Avian encephalomyelitis in naturally infected pigeons in Turkey

Abstract

The pathological and immunohistochemical findings of avian encephalomyelitis (AE) were described in various tissues of naturally infected pigeons of a flock from a outbreak in Turkey. Clinically, paresis, paralysis, circling movement and torticollis of the head associated with nervous signs were marked symptoms among the diseased pigeons. At necropsy, small or large white–greyish foci were detected in the pancreas, and erosive-ulcerative foci along with petechial hemorrhages in ingluves. Histopathologically, lesions in central nervous system, particularly in the cerebullum molecular layer, consisted of non-suppurative encephalomyelitis. Lesions in the pancreas revealed non-suppurative pancreatitis along with acinar degeneration and necrosis and/or lymphoid aggregations. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissues was performed using a direct-fluorescein antibody technique with chicken anti-AE virus serum flourescein isothiocyanate conjugate. Viral antigen was strongly stained in cytoplasm of epithelial cells of the exocrin glands, and neurons of the cerebral hemispheres and midbrain. In addition, viral antigen was also marked in the kidneys and tissues of the digestive system. Consequently, this article is, to our knowledge, the first report of natural AE in pigeons.

Introduction

Avian encephalomyelitis (AE) was first diagnosed and classified as a neurotropic viral infection of young chicks in 1934, and then reported in pheasents, quails and turkeys (Mathey, 1955; Hill & Raymond, 1962; Hohlstein et al., 1970; Deshmukh et al., 1971; Calnek et al., 1997). Pigeons, ducklings and guinea fowls could be infected experimentally (Mathey, 1955; Calnek et al., 1997). The causitive virus (AEV) is in the family Picornaviridae, the sequences of its proteins suggesting its inclusion in the genus Hepatovirus (Marvil et al., 1999).

In young birds typical symptoms are characterized with neurological signs such as ataxia, paresis or paralysis and rapid tremors of the head and neck (Markson & Blaxland, 1958; Maas & Helmboldt, 1962; Itakura & Goto, 1975), whereas in older birds infection is subclinical, resulting in declines in egg production and hatchability (Feibel et al., 1952; Taylor et al., 1955; Calnek et al., 1960; Cheville, 1970). Histologically, AE lesions are of two general types. Changes in the central nervous system (CNS) have been characterized as non-suppurative encephalomyelitis (Mohanty & West, 1968; Yamagiwa et al., 1969; Itakura & Goto, 1975; Hishida et al., 1986). Lesions in visceral organs consist of lymphoid aggregates that are either increased in size or frequency or are found in unusual places (Markson & Blaxland, 1958; Hoekstra, 1964; Springer & Schimittle, 1968; Butterfield et al., 1969; Malik, 1969; Toplu, 2000). Lymphoid aggregates in proventriculus are considered to be pathognomic, especially when coupled with central chromatolysis from lesions of the CNS (Calnek et al., 1997). The aim of this study is to describe the pathological and immunohistochemical findings in naturally infected pigeons in Turkey.

Materials and Methods

Animals

Two 1-year-old-pigeons, one dead (case 1) and one alive showing nervous signs (case 2) from a flock composed of 60 animals were presented in Ankara by the owner for laboratory investigation. These animals that are not included in any group were not vaccinated against any disease. The flock history was that one pigeon had been acquired from another flock in Urfa province and showed nervous signs 1 week later. Then 36 birds in the loft died showing the same symptoms within 20 days. The owner reported that the breeding programme was continued with the surviving 24 birds and new pigeons acquired subsequent to the mortalities. It was also reported that especially young pigeons were affected by the disease. For diagnosis of the disease, we applied the direct fluorescent antibody method with chicken anti-Newcastle disease virus antibody, which is antigenically closely related to paramyxovirus 1, and with anti-AEV antibody. The results were positive for AEV and negative for Newcastle disease virus. Following this, we obtained another nine sick pigeons (cases 2 to 11) from the same flock for detailed examination at various times.

The animals were euthanized and necropsied. Tissue samples were collected from various organs including the brain and medulla spinalis, pancreas, proventriculus, gizzard, intestine, liver, spleen, kidneys and lungs, heart. Then, the tissues were fixed in 10% neutral-buffer formalin solution, embedded in paraffin, sectioned at 4 μm and stained with haematoyxylin and eosin (H&E). The selected brain and medulla spinalis sections were stained by cresyl-violet for Nissl granules (Luna, 1968). The replicate sections were used for immunohistochemical staining.

Preparation of hyperimmun serum and conjugate

The preparation of chicken hyperimmune serum against AEV, and flourescein isothiocyanate (FITC) conjugate was carried out as described by Gialetti et al. (1972) and Johnson et al. (1978). Specific pathogen free chickens, 6 weeks of age, were inoculated intramuscularly with a mixture of 1 ml van Roekel virus strain and 1 ml Freund's complete adjuvant three times at 7-day intervals. The chicks were bled by cardiac puncture 15 days after the last injection and serum was pooled.

