Fatal Avian influenza a H5N1 in a dog.Avian influenza avian influenza: see influenza. H5N1 virus is known to cross the species barrier and infect humans and felines. We report a fatal H5N1 infection in a dog following ingestion ingestion /in·ges·tion/ (-chun) the taking of food, drugs, etc., into the body by mouth. in·ges·tion n. 1. The act of taking food and drink into the body by the mouth. 2. of an H5N1-infected duck during an outbreak in Thailand in 2004. With new reports of H5N1 virus continuing across Asia, Europe, and Africa, this finding highlights the need for monitoring of domestic animals during outbreaks. ********** Highly pathogenic avian influenza (HPAI HPAI Highly Pathogenic Avian Influenza HPAI Hospital Pharmacists Association, Ireland HPAI Hewlett Packard Associates International ) H5N1 has spread across Asia, Europe, and Africa. Not limited to poultry, the virus has also been shown to cross the species barrier infecting humans (1) and felines, including domestic cats (2) and tigers (3-5). Both cats and tigers were reported as becoming infected after eating poultry carcasses harboring HPAI. Here, we report a case of HPAI H5N1 infection in a domestic dog following ingestion of the carcass of an infected duck. The Study In October 2004, the carcass of an a [approximately equal to] 1-year-old dog from Suphanburi Province, Thailand, was submitted for necropsy necropsy /nec·rop·sy/ (nek´rop-se) examination of a body after death; autopsy. nec·rop·sy n. See autopsy. necropsy examination of a body after death. See also autopsy. at the Faculty of Veterinary Medicine, Kasetsart University, in Nakorn Pathom, Thailand. The dog's owner stated that the dog had eaten duck carcasses from an area with reported HPAI H5N1 infections in ducks. Approximately 5 days after ingesting the carcasses, the dog developed high fever, panting panting rapid, shallow breathing, a characteristic heat-losing reaction in dogs; represents an increase in dead-space ventilation resulting in heat loss without necessarily increasing oxygen uptake or carbon dioxide loss. , and lethargy and died on the following day. Within 4 hours of its discovery, the dog carcass was sent to the laboratory. Necropsy findings included bloody nasal discharge; severe pulmonary congestion and edema edema (ĭdē`mə), abnormal accumulation of fluid in the body tissues or in the body cavities causing swelling or distention of the affected parts. (Figure 1A); and congestion The condition of a network when there is not enough bandwidth to support the current traffic load. congestion - When the offered load of a data communication path exceeds the capacity. of the spleen, kidney, and liver. Brain, lung, trachea trachea (trā`kēə) or windpipe, principal tube that carries air to and from the lungs. It is about 4 1-2 in. (11.4 cm) long and about 3-4 in. (1.9 cm) in diameter in the adult. , heart, duodenum duodenum: see intestine; pancreas. duodenum First and shortest (9–11 in., or 23–28 cm) segment of the small intestine. It curves down and then up from the pylorus of the stomach, where chyme enters it. , jejunum jejunum: see intestine. , ileum ileum: see intestine. ileum Final and longest segment of the small intestine. It is the site of absorption of vitamin B12 (see vitamin B complex) and reabsorption of about 90% of conjugated bile salts. , liver, spleen, kidney, pancreas, and urine specimens were obtained separately and processed for virus isolation by injection into 10-day-old embryonated chicken eggs. Forty-eight hours later, allantoic allantoic /al·lan·to·ic/ (al?an-to´ik) pertaining to the allantois. allantoic pertaining to the allantois. allantoic fluid see fetal fluids. fluids harvested from dead embryos that had been injected with supernatants of ground brain, trachea, lung, intestine, liver, and kidney were tested with the hemagglutination hemagglutination /he·mag·glu·ti·na·tion/ (he?mah-gloo-ti-na´shun) agglutination of erythrocytes. he·mag·glu·ti·na·tion n. and hemagglutination-inhibition tests. Influenza virus was isolated from lung, liver, kidney, and urine specimens, and the viral subtype (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. was determined to be H5N1 by reverse transcription (RT)-PCR (6). The 4 tissues that showed virus were also processed for histopathologic and immunohistochemical analysis. Immunohistochemical tests were performed on paraffin-embedded tissues by using a mouse monoclonal antibody anti-nucleoprotein of influenza A H5N1 (B.V. European Veterinary Laboratory, Woerden, the Netherlands) as a primary antibody and a polyclonal polyclonal /poly·clo·nal/ (-klon´'l) 1. derived from different cells. 