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International Journal of Morphology

versão On-line ISSN 0717-9502

Int. J. Morphol. v.28 n.2 Temuco jun. 2010 

Int. J. Morphol., 28(2):627-636, 2010.

Viral Research in Brazilian Owls (Tyto alba and Rhinoptynx clamator) by Transmission Electron Microscopy

Investigación Viral en Buhos Brasileños (Tyto alba y Rhinoptynx clamator) a través de Microscopía Electrónica de Transmisión

*Catroxo, M. H. B.; *Taniguchi, D. L.; *Melo, N. A.; **Milanelo, L.; ***Petrella, S.; **Alves, M.; *Martins, A. M. C. R. P. F. & *Rebouças, M. M.

* Laboratory of Electron Microscopy, Biological Institute of São Paulo, SP, Brazil.

** Tiete Ecological Park, São Paulo, SP, Brazil

*** Adolfo Lutz Institute, São Paulo, SP, Brazil

Correspondence to:

SUMMARY: The barn-owl (Tyto Alba) and striped-owl (Rhinoptynx clamator) belong respectively to the families Tytonidae and Strigidae. Avian paramyxoviruses have been isolated from a variety of species of wild and domestic birds wordlwide causing diverse clinical symptoms and signs. Paramyxoviruses belong to the family Paramyxoviridae and Avulovirus genus, including nine serotypes (APMV 1 to 9). The lymphoid leukosis is a retrovirus-induced neoplasia. The avian retroviruses belong to the Retroviridae family and to the Alpharetrovirus genus. Coronaviruses can cause respiratory and enteric disease in several species of birds. They belong to the Coronaviridae family and to the groups 3a e 3c. In this study, we describe the presence of viruses in four owls, two barn owls (Tyto alba) and two striped owls (Rhinoptynx clamator), rescued from tree-lined streets of Sao Paulo, Brazil and sent to the Recovery Center of Wild Animals of the Tietê Ecological Park, where the animals died. Fragments of lung, liver and small intestine of these birds were processed for transmission electron microscopy utilizing negative staining (rapid preparation), immunoelectron microscopy and immunocitochemistry techniques. Under the transmission electron microscopy paramyxovirus particles, pleomorphic, roughly spherical or filamentous, measuring 100 to 500 nm of diameter containing an envelope covered by spikes, an herring-bone helical nucleocapsid-like structure, measuring 15 to 20 nm in diameter, were visualized in the samples of lung, liver and small intestine of all owls. In small intestine samples of the two striped-owl (owls 3 and 4) it was detected pleomorphic coronavirus particles with a diameter of 75-160 nm containing a solar corona-shaped envelope, with projections of approximately 20 nm of diameter. In liver fragments of one striped-owl (owl 4) pleomorphic particles of retrovirus with a diameter of 80-145 nm containing an envelope with short projections and diameter of 9 nm were observed. The presence of aggregates formed by antigen-antibody interaction, characterized the positive result obtained during the immunoelectron microscopy technique for paramyxovirus, retrovirus and coronavirus. In the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the dense colloidal gold particles over these viruses.

KEYWORDS: Paramyxovirus; Coronavirus; Retrovirus; Owls; Transmission electron microscopy.

RESUMEN: La lechuza (Tyto Alba) y el búho de orejas (Rhinoptynx clamator) pertenecen respectivamente a las familias Strigidae y Tytonidae. El paramixovirus aviario se ha aislado de especies de vida silveste como las aves domésticas por todo el mundo, causando diversos síntomas clínicos. El paramixovirus pertenece a la familia Paramyxoviridae y al Avulovirus genus que incluye nueve serotipos (APMV 1 a 9). La leucosis linfoide es una neoplasia inducida por retrovirus. Los retrovirus aviarios pertenecen a la familia Retroviridae y el género Alpharetrovirus. Los coronavirus pueden causar enfermedades respiratorias y entéricas en varias especies de aves. Ellos pertenecen a la familia Coronaviridae y a los grupos 3a y 3c. En este estudio, se describe la presencia del virus en cuatro búhos, dos lechuzas (Tyto alba) y dos búhos de orejas (Rhinoptynx clamator), rescatados de las calles arboladas de São Paulo, Brasil y enviados al Centro de Recuperación de Animales Silvestres del Parque Ecológico de Tietê, donde hubo murieron los animales. Fragmentos de pulmón, delhígado y del intestino delgado de estas aves fueron procesados para microscopía electrónica de transmisión utilizando tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica. Bajo microscopía electrónica de transmisión, partículas de paramixovirus, pleomórficas, aproximadamente esféricas o filamentosas, de 100 a 500 nm de diámetro con un sobre cubierto por espigas, y nucleocápside helicoidal con características de espiga, midiendo 15 a 20 nm de diámetro, fueron visualizadas en las muestras de pulmón, hígado e intestino delgado de todos los búhos. En muestras de intestino delgado de dos búho de orejas (búhos 3 y 4) se detectaron partículas pleomórficas con coronavirus de un diámetro de 75-160 nm con un sobre con forma de corona solar, con proyecciones de aproximadamente 20 nm de diámetro. En el hígado de un búho de orejas (búho 4) se observaron partículas pleomórficas de retrovirus con un diámetro de 80-145 nm contiene pequeñas proyecciones, con un diámetro de 9 nm. La presencia de agregados formados por la interacción antígeno-anticuerpo, caracterizó el resultado positivo que obtuvimos en la técnica de microscopía immunoelectrónica para paramixovirus, retrovirus y coronavirus. En la técnica de inmunocitoquímica, la interacción antígeno-anticuerpo fue fuertemente reforzada por las partículas de oro coloidal denso en los virus.

