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Short Communication |
1 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany
2 Institute of Infectology, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany
3 PT Multibreeder Adirama Indonesia, Japfa Comfeed Company, Indonesia
Correspondence
Timm C. Harder
timm.harder{at}fli.bund.de
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]. H5N1 virus was recovered from all organs sampled from two apparently healthy pigeons at 3 days post-infection and from the three pigeons which died spontaneously. All surviving birds shed virus via the oropharynx and the cloaca at minimal titres and seroconverted. Sentinel chickens reared in direct contact to the pigeons neither developed clinical signs nor seroconverted to the H5N1 virus. |
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; Isoda et al., 2006; Kaleta & Honicke, 2004). Experimental infection of pigeons with HPAIV of subtype H7 did not uniformly induce clinical signs, virus shedding and seroconversion (Alexander, 2000; Dinter, 1944; Eckert, 1979). There seems to exist an even lower susceptibility of pigeons to HPAIV of subtype H5 (Narayan et al., 1969; Panigrahy et al., 1996). Only one report has described a marginal susceptibility (Slemons & Easterday, 1972). Pigeons were also reported to be fully resistant to experimental infection with HPAIV H5N1 from Hong Kong, isolated in 1997 (Perkins & Swayne, 2002, 2003). Songsermy et al. (2006), however, reported on lethal infections of pigeons with HPAIV H5N1 in Thailand which led to transmission of the infection to domestic cats scavenging on contagious pigeon carcasses. More recently, a number of as yet unconfirmed reports mentioned a possible involvement of feral pigeons in outbreaks of HPAI caused by H5N1 of Asian lineage in northern Iraq, the Arabic peninsula, and Egypt.
Here we studied the susceptibility of pigeons to a recent isolate of H5N1 HPAIV causing devastating losses in poultry in south-east Asia since 2003. A companion paper focussing on the neuropathology of this experimental inoculation experiment has been published by Klopfleisch et al. (2006).
The HPAIV isolate, obtained from chicken organs received from Indonesia, was propagated once in the allantoic cavity of 10-days-old embryonated specific-pathogen-free chicken eggs (VALO SPF; Lohmann Animal Health). The virus has been further characterized by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes (GenBank accession nos AM183669 and AM183681, respectively). H5N1 viruses isolated in Indonesia in 2003 and 2004 belonged to genotype Z (Li et al., 2004). Phylogenetic analysis of the HA gene revealed that the Asian virus isolates from 2003 to 2005 formed distinct clades distinguishing isolates from Vietnam, Thailand, Malaysia, Cambodia and Laos from viruses circulating in China, Indonesia, Japan and South Korea (WHO, 2005). A/chicken/Indonesia/2003 clusters with this second clade (not shown).
Sixteen adult domestic pigeons (Columba livia domestica), clinically healthy and seronegative to avian influenza virus as tested by a competitive ELISA (Starick et al., 2006), were inoculated oculo-nasally with 0.5 ml allantoic fluid containing 108.1 50 % egg-infective dose (EID50) virus in total. Five 12-week-old White Leghorn chickens (VALO SPF) were infected simultaneously and identically as positive controls, but were kept separately. For detection of virus excretion by the pigeons, five further chickens were added as sentinels into the pigeons' aviary 48 h post-inoculation of the pigeons. The sentinel chicken had direct contact to the pigeons' faeces (solid ground, cleaned daily) and, also, a common source of drinking water for pigeons and chickens had been in use. Additionally, three pigeons remained uninoculated and were housed separately from the other birds. All birds had free access to food and water, but separate feeders and food were provided for pigeons and chickens. All experiments were carried out at biosafety level 3 containment facilities complying with German animal welfare legislation.
The birds were monitored daily for a total of 19 days or until death, and clinical signs were recorded. Oropharyngeal and cloacal swabs were taken daily from each pigeon and each chicken until 7 days post-inoculation (d.p.i.), then at 10, 13 and 19 d.p.i. Swabs were collected in 1 ml medium (Dulbecco's modified Eagle's medium supplemented with 5 % fetal calf serum) containing antibiotics and stored at –70 °C until analysed. Two pigeons (number 1 and 2) were euthanized at 3 d.p.i. and likewise all surviving pigeons and sentinel chickens were euthanized at 19 d.p.i. All birds were necropsied and brain, trachea, lung, heart, spleen, kidney, proventriculus as well as intestine (terminal colon and cloaca) were collected for virus recovery and titration. Tissue homogenates and swabs were inoculated into 10-day-old embryonated eggs for virus isolation as recommended (OIE, 2006). Infectivity titres were calculated by the method of Reed and Muench and expressed as EID50 per gram of tissue. Tissue and swab samples received a second egg passage when embryonic death, but no haemagglutination, was detected in the allantoic fluid. Additionally, the swabs were analysed by RT-PCR by amplifying a conserved part of the matrix protein gene as described elsewhere (Starick et al., 2000; Starick & Werner, 2003).
