Classical techniques for aiv

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenza

Istituto Zooprofilattico Sperimentale delle Venezie

OIE/FAO and National Reference Laboratory for Avian Influenza

IZSVe LABORATORY MANUAL

FOR THE DIAGNOSIS OF AVIAN INFLUENZA

Classical Techniques

(for internal laboratory use only)


INTRODUCTION

Aetiology

Influenza viruses are segmented, negative strand RNA viruses belonging to the Orthomyxoviridae

family. At present, four different genera are know: Influenzavirus A, B and C and Thogotovirus.

Only influenza A viruses can cause the disease in avian species. Influenza type A viruses are

divided into subtypes on the basis of two different surface antigenic glycoproteins: the

haemagglutinin (HA) and the neuraminidase (NA). Presently, 16 HA subtypes (H1-H16) and 9 NA

subtypes (N1-N9) have been isolated from wild birds nearly in any combination.

Influenza A viruses have been further grouped into two distinct pathotypes on the basis of the

severity of the disease they cause: the Highly Pathogenic Avian Influenza viruses (HPAIv), and the

Low Pathogenic Avian Influenza viruses (LPAIv) causing a milder infection characterised by

respiratory symptoms and generic depression.

Highly Pathogenic Avian Influenza (HPAI):

The highly pathogenic form of AI has only been associated with some strains of H5 and H7 HA

subtypes. The infection causes a dead-ending disease in poultry (chickens and turkeys)

characterized by very high mortality rate in a very short time, whereas domestic waterfowls and

wild birds may be resistant and may not even show clinical signs. Due to this situation wild birds

and waterfowls are a never ending source of the infection with potential ecological and

epidemiological dangerous implications. Highly Pathogenic Influenza strains belonging to the H5

and H7 subtypes are characterised by the presence of multi-basic amminoacids at the cleavage site

of the precursor of the haemagglutinin molecule (HA0) which attribute confers to the virus the

possibility to be cleaved and therefore activated by all organism’s proteases present in any host

tissue and resulting in a systemic disease.

Low Pathogenicity Avian Influenza (LPAI):

The Low Pathogenic form of Avian influenza is caused by all 16 subtypes (H1-H16) of the virus.

Low pathotypes are responsible of a mild disease, localised to respiratory and digestive apparatus

and resulting in respiratory symptoms, depression and drop in egg production. Low pathogenic H5

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenzaand H7 subtypes lack the multi-basic amminoacids at the cleavage site of the HA0 and for this

reason the virus remains able to infect cells only in the presence of trypsine-like protease enzymes

(digestive and respiratory tract.)

LPAI viruses are maintained in nature with wild bird population since waterfowl represent the

natural hosts for these viruses.

After the introduction into domestic bird populations, these viruses cause low pathogenicity avian

influenza (LPAI).

Phylogenetic studies suggest that HPAI viruses emerge from H5 and H7 LPAIV precursors due to

mutations or recombination of the genomic segments.

This is the reason why, the World Health Organisation for Animal Health considers H5 and

H7 subtypes either belonging to HPAIV or to LPAIV both as Notifiable diseases.

Prevention of Avian influenza: basic principles

The primary introduction of AI viruses in domestic poultry occurs either through direct or indirect

contact with infected birds. This may occur through movements of infected poultry, or contaminated

equipment, vehicles, and fomites. Airborne transmission has not been still demonstrated. For these

reasons, if biosecurity measures are implemented at the farm level AI infections can be prevented.

Big attention should be paid to the correct application of biosecurity measures as part of the disease

prevention strategy. Very basic biosecurity measures such as preventing the introduction of animals

of different origin into a flock, protecting poultry flocks from migratory wild birds contacts, do not

grow different bird species together and other simple rules, must be applied to all rearing poultry

systems (industrial, semi-intensive and backyard farms) to avoid the introduction of the infection

into the commercial circuit.

The risk that rural poultry may become a never-ending source of virus, perpetuating virus

circulation and resulting in the establishment of an endemic situation is particularly expected

where mixed species are reared together and traded through the live-bird market system.

Biosecurity (bioexclusion and biocontainment) represents the first and most important means of

infection prevention. It follows that if biosecurity measures of a high standard are implemented and

maintained, these represent a firewall against the penetration and perpetuation in the industrial

circuit. However, breaches in biosecurity systems do occur and corrective measures should be

subsequently applied. This occurrence indicates the crucial importance of the establishment of an

early warning system and additional control tools for AI.


Epidemiological investigation, necropsy of suspected birds and collection of

pathological samples

Preliminary investigation

Before attempting the diagnosis of Avian Influenza type A and preceding the necroscopy it is very

important to get a complete history of the outbreak and to collect the following epidemiological

data:

Identification of the holding suspected to be the centre of the infection and preliminary

identification of the productive units and subunits of the region involved in the outbreak:

location of the main suspected holding and density of poultry farms in the area, biosecurity

measures applied by each unit and subunits involved, characteristics of the different production

systems (backyard flock or intensive farm), closeness to wet lands attended by resident or

migratory water fowl.

Identification of the staff directly involved with that unit.

Anamnestic data: information to be collected are the feeding and egg production rate of the

domestic poultry (layers, turkey and broiler breeding infected with HPAI virus may at first lay

soft-shelled eggs, but soon stop laying), duration of the clinical signs, number of sick or dead

birds. Interesting information are also the place and the moment of disease of every single bird.

These clues may help to understand the origin of the disease and the diagnose itself.

The clinical investigation must be performed regard every susceptible species present in the farm,

beginning from the most peripheral units to the centre of the outbreak. Particular attention must be

paid if vaccination has been performed. All these information must be reported in the

epidemiological inquiry. An official report must include the identification PER SPECIES of all the

animals present in each unit the starting date and the description of the clinical sings and the

percentage of mortality.

Epidemiological inquiry

The official veterinary (OV) and the laboratory veterinary (LV) have to fill carefully the

epidemiological inquiry form reporting the following information:

Animal movements: animal movements should be recorded up to 20 days prior to the first

clinical signs.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenza People movements: all people (staff, relatives, servicing personnel, veterinarians) who had

access to the farm must be reported.

Vehicles movements: all vehicles, regardless of their contact with animals, which have had

access to the farm must be reported.

The epidemiological inquiry must be sent (possibly faxed) to the competent authorities as soon as it

has been completed.

Clinical examination

It is very important to perform a careful clinical examination on live affected birds before culling, to

identify characteristic neurological signs caused by the very virulent viruses as incoordination,

tremor, torticollis, abnormal gait, paralysis. Besides these symptoms HPAI results in depression,

blindness, severe enteritis with hemorrhagic diarrhoea and respiratory sign.

