2. Avian Influenza Overview
Avian influenza (AI) is a viral infection that affects domestic and wild birds. Clinical signs range in severity from inapparent to acute with high mortality. The severity of the disease changes with the strain of the virus and the avian species affected.
AI is a highly infectious and contagious disease in poultry, characterized by diverse disease syndromes. These syndromes vary from mild (e.g. subclinical or mild respiratory disease) to severe (e.g. loss of egg production or an acute and generalized fatal disease).
"Fowl plague" was first reported and described as a serious disease of poultry in Italy in 1878 by Perroncito. The virus, as the causative agent of fowl plague, was discovered by Centanni and Savunozzi in 1901, but it was not until 1955 that the viruses were characterized and identified as Type A influenza viruses.
Since 1955, all highly pathogenic avian influenza (HPAI) outbreaks have been attributed to viruses of the subtypes H5 and H7. These subtypes have repeatedly demonstrated the tendency to mutate from low-pathogenicity strains to highly pathogenic forms over a period of time, circulating within a poultry population of mainly Galliformes (terrestrial birds such as chickens). This has prompted the World Organisation for Animal Health (OIE) to redefine its reporting requirements for AI.
In 1997, it was reported that a highly pathogenic H5N1 strain of AI virus circulating within poultry populations in Southeast Asia had been transmitted from poultry to humans in Hong Kong. Among those people who had close contact with sick or dead birds, six died. Over the following years, the H5N1 Asian strain continued to cause outbreaks of disease in poultry flocks in Asia, emerging in late 2002 as a global threat. The virus demonstrated high pathogenicity in a number of species, including domestic and wild waterfowl (Anseriformes) and some carnivorous mammals. By the spring of 2006, the H5N1 Asian strain had spread to Western Europe and Africa, apparently through movements of migratory birds. Since January 2006, it is required to report all H5 and H7 subtypes, regardless of pathogenicity on initial diagnosis, to the OIE.
The disease is caused by many different strains of the virus and is also known by the following names:
- highly pathogenic avian influenza (HPAI);
- notifiable avian influenza (NAI); and
- fowl plague.
The causative agents of AI are viruses in the family Orthomyxoviridae. These viruses are pleomorphic, single-stranded ribonucleic acid (RNA) viruses. They have an envelope studded with symmetrical glycoprotein projections that have hemagglutinating (H) and neuraminidase (N) activity that undergo antigenic variation. These two surface antigens, known as hemagglutinating antigen (HA) and neuraminidase antigen (NA), are the basis for describing the serologic identity of the influenza viruses, using the letters H and N with the appropriate numbers in the virus designation, as in H5N2.
There are three antigenically distinct types of influenza virus – Types A, B, and C. In addition, in the Type A influenza viruses, there are now 16 hemagglutinin and nine neuraminidase subtype antigens described. Only Influenza Type A has been isolated from birds.
The type specificity is determined by the antigenic nature of the nucleoprotein (NP) and matrix (M) antigens.
2.2 Susceptible Species
Many domestic and wild avian species can be infected with AI viruses that may or may not produce disease.
- Domestic birds
Turkeys, chickens, and ducks are the most commonly infected avian domestic species under natural conditions. Several other species of birds may be infected with the AI viruses, including ostriches, rheas, emus, guinea fowl, domestic geese, quail, pheasants, partridges, mynah birds, passerines, psittacines, and budgerigars. Pigeons (Columbiformes) are believed to be resistant or minimally susceptible to infection and do not replicate AI viruses, but could serve as mechanical vectors.
- Certain isolates may be pathogenic to turkeys, but not to chickens or any other avian species.
- Wild birds
Among wild birds, AI viruses have been isolated from starlings, waterfowl, mallards, pintails, coots, and rock partridges. AI viruses are frequently recovered from apparently healthy migratory waterfowl, shorebirds, and seabirds throughout the world. The epidemiological significance of these occurrences, relative to outbreaks in domestic poultry, would suggest that waterfowl and other birds serve as a reservoir of AI viruses.
