Use of Transmission Electron Microscopy for Viral Diagnosis in Psittacine Birds

W. L. Steffens

Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388 (USA)

Abstract. Numerous diseases of viral etiology representing at least eleven families of viruses are known to affect psittacine species. In many cases, diagnosis is difficult or uncertain due to lack of specific tests, a wide range of syndromes associated with specific infections, and the problems associated with diagnosis in living birds. Viruses are taxonomically placed into families and sub-families based on a number of inherent features, including but not limited to: a) type and size of genome; b) characteristic morphological features including size and capsid structure; and c) structural protein composition. Although the size of most viruses places them well below the resolving power of the standard light microscope, they are quite readily viewed and identified with any transmission electron microscope (TEM). Since viruses tend to replicate in a site and manner characteristic for the family and transmission of the disease is usually dependent on the shedding of infectious viral particles from the host, the TEM has proven to be a very useful tool for providing rapid ante-mortem and post-mortem diagnoses of viral infections through the examination of appropriately processed lesions, biopsies, and excreta.

Keywords: Virus, TEM, Transmission electron microscope, Negative stain, Adenovirus, Picornavirus, Reovirus, Herpesvirus, Coronavirus, Circovirus, BFD, Polyomavirus, Poxvirus, Paramyxovirus, Diagnosis.

Introduction

Psittacine birds are known to be susceptible to infection by viruses from at least 11 families (Table 1) and many diseases of unknown etiology are suspected to be viral in origin (Ritchie, 1995). Virus taxonomy is based upon several features of the individual viral particles (virions) including: size and nature of the genome (DNA vs RNA, single or double-stranded); architecture of the structure enclosing the genome (protein capsid vs membranous envelope); and nature of the constitutive proteins. In the rare cases where specific clinical tests are available for positive identification of viral agents present in lesions or from body fluids or excreta, these tests are normally based on specific antibodies to viral proteins (immunoprobes) or from complementary polynucleotides developed from specific sequences isolated from the viral genome (molecular probes).

Unfortunately, specific diagnostic tests do not yet exist for most of the viral agents for which psittacine birds are susceptible. The clinician must often rely on his/her recognition of the clinical presentation of the disease or on the findings of the diagnostician in the case where a representative biopsy is available or the bird is presented for post-mortem examination. The pathologist will examine the specimen by light microscopy as prepared histological sections, and if a viral agent is suspected, will look for lesions characteristic of specific viruses. Although viruses have recognizable structural morphologies which are characteristic of their taxon, their size is below the resolving power of the light microscope and is thus not sufficient to allow their identification through this mode. At best the pathologist will be able to find lesions or inclusions which are suggestive of a specific viral infection.

The transmission electron microscope (TEM) is an instrument widely utilized by private, government, and university analytical laboratories for its ability to overcome the magnification and resolution limits of the light microscope. Resolution, the minimum distance which can be discriminated between two points limits the useful magnification range of any microscope and is a function of the wavelength of the illuminating source. Utilizing white light as illumination, the light microscope is capable of resolving at best about 0.25 mm, which gives the light microscope a useful upper magnification of about 1200 X (diameters). The TEM, which utilizes a beam of electrons as the illuminating source is routinely capable of resolving distances of 0.5 nm or less, which gives it a useful upper magnification range of 500,000 X or greater. TEMs are found in most universities, large hospitals, many analytical or research government laboratories, and many private analytical contract laboratories. In biomedical or multidisciplinary facilities, there are often individuals with the necessary experience to accurately identify specific viral agents in appropriately prepared specimens submitted by avian researchers, practitioners, etc. The TEM thus represents a tool whereby a viral agent can in many cases be imaged and identified to family by virtue of its distinctive morphological characteristics.

Sample Collection and Processing

When electron microscopy is selected as a diagnostic tool, the investigator is presented with two options for collecting, processing, and ultimately imaging the specimen:

Negative Stain Method - This technique involves the extraction and crude purification of virus from excreta, lesions, tissue, etc.

In this method, samples suspected of containing virus such as feces, tracheal swabs, small pieces of tissue from biopsy or necropsy, pulp from feather shafts, or skin scrapings are homogenized in a small amount of buffer or sterile water to obtain a viral suspension.

