Post mortem, histopathological, immunohistochemical, transmission electron microscopic and bacteriological examination
Diagnostic examination was carried out usually on 3–5 animals per flock (altogether 120), died during the first days of epidemic, rarely on those in live, moribund state. For detailed post mortem examination brain, heart, pancreas and lung and sometimes trachea, spleen, liver, kidney, small intestine and parts of skeletal muscle (pectoral muscle) and skin taken from 1–3 animals per flock were fixed in 10% buffered formaldehyde solution and embedded into paraffin. Immunohistochemical (IH) examination aiming at the detection of influenza A virus was carried out as described before (30).
For the purpose of transmission electron microscopic examination from the material taken from the carcasses and previously fixed in formaldehyde solution we cut small (1X1 mm2, 3 mm long) pieces from various organs (brain, pancreas, myocardium, kidney, lung) of 2–2 animals of both species. Formaldehyde was washed out from the excised material. The excised parts of organs were prefixed for 2–3 hours at +4 oC in paraformaldehyde of 4% prepared with 0.2 M buffered phosphate solution (PBS) of pH 7.2 and in glutaraldehyde of 0.2%. Fixing preparation was washed out from the prefixed material in 0.2 M PBS for 4X5 minutes. Afterwards, we postfixed it for 2 hours at room- temperature in OsO4 solution of 1% prepared with 0.2 M PBS. Fixing preparation was washed out from the postfixed material in 0.2 M PBS for 4X5 minutes. Afterwards, we dehydrated the material in ascending ethyl alcohol-series (for 10’ in 30%, for 15’ in 50%, for 30’ in 75%, for 2X30’ in absolute ethyl alcohol, for 2X15’ in propylene-oxide). We soaked the dehydrated material with a 1:1 mixture of propylene-oxide-ACM Durcupan (Fluka AG, Switzerland), then with pure Durcupan in a thermostat of 56 oC for 30–30 minutes. Thereafter, the material was embedded into Durcupan in casting mould. The material embedded into Durcupan was placed in a thermostat of 56 oC for 48 hours for polymerisation. We prepared 40–60 nm thick sections from the embedded material by Reichert UM U3 type (Austria) ultramicrotome. The sections were contrasted for 20 minutes in uranyl-acetate solution of 2.5%-prepared with distilled water, then we washed them out once in ethyl alcohol of 50%, then 3 times in changed distilled water. After washing out, for increasing contrast effect, we placed the sections for 10 minutes in Reynolds lead-citrate solution of pH 12, followed by washing 4 times with changed distilled water. The sections were examined in JEOL JEM-1011 (Japan) type transmission electron microscope. The changes found during the evaluation of sections were digitally recorded with the built-in Gatan Bioscan 792 Multi.Scan CCD Camera System device (Gatan GmbH, München, Germany) and worked up with computer controlled image management system (analySIS, Soft Imaging System GmbH, Münster, Germany)
Bacteriological examination were carried out in all cases from the liver and heart blood on conventional agarose slides containing 10% sheep blood, under aerobe conditions at 37 oC. Occasionally there was anaerobe culture from the cerebral ventricles.
Molecular biological examination
We carried out the detection of influenza virus as described by SPACKMAN et al. (24), and the identification of H5 and N1 gene according to conventional agarose gel or real-time RT-PCR protocols, stipulated or recommended by Central Reference Laboratory of Weybridge (9) or WHO (22).
Pathogenicity of the virus established by sequencing the RNS (cDNA) section including protease cleavage site place found in H5 gene. The result of our tests was confirmed in all cases by the Central Reference Laboratory of Weybridge.
Virological examination
Isolation of viruses were carried out by allantoic cavity inoculation of 9–11-day-old embryonated SPF fowl eggs, according to the standards of Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (17). Identification of haemagglutinin subgroup of the isolates was carried out by haemagglutination inhibition test (HAI), by using H5 and H7 polyclonal reference sera (Veterinary Laboratories Agency (VLA), Weybridge, UK). Identification of neuraminidase subgroup was carried out at the avian influenza reference laboratory of EU (Avian Virology Laboratory, VLA, Weybridge, UK).