Traditionally, influenza Type A viruses have been known to cause disease in horses (H3 and H7), pigs (H1 and H3), mink (H3 and H10), whales, and seals (H1, H3, H4, H7, and H13). However, the range of several influenza Type A subtypes is expanding to include other mammalian species. The H5N1 Asian strain has now been shown to infect cats, leopards, tigers, and civets. Cases of canine influenza caused by H3N8 were recognized in the United States in 2005, and both mammalian and avian strains of the influenza viruses have been concurrently isolated from pigs. Swine may play an important role in the epidemiology of infection of turkeys by swine influenza virus when the two animals are housed near to each other. In 2005, turkey breeder flocks in Ontario, Manitoba, and British Columbia became infected with an H3N2 strain that was circulating in local swine barns. The outbreak resulted in a dramatic decrease in egg production, which did not recover.
In 2009, pandemic H1N1 caused an infection in swine in Alberta, an event which showed the zoonotic aspect of this virus and proved human to animal transmission; later in the same year, a turkey breeder's flock showed a drop in egg production that was related to H1N1 and had possible human implication as a virus origin. (Refer to the OIE Immediate Notification Report and Follow-Up Report 1.)
2.3 Global Distribution
The viruses related to the original fowl plague isolates that caused high mortality and morbidity among chickens and other poultry and birds are found in most countries of the world where poultry is produced. Disease outbreaks involving the designated highly pathogenic notifiable avian influenza (HPNAI) virus have been periodically reported in many parts of the globe, including North America, the Middle East, the Far East, Europe, Australia, England, and the former Soviet Union. However, the distribution of AI viruses is clearly influenced by the distribution of both the domestic and wild avian species, the locality of poultry production, migratory routes, season, and the disease-reporting systems used. Accurate prevalence rates of infection are difficult to determine because of the different surveillance systems and procedures used.
AI viruses are worldwide in distribution. These viruses are commonly isolated from the intestinal contents of apparently healthy migratory fowl and shore birds in several areas of the globe. Although the precise distribution and prevalence of AI is difficult to determine, sporadic and infrequent outbreaks of HPNAI in domestic poultry occur worldwide. These outbreaks have occurred in Australia, Canada, Chile, England, France, Italy, Netherlands, Northern Ireland, and Scotland, throughout Southeast Asia, in the United States, and in the former Soviet Union. Since 2000, outbreaks of HPNAI seem to be occurring more frequently, the highly pathogenic H5N1 has ravaged a number of countries in Asia, Europe, and Africa, and it has been responsible for a number of human fatalities. The prevalence of low pathogenicity notifiable avian influenza (LPNAI) (i.e. H5 and H7 subtypes) is difficult to assess, due to the general lack of active surveillance in many countries.
The World Animal Health Information Database (WAHID) Interface provides information on all disease outbreaks reported to the OIE, including LPAI and HPAI outbreaks since 2005, under "Immediate Notifications and Follow-Ups."
In Canada, we have experienced five outbreaks in commercial poultry to date, as follows:
- February 2004 HPNAI (H7N3) in British Columbia
- November 2005 LPNAI (H5 N2) in British Columbia
- September 2007 HPNAI (H7N3) in Saskatchewan
- January 2009 LPNAI (H5N2) in British Columbia
- November 2010 LPNAI (H5N2) in Manitoba.
Countries officially recognized by Canada as being free of Notifiable Avian Influenza are listed on the CFIA website.
2.4.1 Incubation Period – Critical Period
The incubation period for the various diseases and syndromes caused by AI viruses is highly variable, ranging from a few hours in intravenously inoculated birds to three days in naturally infected birds. The incubation period can be up to 14 days in a flock. To facilitate tracing of the source of an infection in a flock, the OIE Terrestrial Animal Health Code 2010 sets the incubation period at 21 days as the critical period, allowing for three seven-day cycles of the virus within the flock. This 21 days is the critical period used by the CFIA to identify the earliest possible date of introduction, when looking for the source of infection during a disease investigation.
The appearance of the initial clinical signs and the length of the incubation period are dependent on the dose and virulence of the virus strain, the route of exposure, the species affected, how the flock is raised (e.g. on litter or in cages), and the ability to detect clinical signs.