The gamma-globulins from the pooled antiserum were fractionated and conjugated with FITC. Gammaglobulins were initially separated using ammonium sulphate prepicipitation. IgG was fractioned using saphadex G-25 (Pharmacia Fine Chemicals) and conjugated with FITC (Fluka AG). This complex was purified.

Immunohistochemistry

The selected sections were placed on glass microscope slides coated with poly-l-lysine. After 2 h incubation at 37°C, sections were deparaffinized in xylene and washed briefly in phosphate-buffered-saline (PBS) solution (pH 7.3). Tissues were digested with 0.1% protease K for 10 min at 37°C. Slides were washed for 15 min in PBS for fluorescein antibody staining. Sections were incubated with chicken anti-AEV serum conjugated with FITC in a 1:16 dilution for 45 min at 37°C, and then washed for 15 min in PBS. They were then washed in PBS for 15 min and mounted in phosphate-buffered glycerin (pH 9.0). For control slides, replicate sections of selected infected tissues were processed, substituting chicken anti-Newcastle disease virus antibody in 1:32 dilution (against La Sota strain) conjugated with FITC for anti-AE serum conjugated with FITC. In addition, the tissues were also stained with an indirect immunoperoxidase method against chicken anti-paramyxovirus 1. The results of the fluorescent antibody reaction were determined using a fluorescent microscope (Leitz-Laborlux D, binocular microscope equipped with a dry darkfield condensor).

Results

Clinical findings

Nervous signs were prominent among the diseased pigeons. These findings were seen in young pigeons (especially aged <1 and 1 to 3 months) rather than adult pigeons. At first, there was depression and anorexia, followed by paresis or paralysis of the extremities and wings (Table 1). In addition, circling movement, opisthotonus, torticollis and tremor of the head, and inability to fly were also seen (Figure 1). Diarrhoea and emaciahia was also noted, especially in adult pigeons. Moreover, a drop in egg production was emphasized by the owner.

Figure 1. Appearance of a naturally affected pigeon, 5 months of age, with torticollis.

Table 1. Clinical signs and necropsy findings in 11 naturally infected pigeons.

Case	Age	Clinical sings	Necropsy findings
1	3 months	Paralysis	Large white–greyish foci in the pancreas and kidneys, erosive-ulcerative foci with petechial haemorrhagia of the crop
2	3 months	Death	No finding
3	20 days	Death	Small white–greyish foci in the pancreas, erosive-ulcerative foci with petechial haemorrhagia of the crop
4	6 months	Torticollis, diarrhoea	Large white–greyish foci in the pancreas, pin-point white foci in the kidneys
5	6 months	Incoordination, head circling	No finding
6	2 months	Paralysis, tremor	No finding
7	2 months	Paresis, diarrhoea	Small white-greyish foci in the pancreas, pin-point white foci in the kidneys
8	5 months	Opisthotonos, tremor	Small white–greyish foci in the pancreas
9	12 months	Incoordination, diarrhoea	No finding
10	4 months	Paralysis	No finding
11	12 months	Diarrhoea	No finding

Necropsy findings

At necropsy gross lesions were detected in the pancreas, kidneys and crop. Lesions in the pancreas of five pigeons had small, roughly circular, white–greyish foci (cases 4, 7, 8, 11), and in some animals (cases 1 and 4) such lesions extended across a lobe (Figure 2). No pancreatic lesions were present in six cases (Table 1). Similar foci were also observed on both kidneys of three birds. Superficial erosive-ulcerative lesions with petechial haemorrhagie into mucosa appeared in crops of two pigeons (cases 1 and 3).

Figure 2. Large white–greyish foci extended across a lobe on the pancreas.

Histopathological findings

The most significant microscopic findings were in the pancreas, brain and kidneys. Pancreatic lesions seen in all cases were of two different types. The first was characterized by non-suppurative pancreatitis along with acinar cell degenaration and necrosis. Exocrine glands were usually destroyed and replaced by mononuclear cell infiltration including lymphocytes, macrophages and plasma cells together with a few heterophils. In these areas, there was cystic dilatation of exocrine glands in which epithelial cells were flattened (Figure 3). In some areas, hydropic degeneration was evident in acinar cells. The second finding was of lymphoid aggregates consisting of well-differentiated lymphocytes (Figure 4).

Figure 3. Mononuclear cell infiltration and cystic dilatation of exocrin glands in the pancreas. H&E, ×20.

Figure 4. Lymphoid follicular aggregations in the pancreas. H&E, ×20.

