108, 400+10X)

 Ascaris lumbricoides: As above with a slightly altered focus.

(DMD-108, 400+10X)

  Ascaris lumbricoides:  Another photo of the Ascaris egg in a fecal concentrate.

(DMD-108, 400+10X)

 Ascaris lumbricoides: As above but with an alternate focus of this fertile Ascaris egg.  I post these alternate focus photos in an attempt to demonstrate the three-dimensional nature of the egg and their complexity.  (DMD-108, 400+10X)

 Ascaris lumbricoides:  Yet another example, 'cause more is better!.

(DMD-108, 400+10X)

 Ascaris lumbricoides: At a higher magnification.  The large single cell interior is clearly visible.

(Nikon, 1000X)

 Ascaris lumbricoides ova:  Okay, more is better, right?  Another photo showing the thick outer albuminoid mammillated layer.  The inner wall often cannot be visualized unless the egg loses this outer layer (decorticated).  The single celled contents are clearly visible.

(Nikon, 1000X)

 Ascaris lumbricoides:   Too many photos?  I have to do something with them!  Another photo of the Ascaris egg however it appears as if the interior cell has divided into four or more individual cells.

(Nikon, 1000X)

 Ascaris lumbricoides: An Ascaris egg with the inset enlargement of the Ascaris egg.  There appears to have been a number of divisions of the original cell contained within the egg.

(Nikon, 250X)

Too many photos?  Just a few more to go..

While the Ascaris lumbricoides ova can be found in iron-hematoxylin stained preparations, the eggs usually stain so intensely that they appear as silhouettes and very little structural information can be discerned.  They may be missed by an inexperienced eye.  A thinner part of the stained slide may offer the best chance of recognizing and finding these eggs.  The sedimentation concentration wet preparation technique is preferable in visualizing these eggs when present.

Note: The following photos are taken from Iron-Hematoxylin stained fecal preparations.

  Ascaris lumbricoides:Two Ascaris eggs are seen in this photo which appear as two large dark masses on either side of the picture.  The dark blob in the lower center is just an artifact present in the stool and clearly does not have the outline of the Ascaris egg.

(Nikon, 250X)

 Ascaris lumbricoides: As with the concentrates, if a good microscopic field is found in the stained preparation, the Ascaris egg exhibits different textures as one focuses through it.

(Nikon, 1000X)

 Ascaris lumbricoides: Same photo as above but with altered focusing.  The mammillated layer appears as a scalloped edge surrounding the single celled contents of the egg.

(Nikon, 1000X)

 Ascaris lumbricoides: In most iron-hematoxylin stained preparations the Ascaris ova will be heavily stained and often appear in silhouette with very little structure revealed.

(Nikon, 1000X)

 Ascaris lumbricoides: As above.  If a thin field can be found, the stained ova may show some detail.

(Nikon, 1000X)

 Ascaris lumbricoides: Okay, last one.  This egg doesn`t look particularly healthy.  The `scalloped`edge of the mammillated coat is somewhat visible along the upper edge.

(Nikon, 1000X)

Diagnostic Problems:

Both fertile and infertile Ascaris eggs concentrate well however well using sedimentation techniques, however, if the laboratory only uses a flotation method for concentrating, infertile eggs may be missed by this technique.

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[i] Pneumonitis refers to an inflammation of the lungs which can be cause by a variety of agents, from allergic, chemical or an infection.  Ascaris pneumonitis is an inflammation caused by the presence of Ascaris lumbricoides migration through the lungs.

[ii] Löffler's syndrome or Loeffler's syndrome is a disease in which eosinophils accumulate in the lung in response to a parasitic infection.

[iii] Oviparous –egg development and maturation occurs outside of the female worm.

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Chrysonilia sitophila

Chrysonilia sitophila -Hyphomycetes (formerly Monilia sitophila)

Ecology:

Chrysonilia species are a cosmopolitan (widespread) saprobe (lives of dead or decaying plant matter).  Chrysonilia may be found within the home as ‘red bread mould’, named for the colour of growth found on contaminated bread.  Chrysonilia is the asexual state (anamorph) of Neurospora, its sexual or (teleomorph) state.  Neurospora’s ascospores are stimulated by applied heat and therefore Neurospora and the Chrysonilia anamorph may be the first organisms to repopulate areas devastated by grass or forest fires.   For the same reason, it is notorious for populating heat sterilized soils such as those found in greenhouses.  Chrysonilia’s teleomorph, Neurospora, is frequently used in the study of genetics and basic eukaryotic cell biology because of its desirable growth characteristics.