Feces can normally be mixed with an equal part of water to form a suspension, then centrifuged at 50,000 xg for 2 minutes for clarification.

Samples containing cellular material or tissue must be adequately homogenized to release virus which might be cell-associated. This can be carried out using a simple ground glass-type tissue homogenizer or a high-speed mechanical type. Once homogenized, the suspension is then clarified through centrifugation as mentioned previously.

If an appropriate cell culture has been inoculated with a sample for virus isolation and CPE is observed, a sample may be obtained from the cell culture. Often it is sufficient to simply withdraw a small amount of the culture medium which will frequently contain the virus in suspension. Culture medium obtained in this way normally will not require clarification. If the results are negative or the virus is believed to be strongly cell-associated, then it becomes necessary to disrupt the cultured cells. This is adequately carried out by exposing the culture to several freeze-thaw cycles, then removing a sample from the culture medium.

The clear sample is then applied to a coated TEM grid. The process is described in detail in Hayat and Miller (1990) and is outlined as follows: a) Put a drop of the suspension of material to be stained on a sheet of Parafilm. b) Float a formvar-carbon coated 400m grid on the drop (filmed side down) for at least 1 minute. c)Remove grid, and drain off excess liquid by touching edge to a piece of clean filter paper. c)Touch the grid (filmed side down) to a drop of 2% Phosphotungstic acid (PTA) (see previous link for formula) stock, and drain off excess as before. d) Allow to dry a few minutes, then examine with the TEM.

Advantages of the negative staining method

Rapid and highly sensitive. Sample preparation time is only a few minutes. If virus is present in sufficient quantity (at least 10⁶ particles/ml), an experienced operator can normally find it within a few minutes of observation.

If no virus can be found, it is possible that the sample concentration is too low. In this case, the clarified sample can be further concentrated through ultracentrifugation or dialysis.

Observed virus can be compared to images of similarly prepared samples in many references and atlases (Palmer and Martin, 1982; Hsiung, 1982; Doane and Anderson, 1987; Hayat and Miller, 1990)

Disadvantages of the negative staining method

Viruses can only be identified to family. For example, a papillomavirus cannot be distinguished from a polyomavirus (both in Papovaviridae). Additional data such as clinical presentation would be needed for a diagnosis.

Artifacts resembling several of classes of viruses may be present. Included here are bacteriophages which are common in feces and often resemble herpesviruses, and membrane fragments which can have surface morphology resembling several families of enveloped viruses

In the case of viruses shed through feces, there is no indication as to the site of viral infection. Many non-enteric viruses are shed through the feces.

The presence of virus may be unrelated to the disease in question. Many viruses may be present in clinically normal birds

Tissue embedment and ultrathin sectioning - This method involves acquiring a sample of diseased tissue from biopsy or necropsy and processing it to be embedded and sectioned for observation in the TEM in much the same way that tissue is processed for histopathology. ). In the normal diagnostic process, the pathologist will first view histopathology slides of the case in an attempt to find lesions. If lesions are found suggestive of a viral etiology, it is often possible to confirm the diagnosis by viewing similar tissue with TEM in an attempt to localize virus in association with the lesion. The preparation process however is time-consuming, laborious and expensive and requires the access to a laboratory which routinely performs this procedure. It is beyond the scope of this article to present the procedure, thus the reader is directed to an appropriate references (Dawes, C. J. , 1971; Hayat, 1989; and Bozzola and Russell, 1992)

Advantages of viewing sectioned material

Virus may be identified within the lesion which characterizes a particular disease.

Site of intracellular viral replication may be observed. This is often useful in identifying the virus family.

Immunolabelling with colloidal gold markers is possible when a specific antiserum is available.

Disadvantages of viewing sectioned material

Time-consuming, laborious, and expensive specimen preparation

Identification of certain virus in cross-sectional profile may be extremely difficult.

Viral morphology is depended on specimen preservation. Poor fixation or high tissue autolysis may render viruses nondescript or may induce changes in otherwise normal cells which resemble certain viral groups.

Some commonly encountered psittacine viruses as seen in both negatively stained preparations and sectioned material are depicted in Figures 1 - 16.