Lesions in the CNS were characterized by non-suppurative encephalomyelitis. The most striking changes were detected in the cerebellum. Purkinje cell degeneration and gliosis were evident in molecular layer (Figure 5). In some areas, Purkinje cells were replaced by glial cells. In cerebral hemispheres, the midbrain, medulla oblongata and medulla spinalis, there was mild perivascular mononuclear cell infiltration and gliosis (Figure 6). Neuronal changes, characterized by chromatolysis and neuronal shrinkage, were other prominent lesions in the CNS. Central or total chromatolysis was best appreciated in large neurons of the midbrain, and motor neurons of the medulla oblongata and medulla spinalis particularly in young pigeons (Figure 7). Some motor neurons were stained poorly with cresyl-violet or eosin, and appeared as ghost-like outlines. In later stages, the neurons lost. No lesion was observed in the CNS of three birds.

Figure 5. Gliosis and Purkinje cell degeneration in the molecular layer of the cerebellum. H&E, ×20.

Figure 6. Perivascular lymphocyte infiltrations and gliosis in the midbrain. H&E, ×20.

Figure 7. Central chromatolysis of the neurons (arrows) in the midbrain. H&E, ×20.

In kidneys of all cases, focal or diffuse non-suppurative interstitial nephritis was seen. Tubules were partly destroyed or absent in areas with diffuse non-suppurative interstitial nephritis. Lymphoid follicular aggregations were found in the spleen and liver. These aggregations in the spleen were generally surrounded with a fibrous band. In addition, necrotic changes characterized with lymphoid depletion were also observed. In the proventriculus and gizzard there were mononuclear cell infiltrations in the lamina propria. In the lamina propria of the small intestine, especially between duodenal crypts, were mononuclear cell infiltrations. In addition, lymphocytic aggregations were localized in muscular layers of the small intestine of two cases. In the crops there were acute erosive-ulcerative changes. The ulcerated fields were filled in intense heterophils and macrophages. In addition, in three pigeons from other cases, mononuclear cell infiltrations in propria mucosa were present. In the bronchi, parabronchi and alveolar areas of three birds, focal lymphocyte infiltrations were seen.

Immunohistochemical findings

In the pancreas, AEV antigen was localized in the cytoplasm of epithelial cells of exocrine glands and in the infiltrated macrophages (Figure 8). Viral antigen was generally more abundant in a section of degenerate necrotic pancreas than in those with lymphoid follicular hyperplasia. AEV antigen in the CNS was seen particularly in the cerebral hemispheres and midbrain. The medulla oblongata and medulla spinalis had a slight antigenic reaction, which was limited in only small neurons. Viral antigen was found in the cytoplasm of neurons of nuclear groups of the midbrain and cerebral hemispheres of diffuse and granular shape (Figure 9). It was also localized in the neuropile and cytoplasm of endothelial cells in these areas. AEV antigen in the cerebellum was detected in neurons of molecular and granular layers, and in substantia alba. No staining was observed in Purkinje cells and ganglion cells in the cerebellum.

Figure 8. Intense FA-positive reactions in exocrin glands of the pancreas. ×20.

Figure 9. Abundant FA-positive neurons in the cerebral hemisphere. ×20.

AEV antigen in the spleen was concentrated in surrounding areas of the lymphoid follicles. In the kidneys, it was in tubular epithelial cells as well as infiltrated mononuclear cells. Viral antigen was found to be disperse in the endothelial cells and Kupffer's cells of liver, and in the infiltrated mononuclear cells. In the proventriculus, gizzard and intestinum, viral antigen was detected especially in mononuclear cells in the lamina propria and intraepithelial lymphocytes in the epithelial layer. In crop sections with erosive-ulcerative changes, AEV antigen had accumulated in the epithelial cytoplasma as well as in the infiltrated mononuclear cells (Figure 10). Occasional positive cells were scattered in the lung sections.

Figure 10. Localizations of AE viral antigen in epithelial cells and infiltrated mononuclear cells in the crop. ×20.

Control slides were negative. Indirect immunoperoxidase staining was also negative for paramyxovirus 1 infection.

Discussion

In this study, clinical symptoms and pathological findings are reported for naturally AEV-infected pigeons. The localization and distribution of AEV antigen was detected in tissue sections by immunohistochemistry.

The most common cause of nervous symptoms and diarrhoea in pigeons is avian paramyxovirus 1 (APV-1) infection or Newcastle disease (Alexander & Parsons, 1984; Eisa & Omer, 1984; Barton et al., 1992). Falluji et al. (1978) determined encephalomyelitis associated with nervous signs in pigeons in Baghdat in 1977, and then a transmisson experiment was carried out with the isolated virus in pigeon. However, it is reported that the isolated virus has not been identified in another study following this (Tantawi et al., 1978). Also, a herpesvirus from pigeons showing a rapidly progresing paralysis has been isolated and the infection was induced after inoculation of this isolate via intracerebrally and orally (Mohammed & Sokkar, 1981).

