Gross- and histopathological lesions caused by highly pathogenic avian influenza virus (H5N1) in mute swans (Cygnus olor)
 
Preliminary report
 
PÁLMAI Nimród, DEIM Zoltán, ERDÉLYI Károly, BÁLINT Ádám, DÁN Ádám, MÁRTON Lázár, GLÁVITS Róbert*
 
Central Veterinary Institute. Tábornok u. 2. H-1149 Budapest. *E-mail: glavitsr@oai.hu
 
Summary
The authors report the results of pathological examinations carried out on 10 mute swans (Cygnus olor) which succumbed to a H5N1 highly pathogenic avian influenza virus (HPAI) infection during an outbreak in Februay 2006 in Southern Hungary.
The most frequently observed macroscopic lesions included haemorrhages under the epicardium, in the proventricular mucosa, pancreas and sometimes skeletal muscles; focal necrosis in the pancreas and liver; myocardial degeneration; acute mucous enteritis; congestion of the spleen and lung and the accumulation of sero-mucinous exsudate in the body cavity.
Histopathological lesions comprised lymphocytic meningo-encephalomyelitis accompanied by gliosis and occasional perivascular haemorrhages; multi-focal myocardial degeneration with lympho-histiocytic infiltration; pancreatitis with focal necrosis; acute desquamative mucous enteritis; lung congestion and oedema; oedema of the tracheal mucosa and in young birds, the atrophy of the bursa of Fabricius as a result of lymphocyte depletion and apoptosis.
The observed lesions and the good to moderate body conditions were compatible with findings in acute HPAI infections of other bird species reported in the literature. Skin lesions and lesions typical for infections caused by strains of lower pathogenicity (LPAI) like emaciation or changes in the reproductive and respiratory organs, sinuses and air-sacs were not observed.
 
The term “influenza” refers to disease of humans, horses, pigs, numerous bird species, mink and sea mammals manifested as an upper respiratory tract infection caused by viruses of the Orthomyxoviridae family (12, 16, 20, 37). The manifestation of influenza virus infections in poultry species ranges from latent infection through upper respiratory disease and lowered egg production to peracute, systemic disease leading to 100% mortality (11). The latter form is caused by highly pathogenic avian infkluenza (HPAI) strains (24,26, 27, 28, 31), belonging to H5 and H7 subtypes of the virus. However not all H5 subtypes are highly virulent to birds.
In wild birds, most infections are inapparent, resulting in a carrier state (31). However HPAI subtypes, like the H5N1, may cause lethal infections in some wild bird species. This HPAI strain emerged from Hong-Kong in 1997 and spread through Korea, Thailand, Indonesia, Laos, Cambodia, Malaysia, Vietnam, Japan, China, Taiwan, The Philippines and then gradually west to Mongolia, Kazakhstan, Russia, Romania, Turkey, Greece, Croatia and very recently to many countries of the European Union, including Hungary (6,14), confirmed mainly in wild birds.
Certain observations on the susceptibility of swans to influenza viruses and their possible epidemiological role were reported from Japan in 1982 (23) and in connection with the present epidemics (6, 14). However, detailed descriptions of the course, pathogenesis and pathological lesions caused by H5N1 infection in swans are not available.
 
Own experiences
The study was conducted on 10 randomly selected mute swan carcasses submitted for post mortem examination to the Central Veterinary Institute during the second half of February 2006. All birds were infected with an H5N1 strain of HPAI and died during the epidemic affecting an area along the left bank of the river Danube in Southern Hungary.
In each case tissue samples were taken from lung, trachea, brain, spinal chord, heart, pectoral and thigh muscles, liver, pancreas, spleen, kidney, proventriculus, small and large intestine and when available, from bursa Fabricii. Tissues were fixed in 10% buffered formaldehyde solution and embedded into paraffin blocks, then cut into 3–4 μm thin sections and stained hematoxilin-eosin (H.-E.).
Virus isolation was carried out by inoculation of embryonated SPF fowl eggs, according to the procedures described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE 2005) (5) and evaluated accordingly. The typing of the hemagglutination activity of the isolates was carried out with H5 and H7 subtype-specific, polyclonal sera (Veterinary Laboratories Agency – VLA, Weybridge, UK). Identification of the N subtype of virus isolates was performed by the OIE Reference Laboratory for Avian Influenza (Avian Virology Laboratory, Veterinary Laboratories Agency, Weybridge, UK).
The results obtained by classical virological methods were also confirmed by molecular methods recommended by the OIE Reference Laboratory. The H5 gene was identified by TaqMan PCR, while the genetic marker of high pathogenicity (protease cleavage site) was identified by sequencing of adequate PCR products recommended by VLA. Results of all our tests were confirmed by the analyses carried out at the VLA, Weybridge.
 
Results
The frequency of observed pathological lesions is presented in the following Table and illustrated with Figures 1–13.
All swans submitted for post mortem examination were in good to moderate body condition, which is an indication of the acute course of the disease. The observed lesions were similar to those described in other bird species (1, 2, 3, 4, 17). However, lesions of skin and head appendices (e.g. cyanosis, haemorrhages, necrosis, periorbital oedema) observed in HPAI infections of domestic fowl were not present. Lesions of the upper respiratory tract, sinuses, air sacs or reproductive organs which are typical for infections by AI strains of low pathogenicity (13, 21) were also absent.
 