Necropsy precautions

All the birds suspected of an HPAI must to be necropsied by the competent authorities with very

rigorous heath precaution. Before the procedure both the carcass and the necropsy table surface

must be wet with soapy water or a disinfectant solution in order to decrease the possibility of

aerosol exposure to potential human pathogens. Resistant rubber glove, respiratory masks with

aspiration valve (FFP-2 or FFP3) and protective visor or protective glasses must be worn.

External examination

During physical examination it is very important to observe the feathers around the vent to

recognise signs of diarrhoea and the unfeathered areas as joints and cutaneous annexes. Birds

affected by HPAI present cyanotic and oedematous combs and wattles, and may have petechial or

ecchymotic haemorrhages at their tips.

Take note either of any swelling on the infraorbital sinuses and cloacal discharges, which, in

particular, should be qualified as to nature, colour, consistency and odour.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaClinical signs in birds infected with HPAI viruses

The incubation period of AI is variable in relation to the different viral strains and to the host.

Usually 5-6 days is quoted, but the range for individual birds is probably from a few hours to about

7 days.

For HPAI the disease may appear suddenly in a flock and a large quantity of birds may die either

without premonitory signs or with non-specific signs as depression, inappetence, ruffled feathers

and fever. The flock mortality rates may arrive up to 100% in few days.

Some species such as chickens and turkeys are very susceptible to HPAI, but the Anseriformes

(ducks and geese) may show a major resistance. Ducks may show no clinical signs when infected

with HPAI viruses, but some strains have been reported to induce depression, inappetence, and

diarrhoea and high mortality in the flock.

Necropsy

Wetting down the plumage with a disinfectant solution is strongly recommend to limit the

distribution of infected dust and feathers. The bird must be placed on its back with the feet towards

the operator. Dislocate the coxo-femoral joint and cut and remove the skin over the abdomen and

examine the superficial breast muscles to recognize if decreased muscle mass, paleness (anemia),

congestion or bruising are present.

Cut the abdominal muscles, ribs and coracoid bone with hard scissors (poultry shears) and

remove the chest to expose the internal organs and the chest cavity. Examine the liver, the

lungs, the heart, and the air sacs. The lungs must be gently removed from the ribcage with the

trachea. Make a longitudinal section of the larynx, trachea, and syrinx for a more accurate

examination.

Cut off the gastrointestinal canal between the esophagus and proventriculus and at the level of the

last tract of the rectum near the cloaca and remove the proventriculus, gizzard and intestine with

pancreas, the liver and the spleen. The spleen is a small, red, round organ located at the junction of

proventriculus and gizzard. Examine the kidneys, which are elongated and lobulated organs

embedded in the vault of the pelvis, and observe the left ovary and oviduct (or paired testes), which

are positioned on the top of the kidneys. Examine, at the end of the necropsy, the interior surface of

the esophagus and crop some of the intestinal mucosa.


Post mortem lesions in birds infected with HPAI virus

Birds that die peracutely may show minimal gross lesions, consisting of dehydration and congestion

of viscera and muscles.

In birds that die after a prolonged clinical course, petechial and ecchymotic haemorrhages occur

throughout the body, particularly in the larynx, trachea, proventriculus and epicardial fat, and on

serosal surfaces adjacent to the sternum. There is extensive subcutaneous oedema, particularly

around the head and hocks. The carcase may be dehydrated. Yellow or grey necrotic foci may be

present in the spleen, liver, kidneys and lungs. The air sac may contain an exudate. The spleen may

be enlarged and haemorrhagic.

AI is characterised histologically by vascular disturbances leading to oedema, haemorrhages and

perivascular cuffing, especially in the myocardium, spleen, lungs, brain and wattles. Necrotic foci

are present in the lungs, liver and kidneys. Glyosis, vascular proliferation and neuronal degeneration

may be present in the brain.

Avian Influenza differential diagnostis.

Symptoms of avian influenza vary a lot on the basis of the pathogenicity of the infecting strain, low

pathogenic avian influenza viruses (LPAIVs) or highly pathogenic avian influenza viruses

(HPAIVs), the species, the age and the immunity competence of the hosts, the risk of super

infections and the rearing manage.

Considering all the reasons mentioned above the avian influenza differential diagnostis is not an

easy task and whereas clinical signs may be suggestive of AI, the diagnosis must be confirmed

through laboratory analysis: virus isolation and identification (LPAI and HPAI) and serologic

testing (LPAI)

Low pathogenic Avian Influenza differential diagnostic:

Generally, low pathogenic Avian influenza, is asymptomatic in wild birds, In poultry LPAI is

responsible of mild to severe respiratory signs. On layers the disease may responsible of egg drop

production and decrease in hatching rate. Chickens and turkeys may present raffled feathers, apathy

and drop in food and water consumption. Acute diarrhoea may be present without lose of weight.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaDiseases which must be considered in the differential diagnosis of LPAI are those causing drop in

egg production and mild respiratory symptoms. These are infectious bronchitis, infectious coryza,

pneumovirosis, laryngotracheitis in its mild form and mycoplasmosis.

It must be taken into account that LPAI is often coexisting to secondary bacterial and micoplasma

infections. In case of a concomitant bacteria super infection, the LPAI may evolve into chronic form

causing lost of weight, infraorbital sinusitis, severe respiratory sings and mortality rate up to 40 -

70%.

Pasteurella multocida and Escherichia Coli when associated to LPAI are often responsible of

macroscopic lesion as fibrin purulent aerosacculitis, pericarditis and abdomen’s egg retention.

Highly Pathogenic Avian Iinfluenza (Avian Plague) differential diagnostic

Infections in birds can give rise to a wide variety of clinical signs that may vary according to the

host, strain of virus, the host's immune status, presence of any secondary exacerbating organisms

and environmental conditions.

HPAI in poultry may cause a sudden high mortality up to 100% even without preliminary symptoms

and birds dying of the peracute form of AI show minimal gross lesions, consisting of dehydration

and congestion of viscera and muscles.

In other cases, symptoms may be observed from the third trough the seventh day, as nervous sings

(ataxia), lethargy, drop in food and water intake and drop in egg production up to stop in the sixth

day.