2.4.2 Persistence in the Environment
AI viruses are relatively sensitive to inactivation by lipid solvents such as detergents. The viruses are easily inactivated by heat, extremes of pH, non-isotonic conditions, and dryness. However, their infectivity, as well as their hemagglutinating (H) and neuraminidase (N) activities, can be maintained for several weeks at 4°C. Storage of the viruses at -70°C or by lyophilization can also maintain the infectivity and other biochemical properties for long periods. In addition, the H and N activities can be maintained, even if the viruses are no longer infectious. Formalin and beta-propriolactone can be used to eliminate the infectivity of the viruses, while preserving H and N activity. In field situations, AI-infected areas can be decontaminated with heat, sodium hypochlorite solutions, formalin, or commercial disinfectants, such as One-Stroke Environ (phenol) and Virkon (potassium peroxymonosulfate). Pathogenic influenza viruses can survive for as long as 105 days after depopulation in a cold (4°C) and moist environment (e.g. liquid manure). Virus infectivity is retained in fecal matter for 30 to 35 days at 4°C and for seven days at 20°C. The virus can survive in poultry carcasses for a few days at room temperature and up to 23 days when refrigerated (4°C).
2.4.3 Modes of Introduction and Transmission
Modes of Introduction
Wild bird populations, especially waterfowl, act as reservoirs and may be the initial source of infection for domestic birds through the contamination of feed and water sources. It also appears that AI viruses are maintained in the wild duck population by passage to susceptible birds, even at a low level, throughout the year. During the breeding season, a new group of susceptible wild duck juveniles preserves the cycle. Before the virus can cause disease in poultry, it must become adapted to the species. Once adapted, the virulence of the virus can develop over time, due to passage from bird to bird in Galliformes (mainly chicken and turkey flocks). The disease is highly contagious, and once a flock becomes infected, high concentrations of virus are present in fecal material (up to 16 x 106 virions/g of feces) and secretions from infected birds. Spread is then mainly mechanical and mediated by humans, with fecal contamination being the main source of spread. The experience from the Fraser Valley HPAI outbreak in 2004 suggests a potential short-distance transport of viral particles; however, more research is required to better define the likelihood of airborne spread of the disease. The sources of primary introduction of AI viruses to domestic poultry are in the following priority order:
- other infected poultry;
- wild birds;
- exotic captive birds;
- human beings; and
- other animals.
Direct contact with excretions, especially the feces and respiratory secretions of infected poultry and other birds, is the principal method of transmission within infected flocks. Once the virus is introduced into a flock of birds, it is transmitted, either from bird to bird or from flock to flock – though some AI viruses spread rapidly through a flock, while others spread slowly. Similarly, the same AI virus may differ in transmissibility in various species of birds. The rate of transmission of an AI virus through a poultry flock is not necessarily dependent on the pathogenicity of the virus. The disease may also be spread by waterfowl and other wild birds, and through contamination of feed, equipment, humans, and other mechanical means. Furthermore, the assessment of transmission in field outbreaks is complicated by the difficulty in distinguishing between direct transmission and spread by an intermediate agent, such as people. Results from studies on shorebirds and gulls indicate that these birds constitute an important reservoir of viruses.
HPAI viruses have also been found in the yolk and albumen of eggs from infected hens. When a hen is infected, the AI virus may be present within eggs, or on their surface. Vertical transmission is not ruled out, but it is unlikely that infected embryos with highly pathogenic strains of the virus could survive or hatch. It is possible that broken, untreated, and contaminated eggs could be a source of infection.
In Galliformes, the infectious process begins with the inhalation or ingestion of the virus. To activate the virus, the surface of the hemagglutinin requires cleaving by enzymes located in the host's cells. The hemagglutinin of LPAI virus, for example, can be cleaved by trypsin-like enzymes in respiratory and intestinal epithelial cells. Consequently, LPAI infections are limited to the respiratory and intestinal tracts. The hemagglutinin of HPAI viruses can be cleaved by ubiquitous furin-like enzymes, thus allowing a pantropic replication. The clinical signs of the viruses, and death, are due to the following: multiple organ failure as a result of direct virus replication in the cells, tissues, and organs; indirect effects from the production of cellular mediators; and ischemia from vascular thrombosis.
The pathogenesis is not as well understood in other birds.
