Abstract

Background and Purpose

Diplomonad flagellates are commonly encountered in the digestive tract of a wide variety of hosts, including fish, amphibians, rodents, and birds; the flagellates may also be found in the blood, and throughout the internal organs. The impact of the infections on the hosts ranges from minimal, to significant morbidity and mortality. Despite the common occurrence of diplomonads, and the associated diseases, many aspects of the infections are poorly understood, including host-flagellate specificity, geographic ranges, and pathogenicity.

Inadequate diagnosis of genus and species of flagellates contributes to much of the confusion in the literature. Many reports, both historic and contemporary, make only a tentative identification to genus and species. In part, this can be accounted for by the small size of the flagellates which are typically 10-20 µm long, and thus their features are at the limits of what can be discerned by light microscopy alone. However, it has now been established that a suite of light and electron microscopic techniques can show characteristics that provide reliable diagnosis to genus and species. A summary of the techniques to be applied, and features to be observed, is presented for the accurate diagnosis of diplomonad infections.

Study Design and Findings

Light Microscopy: Live Flagellates

Diplomonads are typically very active protozoa, and they can therefore readily be recognized in fresh preparations. In order to confirm that a flagellate is a diplomonad of one of the genera Spironucleus, Hexamita or Brugerolleia, it is necessary to observe that the anterior end of the cell bears six flagella, in two groups of three, and that from the posterior end of the cell emerge two trailing flagella. Shape of the flagellate cells is not a reliable characteristic by which to determine genus, due to the fact that diplomonads are pleomorphic; however, length and width do form part of species descriptions.^4,8

Observations of the morphometrics of live diplomonds is made easier by adding a viscous medium, such as Protoslo (Quieting Solution, Carolina Biological Supply Company, Burlington, NC; Tel: 800 547-1733; Item no. 88B51 41) to the preparation. This slows the movements of the flagellates.

Light Microscopy: Whole Stained Flagellates

Use of protargol silver protein stain will show features of diagnostic importance, such as nuclei, microtubules and fibrillar structures. The stain can be applied to Bouin's fixed specimens prepared as a smear,^7,8 or as a suspension and then processed on special filters.^5,6,9 Caution should be exercised in interpretation of nuclear morphology in protargol stained specimens, as this feature can be very variable. Of greater value in protargol stained specimens are the different arrangements of microtubules and fibrillar structures that can be appreciated, since these can be correlated with the internal ultrastructure by which species can be differentiated.

Protargol is the light microscopy stain of choice for elucidation of morphologic features of whole flagellates and ciliates, and is widely used in protozoology.

Scanning Electron Microscopy

Although light microscopy might suggest that diplomonad flagellates are essentially unadorned pyriform organisms, scanning electron microscopy has shown that their surfaces can be very elaborate. Surface ultrastructure provides a suite of characteristics that are emerging as important features contributing to reliable distinction of species.^4,8-10 Of particular interest is the presence or absence of lateral ridges, and the arrangement of contours of the body at the posterior end around the exits of the recurrent flagella.

Transmission Electron Microscopy

Transmission electron microscopy allows definitive diagnosis of diplomonads to genus. The pioneering work of Brugerolle and colleagues has elegantly established that a suite of ultrastructural features are genus specific.^1-3 Of particular note are the following for Spironucleus: elongate nuclei, which taper and wrap around each other anteriorly; and anterior kinetosomes in an anterior-medial position.³ Of particular note for Hexamita are: spherical nuclei which abut medially; and anterior kinetosomes in an anterior-lateral position.¹ The suite of internal ultrastructural features that distinguish Brugerolleia include reniform nuclei, the absence of infranuclear microtubules, and recurrent flagella that are cytoplasmic (contrasting with the ensheathed recurrent flagellae of Hexamita and Spironucleus).⁴

Recent studies on diplomonads from fish by Poynton, Sterud and colleagues have demonstrated that species can be distinguished by their unique combination of surface and internal ultrastructural characteristics. ^8,9,10 Of particular value are arrangements of microtubular and fibrillar elements providing support and adornment to surface ridges, and the patterns of microtubules in the ribbons accompanying the recurrent flagellae through the body.

Although ultrastructure has historically been used primarily by researchers, its use in clinical contexts is becoming increasingly important. For example, the geographic and host reservoir sources of the pathogenic species Spironucleus barkhanus affecting Norwegian salmonids were identified only after ultrastructure was used to describe the diplomonads present in diseased and healthy fish.^10,11

Histologic Sections

Identification of diplomonads in histologic sections is limited, since the full suite of distinguishing features cannot be seen. However it is possible to confirm that flagellates are diplomonads by observing the paired nuclei in the anterior of the cell. Although hematoxylin and eosin is routinely used for histology, the Feulgen stain is recommended for diagnosis of diplomonads, since this gives more distinctive differentiation between nuclei which stain magenta, and cytoplasm which stains turquoise. Longitudinal sections through the flagellates are the most useful for diagnostic purposes, since the shape of the nuclei can be observed, and thus identification to genus can be attempted. Species identification of diplomonads cannot accurately be determined in histologic sections.