PALABRAS CLAVE: Paramyxovirus; Coronavirus; Retrovirus; Buhos; Microscopía electrónica de transmisión.


The barn owl (Tyto alba) and striped owl (Rhinoptynx clamator) species of the order Strigiformes respectively belong to families Tytonidae and Strigidae. They are nocturnal and carnivorous birds of prey, found throughout the Brazilian territory (Sick, 1997). They have been significant in the control of rodent populations for years. Their sensitivity to environmental changes in relation to the other animals in the food chain provides clues about the state of environmental conservation (Motta-Junior & Albo, 2000).

Avian paramyxoviruses have been isolated from a variety of species of wild and domestic birds worldwide (Leeuw & Peeters 1999). These viruses belong to the Mononegavirales order, Paramyxoviridae family. Paramyxovirinae subfamily and Avulavirus genus that include nine serotypes (APMV 1 to 9) and Pneumovirus genus include Avian Metapneumovirus. Paramyxoviruses are pleomorphic, enveloped containing a negative-sense, simple stranded RNA genome (Lamb & Parks, 2007).

The Newcastle disease, caused by paramyxovirus APMV-1 type and highly pathogenic, is one of main diseases affecting the poultry trade. In worldwide economy it can be extremely harmful, producing considerable impact on the poultry industry (Jorgensen et al., 1998).

Psittacidae, passerines and pigeons, although more resistant to this disease, may present diverse clinical signs that include, depression, diarrhea, anorexia, ruffled feathers, conjunctivitis, dispnea, ataxia, tremors, paralysis and death (Richie & Carter, 1995). In several species of owls in the wild or in captivity, the serotype APMV1 was found (Telbis et al., 1989, Lou et al. 1999; Hoffle et al., 2002; Oliveira Jr. et al., 2003; Schettler et al., 2003, Choi et al., 2008) and in other species of wild birds the serotypes APMV 2, 3, 5 were detected (Mustaffa-Babjee, 1974; Nerome et al., 1978; Gougjh et al., 1993, Ritchie et al., 1994; Shihmanter et al., 1998; Beck et al., 2003; Zhang et al., 2006; Jung et al., 2009).

In lymphoid leukosis, caused by retrovirus, neoplastic nodules may develop in the viscera and skin tissue of birds (Harrison & Harrison 1986; Martins & Catroxo, 2009) with a predominance in liver and spleen (Wadsworth et al., 1981; Fowler).

This neoplasm can reach several species of birds. Broilers and laying hens are affected more often, although turkeys and quail are also susceptible. Cases of lymphoid leukosis of passerines and galliform species were also reported (Nobel, 1972; Palmer & Stauber, 1981; Wadsworth et al.; Loupal, 1984; Martins et al., 2004; Pongiluppi et al.; 2006; Hatai et al., 2008).

The clinical manifestation is variable and many times the affected birds are found dead without prior clinical manifestation (Wadsworth et al.; Fowler). Contamination might occur through vertical or horizontal transmission (Ritchie & Cartier, 1995; Fadly, 1997). Studies show that wild birds that harbor the virus can spread it to areas around poultry farms (Varejka & Tomsik, 1974).

Retroviruses are classified into seven designated genus Alpha, Beta, Gamma, Delta, Epsilon retroviruses, Spumavirus and Lentiviruses (Van Regenmortel et al., 2000). Alpha retroviruses (ALV genus) comprise the only genus confined to birds. The ALV members are classified into 10 subgroups (termed A-J) based on their host range, cross neutralization and viral interference (Coffin, 1992). The first four subgroups represent exogenous viruses of chickens, the subgroup E includes a family of endogenous chicken viruses and subgroup F and G include endogenous viruses of pheasants (Goff, 2007).