Respiratory symptoms are less frequent than in LPAI (dyspnoea coughing gasping , expectoration of

bloody exudate )

Due to the systemic characteristic of the disease, extensive subcutaneous oedema, particularly

around the head and hocks is present. Wattles and combs are swollen, petechial and ecchymotic

haemorrhages occur throughout the body, and on serosal and mucasal surfaces of the respiratory and

digestive apparatuses particularly in the larynx, trachea, proventriculus, cecal tonsil and epicardial

and visceral fat.

The age of the animals and the viral strain reflect strongly on the mortality rate which may rise from

50% up to 100%.

Due to the immature immune system chicks are the most sensitive to the disease.

Differential diagnosis of High Pathogenic Avian Influenza:

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaAny circumstance causing sudden high mortality must be considered in the differential diagnosis of

HPAI. Different infectious diseases, velogenic strains of NDV, acute poisoning or bad

environmental conditions should be taken into account. These two last possibilities are mainly due

to inaccurate rearing manage.

Insufficient control of the environmental conditions as heating, ventilation, or humidity, may lead to

very severe consequences when animals are kept in a close rearing system. Bad management of the

temperature regulation may result in the production of carbonic monoxide. On the other hand,

limitation in freshening may overwhelm the heat regulation capacity of the birds and result in high

rate mortality due to dehydration, respiratory deficiency and cardiac arrest.

Also the acute poisoning may produce the sudden mortality of high percentage of the flock in a

short time. The mainly common poisoning reasons may be due to ingestion of pesticides, hyatrogen

intoxication caused by a over dosage of anti-coccidiosis therapy, or chlore intoxication when not

accurately washed away after disinfection of the environment.

A sudden high mortality must also take into account also Botulism. Botulism is an intoxication

caused by the ingestion of the toxins of Clostridium Botulinum.

It has been observed both in poultry and in waterfowls due to contaminated feeding. Signs appear

within a few hours to few days. In chickens symptoms include weakness and progressive loss of

control of the legs, wings and neck. Tremors and paresis progresses to paralysis. Most visible

affected birds dies in few hours.

The main disease to differentiate from high pathogenic avian influenza are listed below:

Newcastle disease.

The most similar disease to Avian Influenza is the Newcastle Disease.

ND is caused by different strains of Paramyxoviruses 1 characterized by different degrees of

pathogenicity (velogenic, mesogenic and lentogenic strains.)

When caused by a velogenic NDV, the disesase is also called avian pseudo-plague.

In chickens the most pathogenic form of Newcastle disease is characterized by short course

followed by death of most affected birds. Velogenic Newcastle strains are responsible of variable

signs according to the tropism of the virus. Sudden onset of clinical signs with marked depression

and prostration. Dyspnoea and violent greenish diarrhoea are often present. There may be swelling

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenzaand cyanosis of the infraorbital region, conjunctivitis with sticky ocular and nasal discharge. Some

birds may show neurological signs as tremor up to paralysis. Mortality can reach 100% in 2-3 days.

Due to the rapid course of the disease, cage birds or wild birds, often reveal no gross lesions or have

mild, non specific lesions. Inflammation of the trachea and air sac is usually severe. Haemorrhages

occasionally occur on the mucosa of the intestine, caecal tonsils, proventriculus and gizzard.

Differentiate a velogenic form of ND from HPAI is impossible without laboratory diagnosis.

Newcastle is a reportable disease. All suspected ND must be notified to animal health authorities

immediately.

Infectious Laryngotracheitis

Avian Infectious Laryngotracheitis (ILT) is a worldwide distributed poultry disease caused by an

Herpesvirus which causes severe respiratory forms.

Incubation period ranges from 5 to 10 days. Up to present only one immunologic strain has been

found although it is considerably variable in pathogenicity. Most strains are markedly virulent.

Accordingly to the virus’s pathotype, the disease may present an acute or a sub acute form.

The acute form spread rapidly into the flock and is characterized sudden high mortality that can

reach 50 % of the birds. Characteristic symptoms are the severe dyspnoea with open-mouth

breathing, loud gasping sounds and coughing up haemorrhagic secretions. Animals may present

blood stained feathers and peak, haemorragic secretions are also found scattered around on the walls

of the premise. Loss in food and water intake and egg drop production are common.

Post-mortem findings are limited to the upper respiratory tract, consisting in haemorrhagic

laryngotracheitis with blood clots and mucoid exudates in trachea.

Cyanosis of the carcass is also often present.

Infectious laryngotracheitis is a reportable disease and when suspected must be notified to animal

health authorities immediately.

Gumboro Disease (IBD)

Infectious bursal disease caused by very virulent strains IBD virus (VVIBDV) belonging to the

family Birnaviridae, is an acute, contagious, viral disease of young chickens. Gumboro Disease is an

infection tipical of poultry speaces, IBV may infect other birds without causing clinical signs.

Morbidity and mortality begins 3 days post infection, peaks and recedes in a

period of 5 -7 days.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaSymptoms are present in chicks after 3 weeks of age, characterised by diarrhoea and dehydration.

Trembling, incoordination and vent picking usually occur. Chicks present also depression, anorexia

and ruffled feathers. The virus severely damages the bursa of Fabricius and lymphoid organs as

thymus, caecal tonsils and spleen. The infection produces variable degrees of immunosuppression

resulting in a high susceptibility to subsequent pathogens. If husbandry is poor or the virus strain is

particularly virulent the mortality in the flock may exceed the 30 %.

Gross lesions are the enlarged and haemorrhagic bursa, which ends to atrophy, increased mucus in

the intestine, swelling of the kidneys with urates deposits and focis of necrosis in the other lymphoid

organs. Petechial haemorrhages may be present in thigh and pectoral muscles and occasionally in

the junction of proventriculus and gizzard.

Infectious Bursal Disease is a reportable disease and when suspected must be notified to animal

health authorities immediately.

Duck virus enteritis (Duck plague)

Duck virus enteritis (DVE) is an acute Herpesvirus infection of ducks causing haemorrhagic

enteritis which may lead to sudden death. Mortality rate may rise up to 100% within 1 to 5 days.

Incubation period is from 3 to 5 days after exposure.

The acute form is characterised by haemorrhagic and watery diarrhoea. Other clinical signs are

dehydration, weakness, lethargy, inappetence and marked egg drop. Tremors and ataxia may appear.

Birds present ruffled and blood stained feathers.

Post mortem findings are severe enteritis, haemorrhages and crustily plaques on the mucosa of all

the digestive apparatus, from the esophagus to the intestinal tract, caeca and rectum included.

Acute fowl cholera

Avian Cholera is a contagious disease resulting from infection by Pasteurella multocida.