In vitro Culture

The development of successful protocols for the maintenance of different diplomonad species in in vitro culture is an important contribution to their characterization. Cultures provide excellent sources of flagellates for ultrastructural studies, since the individuals are typically numerous, and their surface ultrastructure is more easily appreciated, since there is no host material to obscure important details.

In addition, in vitro cultures are an excellent resource for studies of tolerance to such factors as pH and temperature; thus providing insight into some aspects of geographic and host distribution. Cultured diplomonads can also be used for molecular characterization, although this technique is still in its infancy for this group of protozoa from exotic and aquatic animals. Cryopreservation of diplomonads has provided known reference stocks for comparative studies.

Major Conclusions

Diplomonad flagellates can be reliably identified to genus and species by a combination of ultrastructural features visualized by scanning and electron microscopy. Of particular value for determination of genus are the shape of the nuclei, location of anterior kinetosomes, presence or absence of infranuclear microtubules, and presence or absence of a sheath around the recurrent flagella. Species can be determined by a combination of surface and internal ultrastructure, particularly the adornments of the body surface, and the shape of the posterior end of the body.

Light microscopy observations, whether of whole organisms or those in histologic sections can be helpful in confirming the presence or absence of diplomonad flagellates. For whole organisms, use of protargol silver protein stain in recommended, and for organisms in section, Feulgen stain is recommended. Light microscopy observations should be regarded as supplemental to ultrastructural studies for diagnosis, not as an alternative.

Accurate identification of diplomonads to genus and species will contribute greatly to unravelling some of the mysteries of host-flagellate specificity, geographic range, and pathogenicity.

References

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2.  Brugerolle G. 1975. Contribution a l'etude cytologique et phyletique des diplozoaires (Zoomastigophorea, Diplozoa, Dangeard 1910). VI. Caracteres generaux des diplozoires. Protistologica XI:111-118.

3.  Brugerolle G, L Joyon, N Oktem, 1973. Contribution a l'etude cytologique et phyletique des diplozoaires (Zoomastigophorea, Diplozoa, dangeard 1910). II. Etude ultrastructurale du genre Spironucleus (Lavier 1936). Protistologica IX: 495-502.

4.  Desser SS, H Hong, ME Siddall, and J.R. Barta. 1993. An ultrastructural study of Brugerolleia algonquinensis gen. nov., sp. nov. (Diplomonadina; Diplomadida), a flagellate parasite in the blood of frogs from Ontario, Canada. Europ. J. Protistol. 29: 72-80.

5.  Lynn DH. 1992. Protorgol staining. In: Lee, J.J. and A.T. Soldo. Protocols in Protozoology, Society of Protozoologists, Lawrence, Kansas. Pp. C4.1-C4.8.

6.  Montagnes DJS, DHLynn. 1987. A quantitative protargol stain (QPS) for ciliates: method description and test of its quantitative nature. Mar. Microb. Food Webs 2: 83-93.

7.  Nie D. 1950. Morphology and taxonomy of the intestinal protozoa of the guinea-pig, Cavia porcella. J. Morphol. 86: 381-494.

8.  Poynton SL, CM Morrison. 1990. Morphology of diplomonad flagellates: Spironucleus torosa N.Sp. from Atlantic Cod Gadus morhua L., and Haddock Melanogrammus aeglefinus (L.) and Hexamita salmonis Moore from Brook Trout Salvelinus fontinalis (Mitchill). J. Protozool. 37(5): 369-383.

9.  Poynton SL, W Fraser, R Francis-Floyd, P Rutledge, P Reed, TA Nerad. 1995. Spironucleus vortens N.Sp. from freshwater angel fish Pterophyllum scalare: morphology and culture. J. Euk. Microbiol. 42(6): 731-742.

10. Sterud E, TA Mo, TT Poppe. 1997. Ultrastructure of Spironucleus barkhanus N.Sp. (Diplomonadida: Hexamitidae) from grayling Thymallus thymallus (L.) (Salmonidae) and Atlantic salmon Salmon salar L. (Salmonidae). J: Euk. Microbiol. 44(5): 399-407.

11. Sterud E, TA Mo, TT Poppe. 1998. Systemic spironucleosis in sea-farmed Atlantic salmon Salmo salar, caused by Spironucleus barkhanus transmitted from feral Arctic char Salvelinus alpinus? Dis. Aquat. Org. 33: 63-66.