AE is predominantly a disease of 2-week-old to 4-week-old chicks, whereas hosts of APV-1 infection are affected in any period of age. APV-1 infections during this period (2 to 4 weeks old) may cause a problem with differential diagnosis (Calnek et al., 1997). No AE has been reported previously in pigeons. The disease clinically described in the present study was consistent with APV-1 infection rather than AE. For this reason, it is suggested that diagnosis related to nervous symptoms may not be adequate for APV-1 and AE infections.

At necropsy, no gross lesions were seen, aside from occasional white–greyish foci in the pancreas, proventriculus and myocardium of AEV-infected chicks (Burtscher, 1960; Malik, 1969; Springer & Schimittle, 1968; Butterfield et al., 1969). However, in the present study, macroscopic lesions described in the pancreas were frequently a prominent finding, associated with massive mononuclear infiltrations, as reported in chicks (Calnek et al., 1997).

Histopathologically, lesions of the CNS consist of non-suppurative encephalomyelitis both in AEV and APV-1 infection. Certain lesions are peculiar to AE. Vascular changes consist of intense mononuclear cell infiltrations in the perivascular cuff and type gliosis, which are generally severe according to APV-1 infection (Helmboldt, 1972; Calnek et al., 1997). Histopathological changes in the CNS in the present study, however, were mild, and generally limited in the cerebellum and midbrain of most cases.

Neuronal changes in these cases were generally characterized with central or total chromatolysis and neuronal shrinkage. Neuronal shrinkage is also described in natural and experimental AEV infections of chicks (Burtscher, 1960; Maas & Helmboldt, 1962; Mohanty & West, 1968). Toplu (2000) reported neuronal shrinkage to be the most prominent finding according to central chromatolysis in experimental infection of quails. On the other hand, central chromatolysis and lytic alteration of neurons, pathognomic in the diagnosis of AE (Mohanty & West, 1968; Itakura & Goto, 1975; Hishida et al., 1986; Calnek et al., 1997), was not observed in all cases, except for generally in young pigeons. Therefore, the findings in the CNS of the pigeons did not perfectly corroborate histopathologic findings of AE.

Lymphoid aggregations in the pancreas of pigeons are similar to those described by other investigators (Markson & Blaxland, 1958; Butterfield et al., 1969; Malik, 1969). On the other hand, non-suppurative pancreatitis with acinar degeneration and necrosis has not been noted previously in AE, although similar lesions in the pancreas have been described in turkey viral hepatitis from picornaviridae (Guy, 1997). Changes described in pancreas in this study may be considered as the most characteristic findings of AE in pigeons. According to histopathological findings and localization of AEV antigen, it is most possible that AEV affecting pigeons may have a far higher enterotropic than neurotropic character. Therefore, we are going to carry out further studies, transmission and cross-transmission experiments, with brain and pancreas inoculum obtained from pigeons.

The immunohistochemical technique has been used in definitive diagnosis and pathogenesis studies of AE (Mohanty & West, 1968; van der Heide, 1970; Braune & Gentry, 1971; Miyamae, 1983). In natural and experimental AE infections, intense viral antigen has been detected especially in the CNS as well as in samples of pancreas, heart and proventriculus (Miyamae, 1974; Hishida et al., 1986; Toplu, 2000). In our study, immunohistochemistry revealed that AEV antigen was especially found in the pancreas, kidneys and tissues of digestive system from visceral organs. It was also detected in the CNS strongly, despite the histopathological changes of the CNS being generally mild in character. Mohanty & West (1968) suggest that AEV antigen gradually increases with the progression of neuronal degenerate necrotic changes, but with intense inflammation such as perivascular cuff, gliosis and mononuclear infiltration viral antigen decreases until it is not detectable by immunohistochemistry. In contrast, in the present study, inflammatory changes were mild, viral antigen generally limited in small neurons especially of the medulla oblongata, medulla spinalis, and cerebellum. Furthermore, we have also recorded in an another study that there are significant sensitivity differences between fluorescein antibody and avidin–biotin peroxidase methods in detection of AEV antigen in the CNS of naturally infected chickens (unpublished data).

In conclusion, AE in pigeons should be differentiated especially from APV-1 infection, which has frequently been reported in pigeons (Alexander & Parsons, 1984; Eisa & Omer, 1984; Barton et al., 1992). Although clinical and pathological findings are partly useful in diagnosis, AEV antigen is most certainly required to detect for AE of pigeons. On the other hand, further detailed studies on the agent of pigeon AE should be carried out. AEV infection has not been previously reported naturally in pigeons.

Acknowledgements

The authors are grateful to Dr Fethiye Coven, Manisa Research Institute of Avian Vaccine Production and Control, for supplying chicken anti-Newcastle virus and anti-paramyxovirus-1 antibody.