Suspect of avian cholera must be considered when a large number of dead animals are found dead in

a short time. All domestic species are susceptible to the disease, whereas ducks, turkeys and quails

are the most receptive to the bacterium. Acute pasteurellosis infection can result in bird death 6-12

hours after exposition. Susceptibility to infection and the course of the disease is dependent upon

many factors including sex, age, genetic variation, immune status, concurrent infections, nutritional

status and strain virulence.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaSome birds appear lethargic other may show neurological signs as convulsions, swim in circles or

erratic flight efforts due to the pateurellosis common inflammation of the middle ear. Mucous

discharge from the mouth and blood-stained dropping from the nose and ruffled feathers are present.

Sometimes they throw the head back between the wings and dye. Mortality from avian cholera in

poultry may exceed 50 % of the population. Death can be so rapid that birds appear to be in good

flash and gross lesion may be absent. The longer the survival time the more possibility to find signs

of the disease at necroscopy. Commonly observed lesion in birds dying of fowl cholera is the

congested enlarged and haemorrhagic spleen. Frequent findings are also haemorrhages at the

surface of the heart, liver and gizzard. Furthermore on the liver are present area of tissue with

yellow spots, alteration of texture, color and shape.

Pasteurella multocida may cause a form localized to wattles and comb which appear swollen and

edematous.

Avian Cholera is a reportable disease and as soon as identified notification to animal health

authorities must be done immediately.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaLaboratory diagnostic techniques referring to differential diagnosis.

The differential diagnosis, previously considered, must always be supported by the demonstration of

the presence of the causative agent, its antigens or specific antibodies in the serum.

Diagnosis by isolation of the agent and/or by molecular techniques is necessary for the confirmation

of the studied disease.

Isolation and identification techniques adopted may vary from one agent to the other since many

and different laboratory procedures exist. Nucleic acid recognition methods are available in

specialised laboratory with appropriate diagnostic reagents.

All the laboratories techniques described relate to the OIE Manual of Diagnostic Test and Vaccine

for Terrestrial Animals.

The gold standard of virus detection for Avian Influenza and Newcastle viruses is the egg

isolation by inoculation of suspected samples into 9-11 day-old embryonated chicken eggs by the

allantoic sac route. Samples are be obtained from tissue homogenates and respiratory or cloacal

swabs.

All the diagnostic procedures related to AI are accurately described in the following dedicated

section.

Avian Infectious Laryngotracheitis laboratory diagnosis depends on the identification of the

Herpesvirus by detection of its antigenicity or by its isolation .Laryngotracheitis Virus isolation is

performed either in cell culture of chicken embryo liver or kidney cell monolayers, embrionating

SPF chicken eggs. Exudate from trachea is inoculated on the dropped horionallantoic membrane

(CAM) of 10-12 day-old eggs. (Please refer to Annex 1) Incubation at 37 °C and daily candeling, is

performed for up to 7 days. If virus is replicating embryo death may occur from the second day after

inoculation. At the examination opaque plaques on the chorioallantoic membrane are present

resulting from necrosis and proliferative tissue reaction.

The virus may be also observed in the trachea exudate using the electron microscopy. Detection of

its antigens is demonstrated using immunoflorescence assayes, AGID or AC-ELISA.

Histopathology and molecular methods are also available for identifying the viral DNA.

Serological test for ILT antibody detection are virus neutralization, AGID and in direct ELISA.

Diagnosis of Infectious Bursal Disease is most based on the detection of the virus in tissue’s

samples using immunological procedure or by molecular methods. Viral isolation is practised on

SPF chicken embryonated eggs by CAM inoculation of the clinical sample (Please refer to Annex

1). Antibodies detection is also used as routine confirmatory technique.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaIdentification of IBDV is based on the detection of the antigen from the bursa of Fabricius: the virus

target organ. During the earlier and acute stage of the disease, when antibodies are not yet produced,

the bursa is directly used as reagent for agar gel immunodiffusion assay. Small pieces of the organ

are used to fill the wells (of 5 mm of diameter) the AGAR plate against positive serum. Freeze-thaw

cycles may maximize the antigen release.

(Abbreviations: AG positive reference antigen , S+ Positiv regerence serum, Bf 1,2,3,4 Bursa of

Fabricius sample

Identification is also possible with immunoflorescence assay or antigen-capture enzyme-linked

immunosorbent assay (AC-ELISA)

IBD Viral isolation is done by chorioallanoic inoculation (CAM) in 9 to 11 day-old embryonating

SPF chicken eggs but it may present some difficulties.

Dead embryos present typical lesions as dwarfing, subcutaneous oedema, intracranical

haemorrhages, parenchimateous organs are congested and swollen. Viral isolation may be done also

on cell culture of chicken embryo fibroblast. Three blind passages are required to give negative

result.

Serology is based on AGID, virus neutralization test and ELISA.

Pasteurella multocida, causative microrganism of Fowl cholera, is easily isolated from different

kinds of specimens collected from birds died of the acute septicemic form.

The microscopic demonstration of bacterium showing bipolar staining (Wright, Giemsa or

methylene blue stains) may be bases on its direct identification in smears of tissues, such as blood,

liver, or spleen…….

Pasteurella multocida may also be primary isolated on blood agar, or other media, after incubation

at 37°C degrees for 18-24 hours. Identification of the bacterium and differentiation from other

avian Pasteurella spp is based on biochemical tests.

Bf1 Bf2

AG S+ AG

Bf3 Bf4

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaAntigenic characterization of the strain requires the identification of the capsular serogrup (A,B or

D) using haemoagglutination test and somatic serotype by AGID test (up to now16 serotypes has

been isolated from avian hosts.)

Serological tests are not used for the diagnosis of fowl cholera since their lack in sensitivity.

Duck virus enteritis may be isolated by inoculation in 9 to 14 day old embryonated Muscovy duck

eggs in chorioallantoic membrane (Please refer to Annex 1). Dead embryos show extensive

haemorrhages from 4 to 10 days after inoculation. Chicken eggs as well as Pekin duck eggs are not

very susceptible to infection. Isolation may also be achieved on Muscovy duck embryo fibroblasts

or liver cell culture. Identification of the virus is accomplish using serum neutralization test.

Serological tests are not used as diagnostic test for DVE infections.

Laboratory safety measures for avian influenza (AI)

Avian Influenza is a highly contagious disease of birds. This virus is defined as a class 2 biological

agent (WHO classification). This means that it is potentially infectant for humans and constitutes a

risk for workers in particular conditions:

1. direct contact with infected/sick and dead birds,

2. direct contact with surfaces or material contaminated by infected excretions and secretions and

with infected aerosol (i.e. due to improper laboratory management)

The use of safety equipment, combined with good procedures and practices, will help to reduce the

risks involved in dealing with biological hazards. The main safety measures are described below.