Adenoviridae

Fig 1. Adenovirus. Negative stained preparation from liver homogenate. Bar = 100 nm.

Fig 2. Adenovirus. Thin section through nucleus of hepatocyte. Individual virions are arranged in typical paracrystalline array. Bar = 2 micrometer.	Fig 3. Adenovirus. Higher magnification of paracrystalline array seen in fig 2. Bar = 250 nm.

Circoviridae

Fig 4. Negative stain of PBFD virus from fecal extract. Individual virions are profiled on a dark background. Bar = 90 nm.	Fig 5. Negative stained preparation of PBFD virus from sucrose gradient. Tobacco mosaic virus (arrow) is included as a measurement standard. Bar = 50 nm.

Fig 6. Inclusions of PBFD virus (arrows) in a section through skin. The large inclusions have a paracrystalline appearance which is seen in figure 7. Bar = 2 micrometer.	Fig 7. The paracrystalline arrays in the PBFD virus inclusion are observed. This section has been incubated with a specific antibody against PBFD virus. The colloidal gold label appears as dense black spots (arrows). Bar = 500 nm.

Coronaviridae

Fig 8. Avian coronavirus from a fecal extract. The petal-shaped peplomeres (arrow) for the characteristic "corona" around the envelope. Bar = 120 nm.

Herpesviridae

Fig 9. Negatively stained Herpesvirus from a liver homogenate from a conure. The large icosahedral nucleocapsid (arrow) is surrounded by a host-derived membranous envelope (E). Bar = 80 nm.	Fig 10. Herpesvirus inclusion in a section through a hepatocyte nucleus. The nucleocapsids (arrows) appear scattered among clumps of chromatin (C). Bar = 200 nm.

Papovaviridae

Fig 11. Negative stained polyomavirus from cell culture medium. Bar = 110 nm.	Fig 12. Polyomavirus inclusion within a section through a nucleus. Individual virions are evident (arrow) and heterochromatin (H) appears marginated. Bar = 400 nm.

Paramyxoviridae

Fig 13. Negative stained paramyxovirus from fecal extract. The nucleocapsid has the typical "herringbone" pattern and the envelope (E) is covered with spike-like projections. Bar = 100 nm.

Picornaviridae

Fig 14. Negative stained picornavirus (probably enterovirus) from a fecal extract. Bar = 100 nm.

Poxviridae

Fig 15. Negative stained poxvirus extracted from a skin scraping. Bar = 200 nm.

Reoviridae

Fig 16. Negative stained reovirus from a fecal extract. The 2-layered capsid (arrow) is clearly visible. Bar = 125 nm.

References

1. Bozzola, J. J. , and L. D. Russell. 1992. Electron Microscopy. Jones and Bartlett, Boston, MA. 542 pp.

2. Dawes, C. J. 1971. Biological Techniques in Electron Microscopy. Barnes and Noble Inc. , New York. 193 pp.

3. Doane, F. W. , and N. Anderson. 1987. Electron Microscopy and Diagnostic Virology. Cambridge University Press, Cambridge. 178 pp.

4. Hayat, M. A. 1989. Principles and Techniques of Electron Microscopy: Biological Applications. CRC Press, Boca Raton, FL 469 pp.

5. Hayat, M. A. , and S. E. Miller. 1990. Negative Staining. McGraw Hill, New York. 255 pp.

6. Hsiung, G. D. 1982. Diagnostic Virology. Yale University Press. New Haven and London. 276 pp.

7. Palmer, E. L. , and M. L. Martin. 1982. An Atlas of Mammalian Viruses. CRC Press, Boca Raton, FL. 154 pp.

8. Ritchie, B. W. 1995. Avian Viruses: Function and Control. Wingers Publishing, Inc. , Lake Worth, FL. 523 pp.

Acknowledgments

The author wishes to thank the following individuals for providing some of the electron micrographs used in this presentation:

Dr. Kenneth Latimer, Dept. of Pathology, University of Georgia

Mary B. Ard, HT-ASCP, Dept. of Pathology, University of Georgia

Dr. Branson Ritchie, Dept. of Small Animal Medicine, University of Georgia

This Page Last Updated May 15, 1998

^  Top of page