Volume 3. The Flagellates

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Co- Authors: M. Arcari ¹, A. Baxendine ¹ and C. E. Bennett²

1. Intersep Ltd 2. University of Southampton

More information can be obtained on www.diasys.com and www.soton.ac.uk/~ceb/, Ectoparasites and Endoparasites.

CONTENTS

3. Infections through the Gastrointestinal Tract Part Three
The Flagellates
Giardia lamblia
Dientamoeba fragilis 
Trichomonas hominis
Chilomastix mesnili
Enteromonas hominis 
Retortamonas intestinalis

Identifying Flagellates

Table 1. Differential morphology of flagellates
found in stool samples of humans.

Key 1. to identifying stained flagellate trophozoites
Key 2 to differentiating flagellate and amoebic cysts

The Flagellates

The flagellates belong to the Magistophora and possess more than one flagellum. Beating these flagella enable them to move. A cytosome may be present which helps in the identification of the species.

Flagellates possess one advantage over their amoeboid relatives in that they can swim. Therefore, enabling them to invade and adapt to a wider range of environments unsuitable for other amoebae. They are able to change from a flagellated free-swimming environment to a non-flagellated tissue dwelling stage and vice versa.

Flagellates are known to inhabit the reproductive tract, alimentary canal, tissue sites and also the blood stream, lymph vessels and cerebrospinal canal.

There are pathogenic and commensal species of flagellates. The flagellates which are encountered in the intestinal tract are Giardia lamblia, Dientamoeba fragilis, Chilomastix mesnili, Trichomonas hominis, Retortamonas intestinalis and Enteromonas hominis (the latter 2 being less common). The trophozoites are easily recognised in saline preparations by their motility. However, accurate identification is done on a permanently stained faecal smear. Cysts are more commonly seen than the trophozoite.

Giardia lamblia

Introduction

Giardia lamblia is a flagellate of world-wide distribution. It is more common in warm climates than temporal climates. It is the most common flagellate of the intestinal tract, causing Giardiasis. Humans are the only important reservoir of the infection. The infection is most common in parts of the world where sanitation is at its lowest. Giardiasis is an infection of the upper small bowel, which may cause diarrhoea. Only Giardia spreads disease. (Diag 1 illustrates the G. lamblia life cycle)

Morphology of trophozoites

The trophozoites of G. lamblia are flattened pear shaped and are an average size of 15m m long, 9m m wide and 3m m thick. When stained, the trophozoite is seen to have 2 nuclei, 2 slender median rods (axostyles), and 8 flagella arising from the anterior end. They have been described as looking like tennis rackets without the handle (they are often seen has having a comical face-like appearance when looking at the front view). (Diag 2 & Fig 1)

The movement of the trophozoites are described as tumbling leaf motility, using their 4 pairs of flagella for locomotion. They attach themselves to the surface of the jejunal or duodenal mucosa by their disc-like suckers which are found on their ventral surface. They multiply in the gut by binary fission. Once the trophozoites drop off the mucosal surface they are normally carried in the intestinal contents down the gut where they usually encyst.

                 

Diagram 1. Life cycle of Giardia lamblia. (Adapted and redrawn from NCDC)

                   

                                

                  

                       

Fig 1. Giardia lamblia trophozoites trichrome stained from a duodenal aspirate. The nuclei and the flagella are clearly visible. Free living organisms have two nuclei and a large karyosome. The adhesive disc and 4 posteriorly directed flagella are visible in the trophozoite on the left hand side. (8 x 11m m) (www.dpd.cdc.gov)

Morphology of cysts

The cysts of G. lamblia are 8 - 12 m m in length and are ellipsoid in shape. They contain 4 nuclei which tend not to be obvious. Longitudinal fibrils consisting of the remains of axonemes and parabasal bodies may also be seen. Cysts may appear to shrink from the cell wall. (Diag 2 & Fig 2 & 3) The cysts are infective as soon as they are passed.

                                       

                  

Clinical Disease

Giardia lamblia colonises the small intestine where the trophozoites adhere to the mucosal surface by means of their sucking disc. Cysts are produced as the parasites descend the intestinal tract although trophozoites can be passed in the faeces in severe infections. G. lamblia is transmitted through ingestion of cysts in contaminated water or food. Cysts can survive outside the body for several weeks under favourable conditions. The main symptoms are abdominal pain,

flatulence, and episodic diarrhoea with steatorrhea and periodical soreness in severe cases. No blood or mucus is normally seen. However 50% of G. lamblia infections are symptomless, although severe infections may develop in immunocompromised hosts. What determines susceptibility is poorly understood. After swallowing cysts for the first time, symptoms commonly develop 2-6 weeks later.