Discussion
Macroscopic and histopathological changes caused by avian influenza viruses of all pathogenicity levels are widely reported in the literature. Most of these reports present observations on natural and experimental infections of domestic fowl and turkeys (7, 8, 9, 10, 15, 18, 19, 22, 24, 25, 30, 31, 33, 34, 35, 36, 37). There are also a few reports on avian influenza infections in domestic ducks, geese and emus (3, 4, 10, 19, 31, 36).
In chicken and turkeys macroscopic and microscopic lesions caused by HPAI infections are dominated by acute haemorrhages, oedema and necrosis (e.g. oedema in the skin of the head, neck and legs, in lungs; haemorrhages in the skin, under serous membranes, in the mucosa, and pectoral muscles; necrosis in the skin, pancreas, spleen and heart) (1, 2, 3, 4, 17, 31, 35). Specific lesions, apart from signs of circulatory collapse, may be absent in peracute cases. The most important microscopic lesions are lymphocytic encephalitis and meningitis, focal degeneration and focal lympho-histiocytic infiltration of the myocard, focal necrosis in the pancreas, and occasionally in other organs e.g. lungs, lymphatic organs and skeletal muscles. Lesions of the respiratory tract may vary from mild to severe.
Low pathogenic avian influenza strains mainly cause protracted disease accompanied by emaciation and lesions in various organs (13, 21, 29, 31). Gross lesions include sero-mucinous to purulent inflammation in nasal and perinasal cavities, the conjunctiva, trachea and lung, which are frequently accompanied by fibrinous air-sacculitis and peritonitis, inflammation of ovaries, oviducts and enteritis. These latter lesions are usually the result of secondary bacterial infections (e.g. Pasteurella multocida, E. coli). Necrotic pancreatitis is less frequent in chicken than in turkeys.
In Hungary, after 1969 Tanyi (36) diagnosed the occurrence of avian influenza in domestic ducks, muscovy ducks, guineafowl, turkeys and several wild and exotic bird species and isolated several influenza virus strains of various pathogenicity, belonging, among others, to H5 and H6 subtypes.
According to our experiences, gross lesion and histopathological findings – especially lesions in the brain, spinal cord, heart and pancreas – are strongly suggestive of HPAI infection therefore histopathology may be considered an important diagnostic tool.
Our pathological observations suggest that mute swans are highly susceptible to infection with the H5N1 subtype of HPAI, which results in a short illness and high mortality. Therefore, the chances for the development of a protracted disease and a prolonged virus carrier state are unlikely in this species. We believe that these observations provide an important basis for the assessment of the role of swans in the epidemiology of HPAI infections.
 

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Közlésre érk.: 2006. márc. 23.
 


Figure 1. Sero-mucinous exsudate in the body cavity (↑)



Figure 2. Necrotic foci in the pancreas



Figure 3. Irregularly shaped necrotic areas (↑) and haemorrhages in the pancreas



Figure 4. Haemorrhages and areas of focal degeneration in the myocard (↑)



Figure 5. Haemorrhages (↑) on the border between the proventriculus and the gizzard



Figure 6. Spleen congestion (↑) and haemorrhages



Figure 7. Lymphocytic perivascular cuffing, diffuse glial cell proliferation and neuronal degeneration in the brain cortex
H.-E., 200X




Figure 8. Perivascular haemorrhage around blood vessels infiltrated by inflammatory cells
H.-E., 200X



Figure 9. Lymphocytic perivascular cuffing (↑) and oedema in the leptomeninx
H.-E., 200X



Figure 10. Degeneration and necrosis of myocardial cells and lympho-histiocytic infiltration (↑) in the myocard
H.-E., 200X



Figure 11. Necrotic area (↑), vacuolar degeneration (X), and lympho-histiocytic infiltration (L) in the pancreas
H.-E., a – 100X b – 200X



Figure 12. Lung congestion and intestitial oedema (↑)
H.-E., 100X


Figure 13. Oedematous infiltration and widening of the tracheal mucosa
H.-E., 100X


 
Table. Frequency of occurence of macroscopic and microscopic lesions caused by HPAI (H5N1 subtype) infection in 10 mute swans
 

Lesions		Number of affected birds
Body condition	good to moderate	10
Gross lesions
Pancreas	haemorrhages	9
	necrosis	5
Heart	haemorrhages under the epicardium	7
	focal myocardial degeneration	6
Lung	congestion	10
	oedema	10
Air sacs	serosus air-sacculitis	5
Trachea	oedema	6
Proventriculus	haemorrhages	4
Intestines	acute mucous enteritis	9
Spleen	congestion	8
Skeletal muscles	haemorrhages	5
Histopatological lesions
Brain	lymphocytic meningoencephalitis	10
	perivascular haemorrhages	3
Spinal cord	lymphocytic myelitis	6
Heart	focal myocardial degeneration	8
	lympho-histiocytic infiltration	9
Pancreas	necrotic foci	8
	lympho-histiiocytic infiltration	6
Lung	congestion	10
	interstitial oedema	10
Trachea	mucosal oedema	7
Liver	interstitial lympho-histiocytic infiltration	4
	necrotic foci	6
Intestines	desquamative mucous enteritis	8
	haemorrhages	5
Bursa of Fabricius*	lymphocyte depletion	3
	apoptosis	3
Skeletal muscle	focal myocyte degeneration,
	necrosis	4
	haemorrhages	4

* Samples originating from three juvenile birds