2. pertaining to several clones. polyclonal derived from different cells; pertaining to several clones. goat antimouse immunoglobulin G tagged with peroxidase peroxidase /per·ox·i·dase/ (per-ok´si-das) any of a group of iron-porphyrin enzymes that catalyze the oxidation of some organic substrates in the presence of hydrogen peroxide. per·ox·i·dase n. as a secondary antibody (DAKO A/S, Glostrup, Denmark). Diaminobenzidine was used as a substrate. Positive lung tissue from the dog that was incubated with phosphate-buffered saline instead of the mouse monoclonal antibody antinucleoprotein of influenza A H5N1, and tissue from the liver and lung of a cat killed by a car served as negative control (2). [FIGURE 1 OMITTED] Histopathologic examination of the lung showed severe pulmonary edema and interstitial pneumonia with inflammatory cell infiltration. Hemolysis hemolysis (hĭmŏl`ĭsĭs), destruction of red blood cells in the bloodstream. Although new red blood cells, or erythrocytes, are continuously created and old ones destroyed, an excessive rate of destruction sometimes occurs. with brownish black particles was found in the pulmonary parenchyma Parenchyma A ground tissue of plants chiefly concerned with the manufacture and storage of food. The primary functions of plants, such as photosynthesis, assimilation, respiration, storage, secretion, and excretion—those associated with living (Figure 1B), and the liver showed focal necrosis (Figure 1C). The kidneys showed mild nephritis nephritis (nəfrī`təs), inflammation of the kidney. The earliest finding is within the renal capillaries (glomeruli); interstitial edema is typically followed by interstitial infiltration of lymphocytes, plasma cells, eosinophils, and a with tubular degeneration. No microscopic lesions were found in any other organs. On immunohistochemical analysis, positive sites were found in alveolar cells, hepatic cells (Figure 1D), renal tubular epithelium, and glomerulus glomerulus /glo·mer·u·lus/ (glo-mer´u-lus) pl. glomer´uli [L.] a small tuft or cluster, as of blood vessels or nerve fibers; often used alone to designate one of the renal glomeruli. ; none of the remaining organs were positive for H5N1. H5N1 viruses were isolated from the dog's lung tissue and designated A/Dog/Thailand/KU-08/04. Genetic analysis was used to characterize the dog's virus (KU-08), and the sequences were deposited at GenBank under accession number DQ530170-7. Sequencing and phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. analysis of the hemaggluttinin (HA) and neuraminadase (NA) genes of the dog's virus showed that they were similar to those of H5N1 viruses isolated from tigers, chickens, ducks, and humans infected in Thailand during the same time that the dog was infected (Figure 2A and B). In addition, analysis of 6 other genes from KU-08 showed similar results (data not shown). Phylogenetic analysis clearly indicated that all the Thailand isolates were clustered with the Vietnam lineage, which groups separately from the Indonesia lineages and China (Qinghai), Europe, and Africa lineage. Genetic comparisons of the 8 genes analyzed from KU-08 to those of viruses isolated in Thailand from chickens (Jan 04, Jul 04, Oct 05), tigers (Jan 04, Oct 04), humans (Jan 04, Dec 05), cats (Jan 04), and geese (1996, Jun 05) are shown in the Table. The analysis showed that KU-08 was more closely related to the tiger isolate (CU-T3) obtained in Oct 2004, with higher percentages of nucleotide identity (100% identity for 5 genes: H5, N1, matrix [M], nonstructural [NS], polymerase basic protein 