Individual preventive measures:

Vaccination with influenza human vaccine is recommended for the staff that are exposed to the

risk of infection to avoid recombination between avian and human viruses.

Possibility to use antiviral medicines (olseltamivir, zanamivir) depending on the risk of exposure.

The doctor in charge should maintain a stock of medicines for the people at risck of infection and

instructions about the possible use of the antiviral medicines, both for preventive and therapeutic

purpose.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaBiosafety level 2 (BSL2): Include the presence of a class 2 biological safety hood in order to protect the worker, the

surrounding environment and the sample from possible contaminations. The external air is

aspirated and carried in the work zone after being purified by a HEPA filter. Before going out the

air goes through a HEPA filter again.

Each operation (sample container opening, egg inoculation and opening, viral typing, etc….)

must be performed under biological safety cabinet.

Workers must wear protective gears that must be removed before leaving the laboratory.

Biohazard containers should be available for disposal of contaminated materials.

There must be an autoclave in the laboratory or in the building for the purpose of inactivating

laboratory wastes before the disposal.

The biological risk sign must be displayed on the laboratory door.

Biosafety level 3 (BSL3):

The laboratory needs to be physically separated from the others laboratories, should be

isolated by an air lock and provided with a suitably placed shower. The air locks and rooms must

be ventilated by an exhaust air system.

WHO strongly recommends that the BSL3 precautions are adopted and followed for work in BSL2

laboratories with influenza A/H5 virus specimens.

IDP (Individual Disposal Protection)

The following IDP must be worn during each phase of the work in and out of the hood:

Overall (specific for protection from biological agent);

Laboratory shoes;

Disposable gloves (specific for protection from microrganisms). The gloves must be replaced

frequently and removed after their use and before touching any surface or non contaminated

equipment.


Laboratory diagnosis of Avian Influenza and Newcastle Disease

Virological diagnostic procedures for AIV

ISOLATION OF AVIAN INFLUENZA VIRUS

Sample management and preparation

Swabs:

Under sterile conditions, using a laminar flow cabinet, collect 2 ml of PBS solution with

antibiotics from the tube in which the swabs is soaked, if swabs arrive in the lab in such

condition, and put them in another tube.

Add 8 ml of fresh PBS with antibiotics in order to make a dilution of 1:5 of the sample.

Centrifuge the sample at 1000 x g for 10 minutes. This step is necessary to remove rough

particles.

Keep the sample at + 4°C for one night, or at - 80°C until the inoculation in eggs.

Use the supernatant to infect the SPF embryonated eggs.

Organs:

Thaw organs at room temperature or at +4°C.

Write on a tube of 15 ml the identification number of the sample and write out its work sheet.

under a laminar flow cabinet, collect an amount of sample corresponding to 1 cm2 using sterile

forceps and scissors. Organs can be pooled according to the apparatus they belong to.

Homogenise the sample in a mortar, adding sterile quartz sand.

Add 9 ml of PBS with antibiotics.

Decant the homogenate into the 15 ml tube. Leave the sample at + 4°C for 1 night. If the

inoculation in eggs is not performed the day after, keep the sample at –80°C until the starting

date of the test.

Assign an identification number to the sample as it arrives in the lab, write out a work sheet

indicating the identification number of the sample, kind of sample, species and test to perform,

starting and ending date of analysis. This work sheet should be written by the technician who

performs the test.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenza Centrifuge the sample at 1000 x g for 10 minutes.

Use the supernatant to infect embryonated eggs.

Virus isolation

Virus isolation is performed following the protocol of the OIE and European standards (EC,

94/2005).

Candle the 9 day-old eggs to check the embryo viability and mark with a pen the shell of the

egg to delimit the air sac, this will be the inoculation point (Figure 2a).

Write on five eggs the identification number of sample, number of passage (1° or 2°), kind of

sample (lung, cloacal swabs etc), date of inoculation.

Inoculate the clarified supernatant fluid obtained from tracheal and cloacal swabs or organ

homogenised, into the allantoic cavity of five from 9 to 11 days old SPF embryonated fowl’s

eggs Fig. 2b,c).

Inoculate 0.1-0.2 ml of supernatant into each egg.

Incubate the inoculated eggs at 37 C for seven days.

Candle the inoculated eggs daily to check the embryo vitality.

Every day write in the lab hand book the mortality observed for each sample.

Test the allantoic fluid of eggs with dead embryos for haemagglutinating activity as indicated

below (see point 1.3).

If any haemagglutinating activity is detected, identify haemagglutinating agents by means of

the Heamagglutination Inhibition (HI) test as described.

After seven days chill eggs in a refrigerator (+4°C) to end up the first blind passage.

The day after open eggs using sterile techniques under a laminar flow cabinet, and collect

approximately 10 ml of the allantoic fluid.

Test the allontoic fluid for the presence of haemagglutinating activity by “rapid HA test” as

described below.

If at the end of the first blind passage, no heamagglutinating activity is detected, use the

undiluted allantoic fluid, collected from this passage, to perform a second blind passage in

embryonated eggs as described above.

Mortality of inoculated eggs after 24 h post-inoculation can be considered non-specific.

If no haemagglutinating activity is detected, after two passages in eggs are performed the

sample must be considered negative.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenza When an haemagglutinating activity is detected the presence of bacteria must be excluded by

culture.

If bacteria are present, the fluids must be passed through a 450 nm membrane filter, with

further addition of antibiotics and inoculated into embryonated eggs as described above.

A sample resulting positive to the rapid HA test must undergo to the characterisation of the

allantoic fluid by HA performed in microtitre plate (“slow HA”), in order to establish the HA

titre and after perform the HI with reference antisera (against avian influenza viruses H1-H16,

and also for Newcastle disease virus).

If a laboratory does not have the capacity to perform the HI, the haemagglutinating allantoic fluid

should be sent to a National Reference Laboratory or to an International Reference Laboratory to

confirm the diagnosis.

Figure 2. Candling and inoculation of eggs via allantoic cavity.

a. Candling eggs b. The allantoic cavity route c. Inoculation of eggs

Haemagglutination test in Petri dishes (rapid HA test)

This method is based on the reaction between the haemagglutinin protein (HA) and red blood cells

(RBCs). If there is an haemagglutinating agent, a link will be established between them, forming

macroscopical precipitating clusters.