Chrysonilia sitophila was formerly called Monilia sitophila; however the genus Monilia now only encompasses plant pathogens.

Pathogenicity:

Chrysonilia is considered to be a contaminant and is not considered to be very pathogenic.  It has been implicated in peritonitis, eye infections as well as occupational asthma.

Macroscopic Morphology: 

·         Colonies exhibit extremely rapid growth, maturing within 72 hours.

·         Colonies can range in colour from white to a pale pink, a salmon colour to light orange.

·         Texture is very cottony which quickly fills the petrie dish, often referred to as a “lid-lifter” as it crawls up the sides of the dish and presses against the lid.

 Chrysonilia sitophila - 24 Hours growth on SAB agar plate incubated at 30˚C (Nikon)

 Chrysonilia sitophila - This composite shows how quickly this fungus grows.  Within one day the centrally inoculated plate has been filled with the mycelium.  By 4 hours it has already completely filled the standard petrie dish and the areal hyphae are pushing against the lid.  By day three, the slight pinkish to salmon to light-orange colour, characteristic of this fungus is evident.  After a week the growth may have collapsed back on itself and be even be trying to crawl out the sides of the petrie dish.  This organism is notorious for contaminating laboratories with the multitude of spores (conidia) produced. (Nikon)

Microscopic Morphology:  

 ·         Chrysonilia produces smooth-walled, hyaline, septate hyphae.

Sources differ on their description of the conidia produced.  The majority of sources I consulted only mention rectangular ‘arthroconida’, produced as the hyphae disarticulate or break-up.  Yet another source mentions blastoconidia in addition to the arthroconidia.  By definition these should emerge as a bud which is then cleaved off at maturity, often leaving a mark or scar at the point of attachment.  I have pointed these out where I believe they match the description.

·         Simple, poorly differentiated conidiophores can be single or branched.

·         Conidiophores produce branching chains of oval conidia (5-10 µm X 10-15 µm)

·         Mature hyphae break up forming thick-walled rectangular arthroconidia connected by disjunctors.

  Chrysonilia sitophila -a first look at low magnification.

(100X, LPCB, DMD-108)

 Chrysonilia sitophila -septate hyphae are seen with poorly differentiated conidiophores producing chains of rather round blastoconidia.  Rectangular arthroconidia are also evident, produced as the hyphae fragment or disarticulate.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila - another view as above.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -and another.  The rather round Blastoconidia (Bc) are being produced in addition to the rectangular Arthroconidia (Ac).  Are the so called 'Blastoconidia' referring to immature or developing arthrospores in one source?  The blastoconidum shown clearly does not appear to be the product of a fragmenting hyphal element.  The hyphae are septate (S), along the lines where they will disarticulate into separate arthroconidia,

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -Branching hyphae & conidiophores.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -ditto

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -whether these are considered to be blastoconidia or immature arthroconidia, chains are clearly shown to be developing from a branch extending from a main hyphal element.

(400X, LPCB, 400X)

 Chrysonilia sitophila -arthroconidia can be seen joined to each other via a disjunctor cell (Dj) which appears as a small bridge separating one cell from the next.  Various texts show the disjunctor as being more pronounced than seen here.

 (1000X, LPCB, DMD-108)

The photograph show above, as several of the previous, are taken from an adhesive tape preparation.  I have found at times that an adhesive tape preparation preserves features better than a slide culture and sometimes the opposite.  Use whatever technique works best for you.  Overall, I like slide cultures as it generally produces a much clearer photograph.  In tape preps such as this, you  can often detect the unevenness of tape surface and distribution of the adhesive.  Here the background is full of bubbles from trapped air and the adhesive.

 Chrysonilia sitophila -hyphae disarticulating into arthrospores (AC).

(1000X, LPCB, 1000X)

 Chrysonilia sitophila -once again, it appears to me that there is a distinct difference between the blastoconidia (BC) which often appear to develop from the side of the hyphae or rudimentary conidiophore and those of the already rather rectangular arthrospores at the apex of a fragmenting hyphal element. (Dj = Disjunctor)

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

 Chrysonilia sitophila - One last photo again showing what I call a round blastoconidium (BC) on a very basic conidiophore (Cp).  To my eye, these look distinctly different from the rectangular arthrospores forming by the disarticulation of the hyphae which they originate.

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

Physiological Tests:

·         +Growth at 37˚C.

·         +Growth on Cycloheximide Agar

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