Laboratory Diagnosis

Cysts can be found by examination of the deposit of a formol-ether concentrate of a stool preparation. The oval cysts with thick walls serve as characteristic features for these organisms. (Keys 1 & 2) The flagella disintegrate and form a central ‘streak’ which becomes visible when stained with iodine or MIF (merthiolate-iodine-formaldehyde). Cysts may be excreted intermittently, therefore it is important to examine more than one stool. Stools are usually passed 3-8 times / day and are usually pale, offensive, rather bulky and accompanied by much flatus.

Trophozoites are found by examination of saline wet preparations of fresh, diarrhoeic stool, duodenal or jejunal aspirate or in a permanently stained faecal preparation.

Trophozoites can also be found in the jejunal aspirate. These can be recovered by the String Test or Enterotest capsule and the material examined microscopically for motile trophozoites.

Trophozoites and cysts can be found to be scarce in chronic infections. Serological methods of diagnosis are proving to be useful as means of diagnosis. An ELISA to detect IgM in serum provides evidence of a current infection. A polyclonal antigen-capture ELISA can be used to demonstrate submicroscopic infections in faeces and an IgA-based ELISA will detect specific antibodies in saliva. Table 1 (page 17) details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.

Introduction

Dientamoeba fragilis is an amoeba-flagellate with a cosmopolitan distribution. The life cycle is not known.

Morphology of trophozoites

D. fragilis are relatively small, varying from 3 - 22 m m in diameter and there can be considerable variation in size among organisms in the same faecal sample. Diag 3 & Fig 4, 5) The organisms have only a trophozoite stage and in a permanently stained preparation, one, two or rarely three nuclei can be seen, two being the most common. The nuclear chromatin is usually fragmented into three to five granules but these have not been visualised by Giemsa Stain, and there is normally no peripheral chromatin on the nuclear membrane. The cytoplasm is usually vacuolated and may contain ingested debris as well as some large uniform granules. The cytoplasm can also appear uniform and clean with a few inclusions. D. fragilis live in the lumen of the caecum and upper colon.

                                       

                      

                               

                

Pathogenesis

This is a controversial area. The organism has been reported in association with mucous diarrhoea, abdominal pain and tenderness. Nausea, vomiting and low-grade fever have also been reported in a number of cases. The precise role of this organism as a cause of disease remains to be determined.

Laboratory Diagnosis

Diagnosis is dependent on examination of the fresh direct wet preparation or permanently stained smears prepared from unformed or formed stools with mucus. (Keys 1 & 2) It is particularly important that permanently stained smears of stool preparations should be examined, because survival times of the organism in terms of morphology, is very limited and specimens must be examined immediately or preserved in a suitable fixative as soon as possible after defaecation. The recommended stains are Fields’ and Giemsa stain (trophozoites are destroyed in a formol-ether concentrate). Table 1 (page 17) details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.

Trichomonas hominis

Introduction

This flagellate is cosmopolitan in its distribution. It is thought to be non-pathogenic although it has been associated with diarrhoeic stools. It is the most commonly found flagellate next to G. lamblia and D. fragilis. Found in a wide host range including non-human primates, cats, dogs and various rodents. (Diag 4 illustrates the life cycle of T. hominis).

                                  

                  

 

Morphology of trophozoites

Trichomonas hominis do not have a cystic stage. The trophozoites measure from 5-15m m in length by 7-10m m in width. (Diag 5 & Fig 6)The shape is pyriform and has an axostyle which runs from the nucleus down the centre of the body and extends from the end of the body. They also possess an undulating membrane which extends the entire length of the body and projects from the body like a free flagellum (this feature distinguishes it from other trichomonads). The characteristic number of flagella is five, there is some deviation from this number. They also have a single nucleus at the anterior end. Trichomonads swim with a characteristic wobbly movement, which makes them unmistakable during diagnosis.

                       

           

                              

            

Laboratory Diagnosis

In a fresh stool, the flagellates move very rapidly in a jerky, non-directional manner. The axostyle and undulating membrane are diagnostic. (Keys 1 & 2) The flagellates are difficult to stain, however, the axostyle can be seen on a stained preparation and is diagnostic. Table 1 (page 17) details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.