Results
Pathological examination
Pathological examination revealed similar lesions in the goose and duck carcasses but more severe and extent in geese. There was no significant difference in the frequency of changes (Table). In peracute cases the animals were in moderate, while in acute cases in a bit weaker condition. Conjunctiva was congested, eyeballs were hollow and signs of exsiccosis were frequently observed. Around the eyelets and nostrils small amount of serous discharge was visible. In many animals perianal region was contaminated by fluid faeces. No haemorrhages, necrosis in the skin or subcutaneous oedema were detected. Haemorrhages were rarely observed in the muscle. Free pericardial content sometimes increased. Under the epi- and pericardium haemorrhages were frequently visible (Figure 3) and myocardium was greyish-white in strips. Lung was usually congested. Tracheal mucous membrane was usually intact, only occasionally showed congestion or mild oedematous infiltration. Small amount of content was in the digestive tract and there were only occasionally tiny haemorrhages in the mucous membrane. Intestinal tract was always oedematous and covered by catarrhal mucus. In the pancreases haemorrhages and necrotic foci were frequently detected (Figure 4) and (Figure 5). Spleen was intact or sometimes mildly congested. Liver was frequently pale brown and crumbly, the kidneys were congested and rarely swollen.
Histopathological examination
Histopathological examination revealed in almost all cases in both geese and ducks on one or more circumscribed areas (in the brain stem, cortex or cerebellum) lympho-histiocytic perivascular cuffing, focal or extensive glia cell proliferation (Figure 6) , neuronal degeneration and necrosis and focal serous lympho-histiocytic infiltration in the leptomeninx. In some cases there were fresh perivascular haemorrhages (Figure 7). In the pancreas irregularly shaped circumscribed necroses (Figure 8) and in some places of the connective tissue focal lympho-histiocytic infiltration was visible. In some cases there was circumscribed myocardial cell degeneration and lymphocytic and histiocytic infiltration in the myocardium, (Figure 9). In the liver mainly lympho-histiocytic infiltration in the connective tissue and in some cases focal necrosis of the parenchyma was visible. There was congestion, interstitial oedema and rarely, circumscribed catarrhal infiltration in the lung. In the mucous membrane of the intestinal tract catarrhal-inflammatory-cell infiltration, degeneration and desquamation of enterocytes, circumscribed haemorrhages and signs of increase in secretory activity of secretory epithelial cells were observed. In the spleen there was decrease in the number of lymphocytes in Malpighian-corpuscles and frequently haemorrhages. In the kidney there was swelling, degeneration of epithelial cells in some parts of tubules, in the skeletal muscle rarely diapedesic haemorrhages and swelling and blurring of structure of some myofibrils.
Bacteriological examination
Bacteriological examination revealed no causative bacteria in most of the cases. In some cases E. coli bacteria were cultured from the liver and heart blood.
Immuno-histochemical examination
By this method influenza A virus were detected in all of the examined 5 geese, while in none of the 5 examined Pekingese ducks. Presence of the virus was demonstrated in case of four geese only in the brain (Figure 10) , while in one goose from the brain and liver, as well. Virus antigen was present mainly in the nucleus, while it was rarely observed in the cytoplasm. In two geese the virus was present in the cerebrum, brain stem and cerebellum, as well. In the other two birds the virus was demonstrated only in the cerebrum. Cells containing viral antigen were found in the brain of one of the mulard ducks, in one’s lung and in one’s spleen.