Mix in a Petri dish a drop of the allontoic fluid with the same amount of 1%RBC suspension

Wait 30 seconds-1 minute and observe if there are any haemagglutinating aggregates

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaCharacterisation of Avian Influenza viruses

Haemagglutination test in microtitre plate (micro HA test):

1. Dispense 0.025 ml PBS into each well of a plastic microtitre plate (V-bottomed wells).

2. Place 0.025 ml of virus suspension (i.e. allantoic fluid) in the first well.

3. Use a multichannel micropipette to make two-fold dilutions (from 1:2 to 1:4096) of virus

across the plate and discard the last 0.025ml.

4. Dispense 0.025 ml of PBS to each well.

5. Add 0.025 ml of 1 % red blood cells to each well.

6. Mix by tapping gently and place at +4 C or at room temperature (+20-24°C)

7. Plates are read after 30 min (at room temperature) or after 40 min (at +4°C) when the Red

Blood Cell controls have settled. Reading is done by tilting the plate and observing the

presence or absence of tear-shaped streaming of the RBCs. In wells with no

haemagglutination activity RBC should flow at the same rate as RBC in the control wells in

which only PBS and RBC have been dispensed..

The HA titre is the highest viral dilution which causes agglutination of the RBCs. That dilution

contains 1 haemagglutinant unit (HAU). To perform the HI test and characterise the virus subtype,

is conventionally adopted a 4HAU antigen solution, containing 4 times that viral concentration.

For example: if the HA titre obtained is 1:512 (1 HAU), the 4 HAU will be obtained dividing four

times that titre (512:4=128). The dilution of 1:128 of the haemagglutinating allantoic fluid will be

utilised to prepare the antigen solution to perform the HI test.

Haemagglutination inhibition test (Hl test)

This method is based on a simple reaction between the virus and the specific antiserum. When the

antiserum reacts with the virus, it will not allow the viral binding with RBCs consequently the

haemagglutination phenomenon is inhibited and RBCs precipitate as a spot in the bottom of the

microplate wells. By the other hand, when haemagglutination occurs, the antiserum used is non

specific for the unknown virus.


Serum known : use 1 ml of sterile distillate water to reconstitute one bottle of freeze-dried

serum following manufacturer’s instructions. Prepare 16 antisera against all the different

known hemagglutinines antigens.

Antigen unknown : prepare the viral solution with 4 HAU as described above.

Procedure

1. Dispense 0.025 ml PBS into each wells of a plastic microtitre plate (with V-bottomed wells).

(Except the first well of the Virus Control’s row.)

2. Place 0.025 ml of each different serum in the wells of the first column of plate (A to G) and use

the last row (H) to control the 4 HAU of the Virus and the RBCs (Fig. 5).

3. Use a multichannel micropipette to make two-fold dilutions of sera across the plate and discard

the last 0.025ml (Fig. 4).

4. Add 0.025 ml of diluted allantoic fluid containing 4 HAU in each well from row A to row G.

5. In the first two wells of the last row (4 HAU virus control: H1-H6) of each plate, put 0.025 ml

of diluted allantoic fluid containing 4 HAU and make two-fold dilution from the second well to

the sixth well (H2-H6). Discard the last 0.025ml. In the first three wells (H1-H3) it must be

observed total haemagglutination, in well H4 a partial haemagglutination (half drop) and in

wells H5 and H6 no haemagglutination should be observed, correspondingly containing 4

HAU, 2 HAU , 1 HAU , 0.5 HAU, 0.25 HAU, 0.125 HAU.

6. Add 0.025ml of PBS in all wells of virus control and RBCs control row. To complete the

volume amount up to 0.075ml per well.

7. Mix by tapping and place the plate at +4 C for 40 minutes or at room temperature for 30

minutes.

8. Add 0.025 ml 1 % RBCs to all wells.

9. Mix by gentle tapping and place at 4 C or at room temperature.

Plates are read after 30-40 minutes, when RBCs control is settled. This is done by tilting and

observing the presence of tear-shaped streaming at the same rate as in the RBCs control wells.

The virus is identified on the correspondence of the reference antiserum which inhibits its

hemagglutinating activity. The titre of that serum should be equal or one dilution of the

known titre. See figure n°4

The virus control is a HA titration to confirm the presence of the required 4 HAU and should be included in each plate.

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaNeuraminidase inhibition test

The neuraminidase inhibition (NI) test is a laboratory procedure characterising the influenzavirus

neuraminidase. Up to now, nine different neuraminidase subtypes of influenza type A have been

isolated from avian species.

This method is based on the inhibition of the enzymatic activity of the neuraminidase using its

specific antiserum. The remaining neuraminidase activity will detect through the last biochemical

product produced after the addition of severalchemical reagents.

In absence of its specific antiserum the viral neuraminidase reacts with the fetuin substrate releasing

an amount of N-acetyl neuraminic acid.

Subsequently, the addition of periodate oxidises the N-acetyl neuraminic acid into β-formyl pyruvic

acid.

The further addition of thiobarbituric acid develops a chromophore which can be extracted into acid

butanol. Colorimetric reading is performed by using a spectrophotometer. In conclusion if the

antiserum is not specific for the neuraminidase, this protein maintains its enzymatic activity which

leads to the production of a pink coloured solution.

By the other hand the specific antiserum binds to the neuraminidase antigenic protein inhibiting its

enzymatic activity . and the final solution.will be white. .

This test can be performed only after having characterised the subtype by HI, infact all the reference

antisera must have an heterologous haemagglutinin antigen then the virus under test in order to

avoid cross reactions.

Definition: The assay is based on the reaction between the unknown neuraminidase viral type with specific

known antisera able to inhibit its enzyme activity.

Procedure:1. Prepare antisera for each different subtype of neuraminidase (9 in total). The haemagglutinin

of the sera should be different from that of virus under test. Example: the virus under test is

an H7 chose antisera H1N1, H1N2, H5N3, H8N4 etc…try not to chose H7N1 for example.

2. Dilute the antisera 1:5 in PBS pH 5.9

3. Dilute the virus 1:15 in PBS pH 5.9 if the titre is >1:64. Dilute the virus 1:13 if the titre is

≤1: 64


4. Set up the antisera and the sample as suggested in the outline below

N1 N2 N3 N4 N5 N6 N7 N8 N9 V B

5. Dispense 100μl of the diluted antiserum to each appropriate tube (serum anti-N1 in the tube

N1, serum anti-N2 in the tube N2 etc.)