2.6.1 Clinical Signs
Birds affected with AI show a variety of clinical signs that may involve the respiratory, digestive, reproductive, or nervous systems. With LPAI, clinical signs in Galliformes are typically mild and include the following:
- decreased food consumption;
- huddling, depression, closed eyes;
- respiratory signs (coughing and sneezing); and
- decreased egg production.
The signs most commonly observed in Galliformes infected with HPAI are the following:
- sudden death;
- marked depression, with ruffled feathers;
- decreased feed consumption;
- excessive thirst;
- a decrease in or cessation of egg production;
- mild to severe respiratory distress (including coughing, sneezing, and excessive lacrimation); and
- swollen wattles and combs, and watery greenish diarrhea.
Nervous signs are not frequently observed in Galliformes, but when present, they include lack of coordination and an inability to walk and stand.
In Anseriformes, there are no clinical signs associated with LPAI infection. In general, ducks and geese would be more resistant to AI infection; however, the following clinical signs were reported in Anseriformes during the H7N1 HPNAI outbreak in Italy (1999) and the H5N1 HPAI Asian strain outbreak:
- sudden death;
- nervous signs (e.g. lack of coordination, inability to stand and walk); and
- dyspnea, depression, and diarrhea.
The knowledge of pathogenesis and the dynamics of H5N1 Asian strain in domestic Anseriformes is constantly evolving. Nevertheless, the current understanding of H5N1 Asian strain leads the scientific community to believe that the virus would be more pathogenic to geese, while it would cause a silent infection in ducks. Consequently, ducks would be a more likely source of propagation of the H5N1 Asian strain viruses among domestic and wild bird populations. Affected birds may show one or various combinations of the clinical signs. In some cases of HPAI infection, the disease is so acute that birds are found dead without any other observed signs.
2.6.2 Gross Pathologic Lesions
Lesions observed in several of the susceptible avian species are extremely variable with regard to their anatomical location and severity, depending mainly on the species and pathogenicity of the virus strain. Frequently, there are few significant lesions because the disease is mild; for highly pathogenic viruses, there may be no striking lesions because the birds die quickly before any changes can develop.
The lesions most commonly observed in Galliformes infected with LPAI are nasal discharge, sinusitis, and rough and misshapen eggs. The air sacs may also be thickened and covered with caseous or fibrinous exudate. Kidneys can be heavily congested and tubules are usually seen containing white urate deposits (i.e. as a result of dehydration). Galliformes that die with the peracute or acute disease (HPAI including H5N1 Asian strain) may not have prominent lesions, except for those associated with general muscular congestion and dehydration. In less severe cases, the gross lesions are more typical and consistently seen. Subcutaneous edema of the head and neck region with serous exudate is apparent as the skin is reflected. A sero-fibrinous fluid may ooze from the nares and oral opening as the birds are positioned for necropsy. Hemorrhages in various parts of the body are common and particularly striking in the submucosa of the proventriculus and gizzard. The mucosal lining of the gizzard peels easily, and hemorrhages are seen in the submucosal region. Intestinal linings may contain hemorrhages, particularly in the cecal tonsils. Petechial hemorrhages may also be found in the heart, on the intestinal surfaces, on the peritoneum, on the body fat, and inside the keel. Severe congestion is associated with hemorrhages in the conjunctiva. The trachea may be edematous, hemorrhagic, and filled with excessive mucous. In laying hens, the ovary may be hemorrhagic or degenerative with dark spots of necrosis. The peritoneal cavity is usually filled with yolk from ruptured ova, inducing air sacculitis and severe peritonitis.
No gross lesions are associated with LPAI infection in Anseriformes. The following gross lesions have been reported in Anseriformes infected with H5N1 Asian strain: ocular and nasal discharge; conjunctivitis; ecchymotic or petechial hemorrhage of leg and footpad; serous fluid surrounding the heart, pancreas, liver, and abdomen; cyanosis of the oral cavity; pneumonia; pancreatic mottling; splenomegaly; bursal and thymic atrophy; and malacic foci of the cerebral hemispheres.
2.6.3 Morbidity and Mortality
Similar to the clinical signs of the disease, morbidity and mortality are variable because they depend on factors such as the following:
- host susceptibility;
- the pathogenicity of the virus;
- the presence of concurrent disease; and
- farm management.