1 [PB1]) compared to any of the Thailand isolates obtained from early 2004 and late 2005. [FIGURE 2 OMITTED] This finding indicates that the dog's H5N1 infection resulted from the virus circulating during the second wave of H5N1 outbreaks that occurred in Thailand during mid-2004. The HA gene of KU-08 contained multiple basic amino acid insertions at the HA cleavage site (SPQRERRRKKRR), similar to those found at the HA cleavage site for other viruses characterized from Thailand during this time. However, the isolates from the third wave of AI outbreaks that occurred in Thailand in 2005 contained 1 basic amino acid (aa) change at the HA cleavage site (SPQREKRRKKRR) (7). Moreover, the viruses isolated from China (A/Bar-headed goose/Qinghai/65/05, A/Blackheadedgull/Qinghai/1/05) (8), Iran (A/Cygnus cygnus/Iran/754/06), Russia (A/Cygnusolor/Astrakhan/Ast05-2-9/05), and Nigeria (A/chicken/Nigeria/641/06) displayed a different amino acid at the HA cleavage site (SPQGERRRKKRR). The receptor-binding site of the dog isolate still exhibited avian characteristics in that it contained glutamine glutamine (gl  `təmēn), organic compound, one of the 20 amino acids commonly found in animal proteins. (Q) and glycine glycine (glī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Glycine is the only one of these amino acids that is not optically active, i.e. (G) at positions 222 and 224 of the HA gene
(Q222-G224). The NA gene of KU-08 also had 20 aa deletions at positions
49-68 and contained histidine histidine (hĭs`tĭdēn), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. (H) amino acid at position 274, indicating
the absence of an oseltamivir-resistant residue. The NS gene of the
KU-08 isolate contained a 5-aa deletion at positions 79-83, and the M
gene of the KU-08 isolate displayed an amantadine-resistant amino acid
(N31; asparagine asparagine (əspâr`əjēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of mammalian proteins. ). In summary, the viruses from the dog were similar to
the H5N1 viruses isolated in Thailand in 2004 and to the Vietnam lineage
which had been identified as genotype Z (9). A single amino acid
substitution at position 627 of the PB2 gene (glutamic acid [E] to
lysine lysine (lī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. [K]) was observed in KU-08 and had previously been observed in
human, tiger, and cat isolates from Thailand as well as the viruses from
China (Qinghai). The presence of lysine (K) may relate to more
efficiency of viral replication in mammal species (10). On the other
hand, in pigeon isolates from Thailand (KU-03), the PB2-627 aa residue
remained unchanged (E; glutamic acid).This study is the first report of H5N1-related systemic disease in a domestic dog infected during the second wave of outbreaks in Thailand that occurred during October 2004. The most plausible route of the dog's infection was ingestion of infected duck carcasses. Previous studies have shown that avian viruses preferentially recognize [alpha]-2,3 linkage (SA[alpha]2,3Gal) and bind to type II alveolar cells, which are abundant in the lower respiratory tract Noun 1. lower respiratory tract - the bronchi and lungs lung - either of two saclike respiratory organs in the chest of vertebrates; serves to remove carbon dioxide and provide oxygen to the blood of mammals (11,12); these findings support our observations of severe pneumonia with lung edema in the infected dog (Figure 1). Characterization of the H5N1 isolates from the dog showed identical properties to the H5N1 isolates from the Thai epidemic. Moreover, genetic comparison indicated that the dog isolate was similar to the H5N1 viruses recovered from a tiger (CU-T3) in Thailand during the mid-2004 epidemic. Conclusion Our results demonstrate that, as has previously been shown for cats, dogs are at risk for H5N1 infection. Despite the low probability of H5N1 infection in domestic animals, the possibility of humans acquiring H5N1 infection from direct contact with infected cats and dogs Cats and Dogs A slang term referring to speculative stocks that have short or suspicious histories for