Coronavirus infects mainly birds of all ages. It is relevant in the poultry Industry and may cause respiratory and enteric disease in chickens with losses in its production and egg quality in nature hens (Worthington et al., 2008). Wild birds may play a role as both reservoirs, and as the long distance vectors of infectious bronchitis and other coronaviruses (Hughes et al., 2009).

This virus was detected in wild birds species (Catroxo et al., 1996, 2000; Pongiluppi et al., 2004; Jonassen et al., 2005; Woo et al., 2008). Infectious bronchitis is one of the major diseases caused by coronavirus that compromises commercial poultry (Cavanagh & Nagi, 1997; Pennycott, 2000; Guy et al., 1997; Circella et al.; 2007; Pohuang et al., 2009).

Coronaviruses are large enveloped positive-strand RNA. They have a round structure that is often 100 to 160nm in diameter with distinctive long, petal-shaped spikes on the surface (Fenner et al., 1992). Avian coronaviruses belong to the Nidavirales order, Coronaviridae family and to the 3a and 3c groups (Wood et al., 2009). In emergency situations the transmission electron microscopy utilizing negative staining technique is an important tool to identify viruses, due to its speed and its ability to view multiple viral agents (Hazelton & Gelderblom, 2003; Harris et al., 2006).

Considering the lack of literature concerning viruses in Brazilian owls, we have decided by the technique of transmission electron microscopy, to observe the possible presence of viral particles in organ fragments in barn owls (Tyto alba) and striped owls (Rhinoptynx clamator).


Description of the cases.From November 2005 to May 2006, four owls (two barn-owls and two striped-owls) were rescued from tree-lined streets, in the city of São Paulo, SP, and sent to the Center of Recovery of Wild Animals of the Tietê Ecological Park, where they died. Next, they were sent to the Laboratory of Electron Microscopy, Biological Institute of São Paulo, to search for viral agents. During the necropsy, fragments were collected from lung, liver and small intestine of all owls. Later, these fragments were processed for transmission electron microscopy utilizing negative staining (rapid preparation), immunoelectron microscopy and immunocytochemistry techniques.

Negative staining technique (rapid preparation). In negative staining technique, fragments of lung, liver and small intestine were suspended in phosphate buffer 0.1 M,pH 7.0. Drops of the suspensions were placed in contact with metallic copper grids, stabilized with carbon supporting film of 0.5% in collodium amyl acetate. Next, the grids were drained with filter paper and negatively stained at 2% ammonium molybdate, pH 5.0 (Brenner & Horne, 1959; Hayat & Miller, 1990; Madeley, 1997.

Immunoelectron microscopy technique.In this technique, copper grids, previously prepared with collodion film and stabilized with carbon were first incubated with protein A (1µl/ml), placed in contact with a virus-specific antibody. After this, the grids were washed in PBS drops, incubated with the antigen, washed with drops of water and negatively stained with 2% ammonium molybdate, pH 5.0 (Almeida & Waterson, 1969; Derrick, 1973; Berthiaume et al., 1981).

Immunocytochemistry Technique. At the immunolabeling technique with colloidal gold particles for negative staining, the copper grids were placed in contact with viral suspension and, after removing excess with filter paper, the same were put on specific primary antibody drops. After successive washings in PBS drops, the grids were incubated in protein A drops, in association with 10 nm colloidal gold particles (secondary antibody). Grids were then contrasted at 2% ammonium molybdate, pH 5.0 (Knutton, 1995).

All grids submitted to the reactions above described were observed in a Philips EM 208 electron microscope, at 80 kV.


Necropsy. During the necropsy, one barn owl (owl 1) had intestinal bleeding and the other (owl 2) showed bleeding in all organs. The striped owl (owl 3) presented lungs with whitish and autolysate areas of the liver. The presence of the small intestine, containing yellow and watery stools was also observed. In the striped owl (owl 4) it was observed the presence of hemorrhagic lungs, liver and small intestine with watery contents and also yellowish (Table I).

Negative staining technique (rapid preparation). Under the transmission electron microscopy paramyxovirus particles, pleomorphic, roughly spherical or filamentous, measuring 100 to 500 nm of diameter containing an envelope covered by spikes, with characteristic helical herring-bone-like nucleocapsid, measuring 15 to 20 nm in diameter, were visualized in the samples of lung, liver and small intestine of all owls (Fig. 1).