Transmission electron microscope examination
Transmission electron microscope examination in geese and ducks revealed oedema surrounding small vessels in the different organs, lung, airways and neuropil in the brain. Influenza viruses could be found in almost all of the examined organs (brain, myocardium, pancreas, lung and kidney). The viruses were found in all of the organs partly extracellularly, in the intracellular space (occasionally or in large number), and partly in the cavity of vessels (either freely in the blood plasma or freely between endothel cell and blood vessel’s basal membrane), partly in the lung in the cavity of small bronchi (parabronchi) and in the cavity of respiratory capillaries freely in the serum or freely between bronchial, and respiratory epithelial cell and basal membrane (Figure 11) . Influenza viruses were usually round-oval shaped, sometimes in a bit different shape, and their size varied between 80 and 150 nm. Occasionally (in some endothel cells and bronchial epithelial cells) virus form proliferating through cell membrane was also found (Figure 12). In the parabronchi, in case of viruses got between basal membrane and respiratory epithelium, protrusion of the basal membrane close to the virus was also visible (Figure 13). Within the cells – due to advanced autolysis – incomplete forms of influenza viruses (nucleocapsid plexus) were in none of the cases clearly detected.
Virological and molecular biological examination
By virus isolation and by PCR test confirmed also with sequencing we identified highly pathogenic H5N1 strain in all the examined birds (geese, broiler ducks, mulard ducks)
Discussion
Epidemic diseases caused by highly pathogenic strain (H5N1 subtype) of avian influenza have been detected in the countries of Far-East since 1997, and passing gradually into Western direction reached more countries of the European Union, including also Hungary at the beginning of 2006 (7, 18, 19, 23, 33). In Hungary in the first quarter of the year deaths caused by H5N1 virus involved mainly swans (19), while in the second quarter many domestic waterfowl (goose, duck) flocks got also infected. Positive cases occurred in the Southern areas of the central part of the country.
According to previous experiences, highly pathogenic H5N1 strain of avian influenza caused fast, acute disease and death in the different bird species (fowls, turkey). In the dead animals macroscopic and microscopic lesions were reported, accompanied by haemorrhages, oedema and necrosis (oedematous infiltration in the skin of neck, head and legs, pulmonary oedema, haemorrhages in the skin, under serous membranes and in the mucous membranes, in the pectoral muscles, necrosis in the skin, pancreas, spleen and myocardium) (1, 2, 3, 5, 13, 28, 29). In case of peracute course, beside signs of acute circulatory failure, other lesions may have been absent. From histopathological lesions, lymphocytic encephalo-meningitis, focal myocardial degeneration and lympho-histiocytic infiltration and focal necroses are thought to be pathognomic, mainly in the pancreas and occasionally in other organs, e.g. liver, lung, lymphoid organs and skeletal muscles (1, 2, 3, 5, 13, 28, 29). On the basis of observations in transmission electron microscope examinations, distribution and localization of viruses in the organs are typical to disease, when the animals die during viraemic phase, when the body is overrun by viruses.
Reservoirs of influenza viruses are thought to be different wild waterfowl species, including ducks, geese and swans belonging to Anseriformes order, because different virus strains were isolated in large number from the clinically healthy birds (2, 3, 6, 11, 21, 26, 29). It is explained by the fact, that waterfowls, mainly wild species are less sensitive to the different avian influenza virus strains (35).
But during avian influenza epidemic at the end of 2003 and at the beginning of 2004 in South-East Asian countries (Cambodia, China, Indonesia, Japan, Laos, South-Korea, Thailand and Vietnam) H5N1 virus killed the different wild and domestic waterfowls in large numbers. In this area approximately 100 million birds died or were killed (18). This type of H5N1 virus occurred also in Central Asia, Middle-East and in many African countries and caused the death of mainly mute swans and in smaller proportion of other water birds. Birds of prey and gulls were rarely diseased.
In the first quarter of 2006 H5N1 virus occurred also in Hungary causing death of birds of prey. During examinations of swans’ deaths occurred in Hungary, we found such gross pathological and histopathological lesions that were typical to disease of fast course (19).
On the domestic geese and ducks got ill in the second quarter of 2006 acute-subacute course was detected, as well. Neurological signs occurred regularly and in typical form. Pathological-histopathological lesions were similar to those presented in scientific literature in case of avian influenza of fast course (1, 2, 3, 5, 13, 28, 29). However, skin changes (cyanosis, oedema, haemorrhage, necrosis) were absent in waterfowls. In the background of neurological signs, lymphocytic encephalitis was identified.