                                      

Chilomastix mesnili

Introduction

Chilomastix mesnili is cosmopolitan in distribution although found more frequently in warm climates. It is thought to be non-pathogenic although the trophozoite has been associated with diarrhoeic stool. This is the largest flagellate found in man with an incidence of 1-10% being in the large intestine.

(Diag 6 illustrates the life cycle of C. mesnili)

                 

Morphology of the Trophozoite         

The trophozoites of C. mesnili are pear shaped and measure 6-20m m in length. They have 1 large nucleus with a small karyosome and 3 flagella that extend from the nucleus at the anterior end of the parasite. A distinct oral groove or cytosome can be seen near the nucleus with its sides being supported by two filaments. They are known to move in a directional manner. (Diag 7 & Fig 7)

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                

Morphology of cysts

                            The cysts are 6-9m m, they have a large single nucleus with a large karyosome. They also have a prominent side knob giving it a characteristic lemon shape. (Diag 8 & Fig 8) The cytosome is evident with a curved shepherds crook fibril. It also has a characteristically coiled filament which when stained is darker in colour.

                           

                                     

                                

                 

                                 

Fig 8. Chilomastix mesnili cysts are excreted with faeces and constitute the transmission form of the micro-organism. The uninucleated lemon shaped cysts are seen with a little proturberance at one end and a prominent cytostome. (Iodine stained). (6m m) (www.dpd.cdc.gov)

Laboratory Diagnosis

The characteristic lemon shaped cysts can be seen in a formol-ether concentrate. (Fig 8) Motile organisms can be seen in a wet preparation of a fresh stool however the characteristic morphology is evident in a permanently stained preparation. (Keys 1 & 2) Table 1 (page 17) details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.

Enteromonas hominis

Introduction

Enteromonas hominis is a small flagellate and is rarely encountered in man. It is found in both warm and temperate climates and is considered to be non-pathogenic. (Diag 9 illustrates the life cycle of E. hominis)

                 

Morphology of the trophozoite

The trophozoites are oval and 4-10m m in length. They have 4 flagella, 3 anterior flagella and one adheres to the body ending in a tail, producing a jerky rotational movement. They have one nucleus with a large karyosome that is evident in a stained preparation. (Diag 10)

                             

Morphology of the cyst

The cysts are oval and range between 6-8m m in length. They have up to 4 nuclei with a bipolar tendency. (Diag 11)

                              

Laboratory Diagnosis

The cysts are seen in a formol-ether concentrate. The cysts have no distinguishing characteristics and thus can be confused with E. nana or even yeasts. The characteristic trophozoites can be seen in a permanently stained faecal smear. (Keys 1 & 2) Table 1 (page 17) details useful morphological features that are similar between species of flagellate and are used in laboratory diagnosis.

Retortamonas intestinalis

Introduction

Retortamonas intestinalis like Enteromonas hominis is a small flagellate and is rarely encountered. It is found in both warm and temperate climates and is considered to be non-pathogenic. (Diag 12 illustrates the life cycle of R. intestinalis)

                    

Diagram 12. Life cycle of Retortamonas intestinalis

Morphology of the trophozoite

                           

The trophozoite is small, measuring between 4 and 9m m. Its movement is jerky and rotational and has 2 anterior flagella and a prominent cytosome that can be seen in an unstained preparation. It has a relatively large nucleus at the anterior end with a small compact karyosome. (Diag 12)

Morphology of the cyst

The cysts are small and pear shaped. They range in size between 4-7m m with 1 large nucleus frequently near the centre. The fibril arrangement from the nucleus is suggestive of a birds beak. This is characteristic of R. intestinalis cysts. (Diag 13)

                                          

Diagram 13. Systematic diagram of a Retortamonas intestinalis cyst. Seen as a small pear shaped organism. (Adapted and redrawn afterJ.D. Smyth)

Laboratory Diagnosis

The small pear shaped cysts are uncharacteristic in an unstained formol-ether preparation. However, the addition of iodine reveals the characteristic bird beak fibrillar arrangement in the pear shaped cyst.

In a fresh stool preparation, the 2 anterior flagella and cytosome can be seen in the trophozoite. In a permanently stained preparation, the large nucleus with small central karyosome is diagnostic. (Keys 1 & 2) Table 1 (page 17) details useful morphological features which are similar between species of flagellate and are used in laboratory diagnosis.