Blood samples from all pigeons and sentinel chickens were taken before inoculation, at 13 d.p.i. and at the end of the experiment at 19 d.p.i. Detection of haemagglutination-inhibiting (HI) antibodies against the homologous influenza virus has been reported before (Klopfleisch et al., 2006). In addition, the sera were investigated by a competitive ELISA system using a recombinant baculovirus-derived nucleoprotein (NP) fusion protein as antigen (Starick et al., 2006).
The five inoculated control chickens died within 48 h after inoculation. A detailed description of the clinical signs induced is described by Klopfleisch et al. (2006). At necropsy, only mild and inconsistent gross lesions were present, but all pigeons which had developed clinical signs revealed histologic lesions in the brain (Klopfleisch et al., 2006). Virus was detected in all tissue samples of pigeons number 3, 4 and 5 which succumbed (Table 1). Highest viral titres amounting to 108.0 EID50 per gram of tissue were detected in the brain. HPAIV replication in the brain was associated with severe neurological alterations and histologically demonstrable meningoencephalitis and, correlating with Ct values in the real-time RT-PCR (data not shown), a surprisingly high viral load was also measured in the lung, proventriculus and kidneys of these birds in the absence of pathological alterations (Klopfleisch et al., 2006). Clearly, there is a discrepancy between presence of viral antigen in organ-specific cells and detection of replication-competent virus or genomic material in corresponding tissue samples seen in these animals. It can not be excluded that, in pigeons number 3, 4 and 5, viraemia, potentially cell-associated, dominated at time of death and, due to the neurotropic properties of the virus, only the central nervous system has been efficiently colonized extravacularly at that time. In addition, the lower sensitivity of immunohistochemical detection compared to both virus isolation and real-time RT-PCR must be taken into account. No virus was recoverable from any tissues of the birds sacrificed at 19 d.p.i. This included pigeons number 6 and 7 still showing clinical signs and revealing histological lesions in the cerebrum at that time. This is taken as evidence that the lesions were initiated by H5N1 replication, but continued to develop independently from the presence of infectious virus. The two pigeons randomly selected and sacrificed at 3 d.p.i. did not develop any lesions (see companion paper by Klopfleisch et al., 2006), but harboured virus in all tissue samples tested (Table 1). Highest viral loads, in descending order, were measured in the lung, proventriculus, kidney and trachea. The brains of these two pigeons also contained infectious virus but, at a titre that was 4 log10 lower than that of pigeons which succumbed to the disease. Seemingly the virus had established a systemic infection at an early time after inoculation without causing serious alterations or clinical signs. The majority of the inoculated pigeons was obviously able to clear the virus without any disorder.
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) and nucleocapsid-specific antibodies reactive in a competitive ELISA between 3 and 6 log2 at 13 and 19 d.p.i., respectively. Pigeons number 6 and 7, which showed mild clinical signs at 16 and 6 days, respectively, did not develop higher antibody titres than the healthy birds. Sentinel chickens did not seroconvert.
Further evidence for replication of the virus in all inoculated pigeons came from the results of virus recovery from the swabs (Table 2). Virus was isolated at least once from all birds inoculated, but never from non-inoculated pigeons or chickens. Virus was recovered from oropharyngeal swabs three times more often than from cloacal swabs. In apparently healthy pigeons virus was detectable until 6 d.p.i., while some of the diseased birds excreted the virus until 7 d.p.i. In contrast to marked virus shedding by the inoculated chickens (Table 3), only minute titres of virus were detectable in swabs of the pigeons (<100.5 EID50 per ml). In addition, eleven swabs required a second passage until virus was recovered. All 32 samples yielding infectious virus were also positive by RT-PCR. In addition, 24 further samples proved to be positive by RT-PCR while negative by virus isolation.
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). In this part of the study, we showed that, in contrast to previous reports (Kaleta & Honicke, 2004; Li et al., 2004; Narayan et al., 1969; Perkins & Swayne 2002, 2003), replication of HPAIV in all pigeons inoculated was evident according to virus isolation rates at 3 d.p.i. from two randomly selected pigeons and virus recovery from others swab samples until 7 d.p.i. The amount of virus excreted apparently was insufficient to trigger an infection in five sentinel chickens, despite an intense and direct contact of the chickens to the pigeons' faeces. However, with respect to substantial virus titres detected, especially in the brain of diseased pigeons, virus transmission to scavengers such as cats may well be envisaged (Songsermy et al., 2006; Weber et al., 2007).
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REFERENCES |
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Dinter, Z. (1944). Comparative research on atypical and classical fowl plague. Arch Ges Virusforsch 3, 207–219 (in German).[CrossRef]
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Starick, E., Werner, O., Schirrmeier, H., Kollner, B., Riebe, R. & Mundt, E. (2006). Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species. J Vet Med B Infect Dis Vet Public Health 53, 370–375.[Medline]
Weber, S., Harder, T., Starick, E., Beer, M., Werner, O., Hoffmann, B., Mettenleiter, T. C. & Mundt, E. (2007). Molecular analysis of highly pathogenic avian influenza virus of subtype H5N1 isolated from wild birds and mammals in northern Germany. J Gen Virol 88, 554–558.
WHO (2005). Consultation on human influenza A/H5. Avian influenza A (H5N1) in humans. N Engl J Med 353, 1374–1385.
Received 20 April 2007;
accepted 3 July 2007.
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