According to our knowledge, there are only a few data known on the demonstration of avian influenza virus from geese by IH method (20). H5N1 subtype was mainly detected in the central nervous system, accompanied by characteristic histological changes. This corresponds to the virus localization found in the different bird species (duck, crow, pigeon) by others (8, 11, 12, 31) and strengthens further neurotrop characteristic of H5N1 subtype (8, 11, 12, 30, 31). Earlier, in mute swan (25) we also detected avian influenza virus mainly from central nervous system and from the changed part of other organs (pancreas, lung, liver, spleen, intestines). However, by this method the virus was not found in Pekingese duck, while in mulard duck it was found in small quantity in some infected cells, in spite that H5N1 virus was isolated from them and histological lesions (encephalitis) caused by it could be demonstrated, as well. Histological lesions detected in the brain were, however, milder, compared to those in geese. Lower sensitivity of Anseriformes to H5N1 subtype was observed by others, as well (8, 21).
American authors have examined the sensitivity of different species to H5N1 virus strain isolated in Hong Kong in 1997. According to their findings, the virus strain killed 100% of gallinaceous (birds of 7 different species), while 75% of parrots. From the other examined species it has not killed, however, geese, ducks and many other wild bird species (emu, gull, pigeon, starling, sparrow). From them, geese, ducks, gulls, emus and sparrows have got ill and avian influenza virus caused pathological lesions, while pigeons and starlings have not got ill and there was no lesions found in their organs.
During the epidemic of 2003–2004 in South-East Asia and also in 2006 in Hungary H5N1 virus killed with subacute course the different waterfowl species (18, 19). During our domestic examinations the pathological and histo-pathological changes corresponded to the lesions described as the effect of highly pathogenic avian influenza strains, and in all cases neurological signs and lesions developed, as well. The establishment of morbidity and mortality data at flock level of the diseases in the Hungarian waterfowl (goose, Pekingese duck, mulard duck) flocks was not possible because of official measures (killing). We have not carried out infection trial yet. There was no human case.
From the applied diagnostic tests, classical histopathological examination (detection of encephalitis) and virus isolation and PCR gave the fastest positive result. Immuno-histochemical, RT-PCR, in situ hybridization and transmission electron microscopic examinations can give the opportunity to determine virus’ organ-tropism and the quantity of virus detectable in the organs and to study the interaction between the virus and cells, and subcellular changes in cells.
Figure 1. Nasal discharge and lacrimation
(duck infected by H5N1 virus)
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Figure 2. Neurological signs (deviation of the head)
(duck infected by H5N1 virus)
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Figure 3. Hydropericardium, haemorrhage under epicardium
(duck infected by H5N1 virus)
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Figure 4. Necroses and haemorrhages in the pancreas
(goose infected by H5N1 virus)
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Figure 5. Necroses and haemorrhages in the pancreas
(goose infected by H5N1 virus)
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Figure 6. Lymphocytic perivascular cuffing and glia cell profileration in the brain
(goose infected by H5N1 virus)
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Figure 7. Haemorrhages around the damaged vessels in the brain
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Figure 8. Circumscribed necrosis in the pancreas
(duck infected by H5N1 virus)
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Figure 9. Focal myocardial cell degeneration and lymphocytic infiltration
in the myocardium (goose infected by H5N1 virus)
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Figure 10. Goose, cerebellum. Strong staining indicates the presence of
influenza virus in Purkinje cells and in wide area of molecular and granular layer
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Figure 11. Goose, lung. Parabronchus. Influenza viruses between two bronchial epithelias cells (intercullalarly)
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Figure 12. Goose, lung. Parabronchus. Influenza virus proliferating on the basal membrane of bronchial epithelial cell
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Figure 13. Goose, lung. Parabronchus. Basal membrane of the bronchus widens out before influenza viruses got between bronchial epithelial cell and basal membrane
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