sales, earnings, dividends, etc. Notes: In a bull market analysts will often mention that everything is going up, even the cats and dogs. warrants concern and highlights the need for monitoring domestic animals during H5N1 outbreaks in the future. Acknowledgments We thank the staffs of the small animal hospital, Kasetsart University, Kumpansan Campus, Nakhon Pathom, Thailand, and of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, for their assistance. We also thank Petra Hirsch for editing the article. Genomic research was supported by the Thailand Research Fund, Senior Research Scholar, and the Center of Excellence in Viral Hepatitis Research. References (1.) Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, Chunsuthiwat S, Sawanpanyalert P, Kijphati R, et al. Human disease from influenza A (H5N1), Thailand, 2004. Emerg Infect Dis. 2005;11:201-9. (2.) Songserm T, Amonsin A, Jam-on R, Sae-Heng N, Meemak N, Pariyothorn N, et al. Avian influenza H5N1 in naturally infected domestic cat. Emerg Infect Dis. 2006;12:681-3. (3.) Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RA, Amonsin A, Payungporn S, et al. Avian influenza H5N1 in tigers and leopards. Emerg Infect Dis. 2004;10:2189-91. (4.) Thanawongnuwech R, Amonsin A, Tantilertcharoen R, Damrongwatanapokin S, Theamboonlers A, Payungpom S, et al. Probable tiger-to-tiger transmission of avian influenza H5N1. Emerg Infect Dis. 2005; 11:699-701. (5.) Amonsin A, Payungporn S, Theamboonlers A, Thanawongnuwech R, Suradhat S, Pariyothorn N, et al. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression . 2006;344:480-91. (6.) Poddar SK. Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) and agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). . J Virol Methods. 2002;99:63-70. (7.) Chutinimitkul S, Bhattarakosol P, Srisuratanon S, Eiamudomkan A, Kongsomboon K, Damrongwatanapokin S, et al. H5N1 influenza A virus and infected human plasma. Emerg Infect Dis. 2006;12:1041-3. (8.) Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang XW, et al. Highly pathogenic H5N1 influenza virus infection in migratory birds. Science. 2005;309:1206. (9.) Li KS, Guan guan: see curassow. Y, Wang J, Smith G J, Xu KM, Duan L, et al. Genesis of a highly pathogenic and potentially pandemic pandemic /pan·dem·ic/ (pan-dem´ik) 1. a widespread epidemic of a disease. 2. widely epidemic. pan·dem·ic adj. Epidemic over a wide geographic area. n. H5N1 influenza virus in eastern Asia. Nature. 2004;430:209-13. (10.) Shinya K, Hamm S, Hatta M, Ito H, Ito T, Kawaoka Y. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism tropism (trōp`ĭzəm), involuntary response of an organism, or part of an organism, involving orientation toward (positive tropism) or away from (negative tropism) one or more external stimuli. , of Hong Kong H5N1 influenza A viruses in mice. Virology. 2004;320:258-66. (11.) van Riel ri·el n. See Table at currency. [Origin unknown.] Noun 1. riel - the basic unit of money in Cambodia; equal to 100 sen D, Munster VJ, de Wit E, Rimmelzwaan GF, Fouchier RA, Osterhaus AD, et al. H5N1 Virus attachment to lower respiratory tract. Science. 2006;312:399. (12.) Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza virus receptors in the human airway. Nature. 2006;440:435-6. Thaweesak Songserm, * Alongkorn Amonsin, ([dagger]) Rungroj Jam-on, * Namdee Sae-Heng, * Nuananong Pariyothorn, ([dagger]) Sunchai Payungporn, ([dagger]) Apiradee Theamboonlers, ([dagger]) Salin Chutinimitkul, ([dagger]) Roongroje Thanawongnuwech, ([dagger]) and Yong Poovorawan ([dagger]) * Kasetsart University, Nakorn Pathom, Thailand; ([dagger]) Chulalongkorn University, Bangkok, Thailand Address for correspondence: Yong Poovorawan, Center of Excellence in Viral Hepatitis Research, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, 1873 Rama IV Rd, Patumwan, Bangkok 10330, Thailand; email: Yong.P@chula.ac.th Dr Songserm is a veterinary pathologist at the Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen Campus, Nakorn Pathom, Thailand. His research interests include avian pathology, duck and goose diseases, and emerging diseases in animals.