6. Add 100μl of diluted virus in all tubes except from tube B (blank)

7. Add

100μl of

PBS pH

5.9 in the

tube V (virus control) and 200μl in the tube B (reagents control)

8. Close each tube with the plug and incubate them at room temperature for 30 minutes.

9. Add 300μl of standard fetuin to each tube. Shake carefully the tubes for 15 seconds in order

to mix the reagents

10. Close the tube with the plug and incubate at 37°C for 16-20 hours

11. Add 200μl of sodium periodate to all the tubes. Shake carefully the tubes for 15 seconds in

order to mix the reagents.

12. Close the tube with the plug and incubate at 37°C for 30 minutes

13. Add 200μl of sodium arsenate to all the tubes. Shake them carefully to mix the reagents until

the brown colour generated by the reaction has not been completely vanished (now is

possible to stop the assay by storage the tubes at +4 °C)

14. Add 2 ml of thiobarbituric acid to each tube. Shake carefully the tubes for 20 seconds to mix

the reagents.

15. Remove the plug and immerse the tubes in boiling (100°C) water for 7 minutes and 30

seconds

Results:

Symbols:

N: Neuraminidase

B: Blank (reagents control)

V: Virus (Virus control)

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaTubes containing the pink coloured solution = no inhibition has occurred. The

antiserum used is not specific for the neuraminidase.

Tube containing transparent or white coloured solution compared to virus control

tube = total inhibition. The antiserum used is specific to the neuraminidase.

For example A white solution has been obtained using antiserum against N9: the

neuraminidase is type 9.


SEROLOGY

Domestic and wild birds infected with Avian Influenza Viruses (AIVs) through natural exposure or

vaccinated against AIV develop antibodies (Ab). These antibodies can be detected by means of

serological tests: Agar Gel Immunodiffusion (AGID), Enzyme Linked Immuno-sorbent Assay

(ELISA) and Haemagglutination Inhibition (HI) test.

Detection of Antibodies against the Type A AIV group antigens

The diagnostic tests that detect the Ab against group A AI can only give information on:

Infection of animals with a Type A AIV

Antibodies induced by vaccines with the exception of live Fowlpox vectored vaccines or other

engineered vaccines that do not induce Ab against the group A antigens (nucleoprotein and

matrix protein)

No additional information can be deduced about the viral subtype circulating.

Agar Gel Immune Diffusion AGID

This test is widely and routinely used to detect the presence of antibodies against Influenza A virus

in serum of birds. It is very specific but is of limited sensitivity (i.e. can recognize almost all the

negative samples but in turn it will miss some positive samples). For this reason it must be used as a

diagnostic tool on a flock basis. It has not been fully validated in other avian species with the

exception of chickens and turkeys and it is not applicable to waterfowl sera (ducks and geese) as

they lack precipitating antibodies.

Antibodies against Influenza A viruses are detected by lines formed by the precipitation of the

immune complex composed by the link between the Ab of the tested serum and reference antigen

(Ag).

Preparation of agar dish:

1. Dissolve 8g of NaCl in 100 ml of distilled water in a volumetric flask,2. Add 1.25g of Noble Agar and mix gently


3. Dissolve the agar by immersing the flask in a boiling water bath until the agar is completely

dissolved

4. Transfer 15ml of the agar solution per Petri dishes

5. Allow the agar to cool at room temperature without covering the dishes

6. Identify the batch of agar dishes by writing the production date on each cover then close the

dishes in a sealed plastic bag

7. Store the agar dishes up to 15 days at +4°C inverted in order to avoid the condensation on

the lid

Testing procedure:

1. Write on the dishes the identification number of the samples.

2. Punch wells with the agar punch as shown in the scheme below. Remove the agar plugs with

a steel tip or a Pasteur pipettes attached to a vacuum pump.

3. Place 30 μl of Ag into the central well (see scheme 1).

4. Add 30μl of positive antiserum (S+) into two wells that are directly opposite from each other

as shown in scheme below.

5. Place 30 μl of the serum under examination (SE) in the remaining wells. The disposition of the

reagents envisages that each suspected serum must be adjacent to a S+ and Ag.

6. Incubate the dishes in a humid chamber at room temperature for 48 hours.

Interpretation of results:

1.After 48 hours incubation observe the dishes using a diffuse light source.

2.The test is valid when a precipitation band is seen between the well containing the S+ and the

central well containing the Ag.

3.The sample is positive when a precipitation band is observed between the well containing the

SE and the central well containing the Ag.

4.The sample is negative when no precipitation band is seen between the well containing the SE

and the central well containing the Ag.

Scheme 1. Agar Gel Immunodiffusion plate: position of reagents in the plate


ELISA (Enzyme Linked Immuno-sorbent Assay) test for antibodies against Avian

Influenza VirusELISA tests are useful and sensitive for gross serological screenings. They are difficult to be set up,

but several kits are available on the market.

The majority of the commercial kits can detect Ab against Type A Influenza Virus.

Specifically they can detect the presence of Ab against the nucleoprotein (NP) and the matrix

protein (M) that are conserved in all the AIV subtypes.

Several test and antigen preparation methods are used. Such tests have usually been evaluated and

validated by the manufacturer, and it is therefore important that the instructions specified for their

use must be followed carefully.

Despite their large diffusion, not all the tests have been validated and standardized by OIE.

There are two kinds of ELISA tests: competitive and indirect ELISA tests.

Competitive ELISA tests have the advantage that sera of any avian species can be examined, on the

contrary, Indirect ELISA tests can be used only for the detection of antibodies against the species

indicated by the manufacturer .

Preparation of Red Cell Suspensions

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A 1% Red Blood Cell Suspensions is used in HA and HI assays on chicken sera, while a 10% RBCs

suspension is used in the pre-treatment of sera, coming from different species than chickens. Non

specific reactions, due to the different species provenance of the blood may appear during HI assay.

To avoid this inconvenient a previous contact of that serum with the chiken RBCs exhausts non

specific hemagglutinant agents.

SE SE

S+ AG S+

SE SE


Procedure

From SPF chickens, collect 5 ml of blood with a syringe containing the same quantity of Alsever's

solution to give a ratio of 1:1.

Pool the syringe together and centrifuge the blood suspension at 2,500 rpm for 10 minutes and

discard the Alsever's solution or supernatant.

Wash the RBC's two times in PBS solution, centrifuging at 2,500 rpm for 10 minutes after each

washing.

The supernatant fluid is removed with a pipette and the packed RBC are ready

to prepare a Red Cell Suspensions of appropriate concentration for a given test.