3.1 Identifying Flagellates

It is important to know and understand the morphological features which differentiates each species of flagellate from an other. Table 1 details the important features that are used when identifying flagellates found in human stool samples. Trophozoites and cysts can be seen in saline mounts of fresh faeces. On occasions, species identification may require stained preparations.

Species	Size (length)	Shape	Motility	Number of Nuclei	Number of Flagella*	Other features
Trichomonas hominis	8-20m m Usual range, 11-12m m	Pear Shaped	Nervous Jerky	1 Not visible in unstained mounts	3-5 anterior. 1 posterior.	Undulating membrane extending length of body.
Chilomastix mesnili	6-24m m. Usual range, 10-15m m	Pear Shaped	Stiff Rotary	1 Not visible in unstained mounts	3 anterior. 1 in cytostome	Prominent cytostome extending 1/3 –1/2 length of body. Spiral groove across ventral surface.
Giardia lamblia	10-20m m. usual range, 12-15m m.	Pear Shaped	"Falling Leaf"	2 Not visible in unstained mounts	4 lateral. 2 ventral. 2 caudal.	Sucking disc occupying 1/2 -1/3 of ventral surface.
Enteromonas hominis	4-10m m. usual range 8-9m m.	Oval	Jerky	1 Not visible in unstained mounts	3 anterior. 1 posterior.	One side of body flattened. Posterior flagellum extends free posteriorly.
Retortamonas intestinalis	4-9m m. Usual range, 6-7m m.	Pear Shaped Or oval	Jerky	1 Not visible in unstained mounts	1 anterior. 1 posterior.	Prominent cytostome extending approx 1/2 length of body

* Not a normal feature for identifying species in routine stool samples

Table 1. Differential morphology of flagellates found in stool samples of humans. (www.dpd.cdc.gov)

Flagellate trophozoites are best identified in fresh saline mounts, allowing you to observe the way that they move. Use Key 1 to help to identify stained flagellate trophozoites.

          

Iodine solutions are used primarily to stain flagellate cysts, this makes it possible to see the structure of the nuclei.
Use Key 2 to help to identify amoebic and flagellate cysts:


Key 2. Key to differentiating flagellate and amoebic cysts. (Adapted and redrawn, WHO, 1991)

References

Murray, PR, Drew, WL, Koyayashi, GS & Thomson, JH: Medical Microbiology. Mosby Books Inc., New York (1990)

Peters, W & Gilles, HM: Tropical Medicine & Parasitology. Wolfe Medical Publications Ltd.

Jeffrey & Leach: Atlas of Medical Helminthology and Protozoology. E & S Livingstone Ltd.

Ash, LR & Orihel, TC: Atlas of Human Parasitology. ASCP Press, Chicago.

Garcia, LS & Bruckner, DA: Diagnostic Medical Parasitology. Elsevior Science Publishing Co. Inc.

Muller, R & Baker, JR: Medical Parasitology. Gower Medical Publishing.

Snell, JJS, Farrell, ID & Roberts, C: Quality Control, Principles and Practice in the Microbiology Laboratory. Public Health Laboratory Service. ISBN 0 901 144 312.

World Health Organisation: Basic Laboratory Methods in Medical Parasitology. ISBN 92 4 154410 4. (1991)

Smyth, J.D: Introduction to Animal Parasitology. Cambridge University Press (1994)

Pennell, DR et al: Advances in Giardia Research. p211-213. University of Calgary Press.

Samways, KL et al: Assessments of ParasepÒ , a novel parasite egg retrieval system; use in faecal and waste water testing. Presented to Royal. Soc.Trop.Med.

Howells, H: A Critical evaluation of methods available for diagnosis of Cryptospridium parvum and Giardia lamblia in faecal samples. St Mary’s Hospital Portsmouth.

Juranek, DD. Cryptosporidiosis. In: Hunter’s Tropical Medicine. 8^th edition, Strickland, GT (editor).

Brown, VC. A Longitudinal study of the prevalence of intestinal helminths in baboons (Papio doguera) from Tanzania. (1994) Thesis, Liverpool.

We would like to thank the authors of the following web sites:
www.cdfound.to.it/atlas.htm, www.udel.edu, www.ferris.edu, www.jeflin.tju.edu
www-medlib.med.utah.edu/parasitology, www. dpd.cdc.gov

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