Fig. 1. Negatively stained paramyxovirus particles, pleomorphic, roughly spherical
(minor arrow) or filamentous (big arrow), containing an envelope covered by spikes,
with characteristic helical herring-bone-like nucleocapsid. Bar: 320 nm.

In liver and small intestine fragments of one striped-owl (owl 4) pleomorphic particles of retrovirus with a diameter of 80-145 nm and an envelope containing short projections with a diameter of 9 nm (Fig. 2) were observed.

Fig. 2. Negatively stained pleomorphic retrovirus particles with
an envelope containing short projections. Bar: 110 nm.

In two small intestine samples of two striped-owls (owls 3 and 4) it was detected pleomorphic coronavirusparticles with a diameter of 75-160 nm containing a solar corona-shaped envelope, with projections of approximately 20 nm of diameter (Fig. 3).

Fig. 3. Negatively stained pleomorphic coronavirus particles,
with a solar corona-shaped envelope. Bar: 120 nm.

Imunoelectron microscopy Technique. The presence of aggregates formed by antigen-antibody interaction, characterized the positive result obtained, at the immunoelectron microscopy technique for paramyxovirus (Fig. 4), retrovirus (Fig. 5) and coronavirus (Fig.6).

Fig. 4. In the immunoelectron microscopy technique the paramyxoviruses particles
were aggregated by antigen-antibody interaction. Bar: 70 nm.

Fig. 5. Retroviruses particles aggregated by
antigen-antibody interaction. Bar: 120 nm.

Fig. 6. Coronaviruses particles aggregated by
antigen-antibody interaction. Bar: 200 nm.

Immunocytochemistry Technique. In the immunocyto-chemistry technique, the antigen-antibody interaction was strongly enhanced by the dense colloidal gold particles over the paramyxovirus (Fig.7), retrovirus (Fig. 8) and coronavirus particles (Fig. 9).

Fig. 7. In the immunocytochemistry technique, the antigen-antibody
interaction was strongly enhanced by the dense colloidal gold particles
(arrow) over the paramyxovirus particles. Bar: 140 nm.

Fig. 8. Retroviruses particles strongly enhanced by
colloidal gold particles (arrow). Bar: 180 nm.

Fig. 9. Coronaviruses particles strongly enhanced by
colloidal gold particles (arrow).Bar: 100 nm.


In this study by means of the negative staining technique, viral particles with morphology similar to paramyxovirus were identified in suspensions of fragments of lung, liver and small intestine of two barn-owls (Tyto Alba) and two striped owls (Rhinoptynx clamator).

Other experiments using serological and molecular tests detected paramyxovirus APMV type 1 in several species of wild and captive owls (Telbis et al.; Gohm et al., 1999; Höffle et al.; Oliveira Junior et al.; Schettler et al.; Choi et al.).

APMV 1, 2, 3, 5 types occur more often among free-living birds, showing variable symptoms or remaining asymptomatic (Shihmanter et al.; Grund et al., 2002; Beck et al.; Greenacre, 200; Zhang et al.; Jung et al.).

Other researchers failed to detect paramyxovirus in owls (Catroxo et al., 2000; Shin et al., 2000; Sousa et al., 2010).

At least, 236 species of birds are susceptible to the paramyxovirus (Kaleta & Baldhauf, 1988), and the wild birds are an important reservoir and disseminator (Oliveira Júnior).

The owls in our study had no symptoms or clinical signs of the disease. This asymptomatic state was also reported by other authors in poultry research. (Gohm et al.; Hoffle et al.; Oliveira Júnior et al.; Schettler et al.).

The intestinal hemorrhage was a common finding in the animals autopsied. This change characterizes the Newcastle as viscerotropic disease (Alexander, 2003).

Other types of paramyxoviruses (APMV 1, 2, 3 and 5) occur more frequently among birds in the wild, at times causing varied clinical symptoms (Shihmanter et al.; Grund et al.; Beck et al.; Greenacre; Zhang et al.; Jung et al.).

The morphological paramyxoviruses characteristics described by us were similar to those found in owls by Kou et al. and in other birds species (Gough et al., 1983, 1993; Catroxo et al., 2000; Chang et al., 2001; Grund et al., Zhang et al.).

By agglutination of a great number of paramyxovirus particles during the reaction of immunoelectron microscopy, we confirmed the presence of this virus in animals. The same technique was employed by Catroxo et al. (2003) who confirmed it in canine distemper.

Likewise, in the immunocytochemistry technique, the paramyxoviruses were sharply marked by colloidal gold particles. In previous studies, the use of this technique allowed the observation of avianpox (Catroxo et al., 2009).