However, for HPAI, the morbidity and mortality can reach up to 100% in Galliformes.
2.6.4 Laboratory Diagnosis
A tentative diagnosis of AI may be possible, if the investigating veterinarian obtains a complete medical history from an animal's owner and carefully observes the clinical signs and necropsy lesions. The presence of a wide range of clinical signs makes clinical diagnosis difficult, except in an HPAI epizootic situation. In an AI initial investigation, obtaining a virus isolate that could be fully characterized would be ideal; however, this may not always be possible. In these situations, a combination of other tests, such as real-time reverse transcription polymerase chain reaction (RRT-PCR), sequencing, and serology would be used to characterize the virus as much as possible.
A diagnosis of NAI, for the purpose of immediate field action, can be made by an approved Canadian Animal Health Surveillance Network (CAHSN) laboratory (AI network laboratory: AI-NL). These laboratories are certified by the National Centre for Foreign Animal Disease (NCFAD) to perform AI diagnostic testing on behalf of the CFIA. The results of these tests performed in the AI-NL must be reported through an agreed-upon communication plan. All samples at an AI-NL lab that have positive results must be forwarded to NCFAD for confirmatory testing, as required under international agreements. NCFAD acts as the AI Reference Laboratory (AI-RL) for Canada.
Laboratory tests for AI diagnosis in Canada include the following:
Virus Isolation – to indicate an active infection
Isolation and identification of the AI virus from tracheal or cloacal swabs, feces, or internal organs is the gold standard method of diagnosis. To attempt virus isolation, specimen samples for laboratory submission should be collected from several birds; it is not unusual for many specimens to fail to yield virus. Embryonated chicken eggs are inoculated with swab or tissue specimens diluted in virus diluent. The AI virus normally kills embryos within 48 to 72 hours. However, confirmation of an egg inoculation as negative can take up to 14 days, as prescribed by laboratory procedures. The egg allantoic fluid harvested from dead embryos is tested for the presence of hemagglutinating activity using chicken erythrocytes. It is important to determine whether the H activity in the allantoic fluid is due to influenza virus or other H viruses, such as Newcastle disease virus (NDV). A battery of tests to confirm the virus identity and type specificity may also be completed. Once an AI virus is isolated and determined to contain the H5 or H7 HA, an intravenous pathogenicity index is performed to determine the virulence of the virus. In addition, molecular pathotyping of the virus by the RRT-PCR assay, and nucleic acid sequencing, will be done.
Rapid Molecular Diagnostic tests – to indicate an active infection
These tests include RRT-PCR for detection of AI virus matrix, and H5 and H7 RNA. The specimens required are oropharyngeal swabs, cloacal swabs, and tissues. The RRT-PCR can also be performed on allantoic fluid during virus isolation. Although the technique can be performed within three hours on individual samples, for multiple samples, it is expected that a minimum of 24 hours (one day) will be required, from the moment the samples are received by the lab, before a result becomes available.
Antigen Capture Tests – to indicate an active infection; used in an outbreak situation as a field test
The specimens required are oropharyngeal and cloacal swabs. These tests are most useful for clinically ill birds (i.e. acute phase of the disease). The following antigen capture tests are designed to detect only influenza Type A and can give both false positive and false negative results:
- Directigen Flu; and
- Flu Detect (not currently available in Canada).
These tests still require thorough evaluation by the Canadian Centre for Veterinary Biologics (CCVB) and NCFAD to determine the usefulness of these tools in an outbreak situation.
Serological tests – to indicate a past infection
Serological tests are used to demonstrate the presence of antibodies that may be detected five to seven days post-infection. Presently, serum specimens are required; however, work is being done to validate the use of egg (yolk) as a possible source for enzyme-linked immunosorbent assay (ELISA) testing. The most commonly used techniques are the hemagglutination inhibition (HI) tests to detect antibodies to the HA and double immunodiffusion to detect antibodies to the nucleoproteins (NP). Other serologic tests used to detect antibodies are the virus neutralization (VN) and neuraminidase inhibition (NI).