Table. Genetic comparison of 8 gene segments of the H5N1 influenza
virus isolated from the dog's lung (KU-08) to those of H5N1 isolates
from Thailand *
% nucleotide identity
KU-08: GD-1: CU-K2: SP-33:
dog goose (1996) chicken human
(Oct 04) Region of ([double (Jan 04) (Jan 04)
([dagger]) comparison dagger]) ([section]) ([paragraph])
HA 46-1623 96.0 97.1 99.0
NA 28-1296 91.6 99.3 99.3
M 1-801 96.1 99.5 99.6
NS 37-789 68.5 99.0 99.2
NP 58-1428 92.6 99.7 99.7
PA 28-2118 93.5 99.4 99.3
PB1 67-2229 93.4 99.6 99.8
PB2 88-2220 94.0 99.4 99.3
% nucleotide identity
CU-T3:
CU-23: tiger (Oct
KU-08: Ti-1: chicken 04) ([double
dog tiger KU-02: (Jul 04) dagger]
(Oct 04) (Jan cat (Jan ([dagger] [double
([dagger]) 04) # 04) ** [dagger]) dagger])
HA 99.6 99.6 99.6 100.0
NA 99.5 99.5 99.9 100.0
M 99.4 99.9 99.9 100.0
NS 99.3 99.5 99.3 100.0
NP 99.6 99.6 99.8 99.9
PA 99.5 99.4 99.5 99.7
PB1 99.8 99.8 99.9 100.0
PB2 99.4 99.7 99.8 99.8
% nucleotide identity
CK-160: NK-165:
KU-08: chicken human QH-65:
dog (Oct 05) (Dec 05) goose
(Oct 04) ([section] ([paragraph] (Jun
([dagger]) [section]) [paragraph]) 05) (##)
HA 99.2 98.9 96.8
NA 98.5 98.8 97.2
M 99.4 99.3 98.1
NS 99.1 99.0 96.4
NP 99.1 99.1 98.1
PA 99.2 98.8 97.7
PB1 99.1 98.8 97.9
PB2 98.9 98.9 97.5
* HA, hemagluttinin; NA, neuraminadase; M, matrix; NS, nonstructural;
NP,nucleoprolein; PA, polymerase A; PB, polymerase basic protein.
([dagger]) A/dog/Thailand-Suphanburi/KU-08/04 (DQ530170-7).
([double dagger]) A/Goose/Guangdong/1/96 (AF144300-7).
([section]) A/chicken/Nakorn-Patom/Thailand/CU-K2/04 (AY590567-8,
AY590578-82, AY551934).
([paragraph]) A/Thailand/SP-33/04 (AY555152-3, AY627893-8).
(#) A/tiger/Suphanburi/Thailand/Ti-1/04 (AY646167-74).
** A/cat/Thailand/KU-02/04 (DQ236077-84).
([dagger][dagger]) A/chicken/Ayutthaya/Thailand/CU-23/04
(AY770991-AY7709918).
([double dagger][double dagger]) A/tiger/Thailand/CU-T3/04
(AY907672-3, AY842935-6, AY972547-50).
([section][section]) A/chicken/Thailand/Kanchanaburi/CK-160/05
(DQ334757-64).
([paragraph][paragraph]) A/Thailand/NK165/05 (DQ372591-8).
(##) A/Bar-headed Goose/Qinghai/65/05 (DQ095622, DQ095642, DQ095662,
DQ095682, DQ095702, DQ095722, DQ095742, DQ095762).
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