When the suspension is prepared keep it for 7 days at +4C

10% Red Blood Cell Suspensions

Prepare 9 ml of 0.05% Bovine Albumine PBS solution and add 1 ml of packed RBCs

1% Red Blood Cell Suspensions

Prepare 90 ml of 0.05% Bovine Albumine PBS solution and add 10 ml of packed RBCs

The correct percentage should be controlled with one of the following methods.

1-Spectrophotometer.

Set up two cuvettes with 3.6 ml of distilled water and 0.4 ml of the suspension to be read in the

spectrophotometer at the 545 nm length.

Refer to the PBS 0.05% albumin solution as the blank.

RBCs 1% suspension should result an O..D. of 0.250 nm.

2- Microhematocrit tube method

The hematocrit may be measured manually by centrifugation. A thin capillary tube called a

microhematocrit tube is filled with blood and sealed at the bottom. The tube is centrifuged at 10,000

RPM (revolutions per minute) for five minutes. The RBCs have the greatest mass and are forced to

the bottom of the tube. The height of the red cell column is measured as a percent of the total blood

column. The higher the column of red cells, the higher the hematocrit and the percentage.

3- Cell counter chamber ( Thoma , Burker , Malassez)

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian Influenza75 millions – 80 millions of red blood cells in 1 ml correspond to a concentration of 1%.

Detection of specific subtype Antibodies

Haemagglutination Inhibition test – (HI):

Before performing an HI test, titration of the reference Ag is necessary in order to prepare the 4HAU

antigen solution. (For this procedure please refer to § 3.5.)

This method is based on a simple reaction between virus and specific antiserum. When the

antiserum reacts with the virus, it will not allow the viral binding with RBCs consequently the

haemagglutination phenomenon is inhibited and RBCs precipitate as a spot in the bottom of the

microplate wells. This phenomenon is specifically called “haemagglutination inhibition” (HI). By

the other hand, when haemagglutination occurs, the diagnostic serum does not contain specific

antibodies against the reference antigen.

Reagents:

1. Phosphate Buffer Saline solution (PBS)

2. PBS and albumin (PBS/albumin 0.05%)

3. Freeze-dried reference antigen diluted with PBS in order to obtain 4 UHA per 0.025 ml

4. 1% chicken RBCs suspension

5. Negative control chicken serum

6. Positive control chicken serum

See the composition of reagents in Annex 2.

* Reconstituted reference sera must be store at -20°C and reconstituted reference antigens at -80°C.

HI -Test for chicken sera

Dispense 0.025ml PBS into all the wells of a microtitre plate with the exception of the H1

well (see figure 3, page 51 )


Dispense 0.025ml of serum into the first wells of the microtitre plate (column 1). Add

0.025ml of the positive control serum (with known HI titre) in the F1 well and 0.025ml of

negative control serum in the G1 well. Repeat the positive and negative control sera at least

after every 10 microplates. Keep the 4HAU control virus and the RBCs control in all the

microplates.

Using a multichannel micropipette make two-fold dilutions of the sera (A1-A12) across the

plate. Discard the last 0.025ml

Add 0.025ml of antigen suspension containing 4UHA across the plate with the exception of

row H.

Add 0.025ml of antigen suspension containing 4HAU in the first two wells of raw H (4HAU

control from H1 to H6), make two fold dilution from H2 to the H6 and discard the last

0.025ml in order to obtain 4, 2, 1, 0.5, 0.25, 0.125 HAU. This control must be included in

each microtitre plate.

Add 0.025ml of PBS + albumin 0,05% in all wells of the H row.

Mix by gentle tapping and place the plate at +4 C for 40 minutes or at room temperature

(+20-24°C) for 30 minutes.

Add 0.025ml of 1 % RBCs suspension into all wells.

Mix by gentle tapping and incubate at +4 C for 40 minutes or at room temperature for 30

minutes.

Read the plates after 30-40 minutes, when RBCs control is settled. This is done by tilting

and observing the presence or absence of tear-shaped streaming at the same rate as control

wells containing RBCs (0.025 ml) and PBS (0.05 ml) only.

The HI titre is the highest dilution of serum causing the complete haemagglutination

inhibition

The test is valid if:

the negative control serum has a titre of less than 2³ for 4HAU.

the positive control serum has a titre which coincide with its declared titre or with one lower or

higher dilution step.

Complete haemagglutination is observed in the first three wells (H1-H3) of the 4UHA control row

(containing respectively 4, 2, 1 UHA)

Istituto Zooprofilattico Sperimentale delle Venezie OIE/FAO National Reference Laboratory for Avian Influenza and Newcastle Disease Training in laboratory diagnosis for Avian InfluenzaInterpretation of results

1. When the HI titre is < 1:8, the sample is considered negative. This means that the bird is not

probably immunized or infected.

2. When the HI titre is ≥ 1:16, the sample is considered positive. This means that the bird is

immunized or infected.

HI Test for different avian species than chickens

Non-specific reactions frequently occur when sera samples coming from other avian species than

chickens get in touch with with the 1%RBCs suspension in ordinary HI test. In order to reduce these

inconvenient a pre-treatment with a 10% RBCs suspension is necessary.

It is also suggested to inactivate the non specific hemagglutinating agents in the serum of hunting

birds (pheasant, partridge, etc.), quails, ostriches and guinea fowl with a heat treatment in a water

bath at 56°C for 30 minutes.

Pre-treatment procedure:

Dispense 0.05ml of PBS into the first column wells of the microplate (wells A1- E1).

Do not dispense anything in the second column wells (A2- E2).

Dispense 0.025ml PBS into all the other wells of the microtitre plate. (respecting the

exclusion of column 2)

Add 0.025ml of serum into the first wells of the microplate (column 1).

Add 0.025ml of the 10 % RBCs suspension to the first wells (column 1).

Incubate the plate for 30-40 minutes at room temperature (+20-24°C). waiting the 10%

RBCs suspension to settle.

Transfer 0.025ml of the supernatant from the wells of the first column to the wells of the

second column.

Transfer other 0.025ml of the supernatant from the wells first column to the wells of the third

column. Make two-fold serial dilution of the sera from the third column to the last column (12).

Discard the last 0.025ml.


ELISA

Infection with other subtypes

AGID

Wild birds

HI for H5 and H7

-+

Negative to AIV infection

- +Seropositive for

AI

Seronegative for antibodies to AI subtype

Domestic poultry

HI for other subtypes

Guidelines of correct applications of serological test in the practice

Science

Classical techniques for aiv