In serologic surveillance programs, the test to detect the anti-NP antibody is the method commonly used, because it detects antibodies to a cross-reactive antigen shared by all influenza Type A viruses. Nevertheless, caution is important because there is significant variation in the immune reaction among various avian species. These are the serological tests used:
- Competitive-ELISA (C-ELISA) for detecting antibodies to influenza Type A (one-day test); and commercially available C-ELISA kit for detecting antibodies to influenza Type A. These tests, which are not recommended for species other than Galliformes, require one day. Although these tests are circulating in Canada, they are not approved for use by the CAHSN labs.
- Agar gel immunodiffusion (AGID) test for detecting antibodies to influenza Type A (one-day test). This test is not recommended for Anseriformes, as they are poor producers of precipitating antibodies.
- HI for detection of subtype-specific antibodies. This test requires two days. It can also be used for virus identification on allantoic fluid.
2.6.5 Differential Diagnosis
Depending on the clinical picture and species affected, the differential diagnosis should include systemic diseases for HPAI and respiratory diseases for LPAI. The HPAI may resemble acute fowl cholera (Pasteurella spp.), velogenic viscerotropic Newcastle disease (i.e. Paramyxovirus PMV-1), and intoxication (i.e. from food or water). In the milder forms of AI, it may be confused with other common viral respiratory diseases, such as infectious bronchitis, infectious laryngotracheitis, and avian paramyxovirus infections. Bacterial infections (e.g. Mycoplasma spp., coryza [Haemophilus paragallinarum], Ornithobacterium spp., Bordetella avium, and fowl cholera) and fungal infections (e.g. Aspergillus spp.) should also be included in the differential diagnosis for a respiratory disease. Concurrent infections with influenza viruses and mycoplasma species or other bacteria have been a common occurrence.
Infection with, or exposure to, AI viruses, as well as immunization with vaccines, stimulates an antibody response at both the systemic and the mucosal levels. A systemic Immunoglobin M response by five days post-infection is followed shortly by an Immunoglobin G response. The intensity of the antibody response varies with bird species, in the following order (from most intense to least):
- quail; and
Antibodies against the surface proteins are neutralizing and protective. Protection has been primarily associated with antibodies directed to the HA protein; however, the presence of either HA or NA antibodies, or both, prevents clinical signs and death following challenge with HPAI viruses having homologous HA or NA subtypes. The level of protection against mucosal infection and subsequent shedding of the challenge virus may depend on the degree of sequence similarity in the HA of vaccine and challenge virus. The duration of protection is variable and depends on many factors, but in laying hens, protection against clinical signs and death has been demonstrated to be at least 30 weeks following a single immunization.
Immune response against internal proteins has not been shown to prevent clinical signs or death, but may shorten the period of virus replication and consequently reduce the shedding.
Studies on protection by maternal antibodies from homologous HA or NA have not been reported, but based on the available information about other viral avian diseases, protection against clinical signs and death from a homologous AI viral challenge is probable for the first two weeks after hatching. For surveillance purposes, the OIE suggests that maternal antibodies derived from a vaccinated parent flock are usually found in the yolk and can persist in progeny for up to four weeks.
The modernized approach of the OIE and the scientific community regarding AI vaccination makes vaccine use more acceptable. Vaccination has been used in various poultry species, and its effectiveness in preventing clinical signs and mortality is well documented. Developed countries should aim for eradication without the use of vaccines when facing a NAI outbreak. As part of preparedness for a disease outbreak, countries should identify available sources of NAI vaccines in advance.
2.8 Public Health
AI viruses could be involved in the development of new mammalian strains through genetic reassortment, which is a feature of Type A influenza viruses. Direct transmission of AI viruses between avian species and humans has occurred with the HPAI H5N1 Asian strain and the H7N7 strain in the Netherlands. In fact, there is data to prove that swine influenza virus (H1N1) is involved in inter-species transmission. Therefore, a swine-avian-human association could conceivably have public health relevance.
AI viruses may play a significant role in the emergence of new human influenza virus strains by contributing viral genes to human strains through genetic reassortment. The potential for avian and mammalian viruses to infect humans exists, due to reassortment and the resultant mutation of genes in the influenza viruses.
For this reason, all investigations into possible NAI outbreaks require the use of personal protective equipment (PPE) to prevent zoonotic infections.
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