The use of negative staining technique also helped in the discovery of coronavirus in samples of small intestine of two striped-owls (owls 3 and 4). During necropsy of these animals, the feces were watery and yellowish, giving evidence of diarrhea.

Some studies do not reported the existence of coronaviruses in other species of owls, such as burrowing owl (Speotyto cunicularia), tropical screech-owl (Otus owl) and barn owl (Tyto alba), but it was found in peregrine-falcon (Falco peregrinus ) (Catroxo et al., 2000; Pongiluppi et al., 2004; Sousa et al.).

During diarrhea outbreaks, coronavirus can be detected in species such as, rhea (Catroxo et al., 1996), turkey (Guy et al.; Breslin et al., 1999), quail (Circella et al.) and in pheasant (Gough et al., 1996; Pennycott), peacock (Liu et al., 2005), pigeon (Jonassen et al.) and psitacids (Gough et al., 2006) with concomitant respiratory problems.

The negative staining technique allowed us to observe viral particles with coronavirus features. Many authors have described similar particles by the same technique in birds (Dea et al., 1990; Catroxo et al., 1996, 2000; Pongiluppi et al., 2004; Liu et al.; Gough et al.; Circella et al., 2007).

In this study, positiveness was obtained by the immunomicroscopy method and sharp marking of this antigen with colloidal gold particles. Other authors confirmed the presence of coronavirus in birds using these techniques. (Dea & Tijssen, 1989; Dea & Garzon, 1991; Gough et al., 2006).

The presence of infectious bronchitis virus in wild and exotic birds can be explained by the interaction between species or by its close proximity with commercial poultry farms (Sousa et al.). Among all hosts, the diversity of coronaviruses is more evident in bats and poultry, as a result of species diversity, ability to fly, environmental pressure and habits of roosting and flocking (Woo et al., 2009).

In addition to paramyxovirus and coronavirus particles, the retrovirus was found in samples of liver and small intestine of one striped-owl (owl 4). The literature reports early findings of this virus in the small intestine of pale-breasted thrush (Turdus leucomelas) (Catroxo et al., 2006;), in nodules of gizzard ruddy ground-dove (Columbina talpacoti) (Pongiluppi et al., 2006) and roller canaries (Serinus canarius) (Martins et al.).

The retrovirus was observed in cases of lymphoid leukosis in wild bird columbiformes, psittaciformes and passerines (Nobel; Palmer & Stauber, Wadsworth et al.; Loupl; Martins et al.; Catroxo et al., 2006; Pongiluppi et al., 2006). Free-living birds as the house sparrow (Passer domesticus) may harbor the leukosis virus, acting as disseminators especially in areas near poultry farms (Varejka & Tomsik).

Although in this disease, nodules may develop in any visceral area or skin of the animal (Harrison & Harrison), we did not check the presence of these in the organs examined from the two striped-owls (owls 3 and 4).

A published article reported the presence of nodules in the gizzard of ruddy ground-dove (Columbina talpacoti) (Pongiluppi et al., 2006).

Although the four owl died without preliminary presentation of clinical signs, according to Wadsworth et al. the manifestation of these signs is variable and many times the affected birds are found dead without prior disclosure of clinical disease .

The positive results we obtained from the reaction of immunoelectron microscopy for retrovirus was signaled by the presence of aggregates formed by antigen-antibody interaction. Valicek et al. (1985) applying this technique observed retrovirus particles in enzootic bovine.

These retroviruses were also intensely labeled by colloidal gold, when we applied the immunocytochemistry method, also used to detect porcine endogenous retroviruses (Fischer et al., 2003).

The techniques used allow a quick visualization of viral particles. They are extremely effective in routine diagnosis of several avian viral pathogens. Additionally, it is worth emphasizing that the negative staining technique is one of the tests required by the OIE for the diagnosis of avian infectious bronchitis virus (OIE, 2008).

The accurate knowledge of viral diseases affecting birds of prey is of great importance, since some species, like the falcon peregrinus are constantly threatened by these viruses (Sander, 1995; Schettler et al.).

Owls deserve special attention in their preservation , their importance in the environment is vital, as is their cultural significance. (Sick, 1997).


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Correspondence to:

Prof. Dr. Marcia Catroxo
Electron Microscopy Laboratory
Research and Development Center in Animal Health
Biological Institute of São Paulo
Av. Conselheiro Rodrigues Alves, 1252
CEP 04014-002
Vila Mariana, São Paulo, SP


Received: 11-03-2010
Accepted: 04-04-2010

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