Avian Influenza Massi Paola Sezione di Forlì IZSLER Santa Sofia,

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Dal 2003 a giugno 2006 registrati presso l’OMS 205 casi di infezione con 113 decessi

La letalità media del virus dell’influenza aviaria H5N1 (OMS) L’OIE segnala focolai epizootici negli uccelli domestici e selvatici in una cinquantina di Paesi

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Per il 50% i casi di contaminazione sono stati registrati nei soggetti con meno di 20 anni, il 90%in quelli con meno di 40 anni

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Quanto ai bambini colpiti dall’infezione, 21 avevano meno di 5 anni e 32 tra 5 e 9 anni.

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Il fatto che la maggior parte dei casi interessa soggetti di età compresa tra i 10 e i 29 anni potrebbe spiegare la loro presenza nei Paesi dove la popolazione è giovane

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Per esempio, Egitto e Indonesia, nel 2005, dove rispettivamente il 34 e il 28% della popolazione ha meno di 15 anni.

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Inoltre, i comportamenti legati all’età o al sesso (es.lo spiumaggio,la macellazione e la preparazione degli alimenti vengono effettuati dalle giovani donne e i bambini giocano con volatili infetti) aumentano il rischio di esposizione prolungata e di stretto contatto con i volatili infetti.

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Tuttavia, nessuna differenza statisticamente significativa è stata evidenziata fra gli uomini e le donne quanto al rischio dell’infezione

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Il livello generale di mortalità si assesta al 56%.E’ elevato per tutte le età anche se raggiunge il 73% dai 10 ai 39 anni Il livello più basso (18%) si registra negli over 50. Il livello di letalità differisce da quello dell’influenza stagionale”tradizionale”per la quale la mortalità più elevata si registra nelle persone più anziane.

La letalità media del virus dell’influenza aviaria H5N1 (OMS) Il livello generale più elevato di mortalità si è raggiunto nel 2004 con il 73% Durante gli ultimi tre anni, l’incidenza di mortalità ha raggiunto il picco nel corso del periodo inverno-primavera dell’emisfero nord

DIAGNOSIS Clinical, gross amd microscopic findings Laboratory diagnosis

Meat-type and breeder turkeys

Before 1999 We known H6 and H9 in turkeys Respiratory signs Inactivated vaccines

H7N1 LPAI 1999 Year

Low pathogenicity avian influenza (LPAI) Clinical findings Mortality rates ranging from 5 to 97% Depending of the age, on a series of environmental factors (temperature,ventilation,hygienic conditions) and the presence of infectious agents

Low pathogenicity avian influenza (LPAI) During the fase acute of the disease, depression,ruffled feathers and congiuntivitis Swelling of infraorbital sinuses with caseous clots Egg production dropped from 30 to 80% 3dispnea

3Swelling of infraorbital sinuses

Low pathogenicity avian influenza (LPAI Swelling of infraorbital sinuses with caseous clots

Low pathogenicity avian influenza (LPAI Haemorrhagic tracheitis

Lungs congested and haemorragic

H7N1 HPAI 2000 Year

Severe depression in preagonic phase and dead birds on their back due to nervous signs and spastic contractions prior to death

Highly pathogenic avian influenza (HPAI) In meat-type and breeder turkeys, 100% mortality hours from the onset of the first clinical signs. Drop in food consumption and nervous signs

Highly pathogenic avian influenza (HPAI) Post mortem findings

Haemorrhagic tracheitis with caseus clots

Pancreatitis and duodenitis

Necrosis of pancreas

Haemorrhages on the caecal tonsils

3Polmonite acuta

Chicken

LPAI Broilers and broilers breeders Clinical findings In the majority of flocks, LPAI did not cause any clinical signs In a limited number of outbreaks was characterised by anorexia and mild respiratory signs, with low mortality, in the order of 2-3%

LPAI Post mortem findings Polmonary and tracheal congestion with cattarrhal tracheitis Ovarian follicles haemorrhagic and oedematous

LPAI Caged layers Clinical findings Only 10-20%of the birds with loss of appetite and depression, with very mild respiratory signs and congestion of combs Drop in egg from 2 to 10% until 30% Mortality between 0.5 and 2%

LPAI Post mortem findings The lungs and tracheas were congested The ovary and oviduct edematous and haemorrhagic

Lungs and trachea congested

HPAI Chickens reared on litter Clinical findings 100% mortality h.from the onset of the first clinical signs Anorexia, depression and cessation of egg- laying were followed by complete reluctance to move and tremors of the head and paralysis of the wing and legs

HPAI Caged layers Clinical findings The disease moved more slowly within the flock Sonnolence, cessation of egg-laying and feed consumption. Cianotic coombs with tremors of the head

HPAI Post-mortem findings Pancreatitis, caecal tonsils haemorrhagic Internal organs appeared congested Urate deposits in the kidney

Duck and goose

HPAI Clinical findings Incoordination and tremors and mortality % Post mortem findings Pancreatic lesions Heart congested Duodenum congested

Japanese quail (coturnix coturnix japonica)

HPAI Severe respiratory condition, prostration and diarrhoa Nervous signs with torticollis and opistothonus High mortality

Ostrich (struthio camelus)

Clinical signs were observed only in juvenile (7-9 months of age) anorexia, depression Feed consuption dropped Nervous signs haemorrhagic faeces Mortality 1-20%

H7N3 LHAI 2002 year

Laboratory diagnosis

A presumptive diagnosis can be made by Detecting antibodies

A definitive diagnosis of AI is established by: 1.Direct detection of AI viral proteins or genes in specimens such as tissues, swab, cellculture or embryonating eggs 2.Isolation and identification of AI virus

Sample selection and storage Tracheal and cloacal swabs or tissues collected If the samples can be tested within 48h. After collection they may be kept at 4°C; if over h., storage at -70°C

Direct detection of AI viral protein or Nucleic acid Antigen capture ELISA to detect viral antigens in samples Elisa sandwich virologica Con Mab anti NPA (HB65)

Direct detection of AI viral protein or Nucleic acid a human influenza test (Directigen Becton- Dickinsos) used to detect influenza viral antigen This antigen capture enzyme immunoassay was found to be specific and sensitive in tracheal swabs

KIT IMMUNOENZIMATICO

3Kit immunoenzimatico

3Tamponi ed estratti d’organo per kit rapido

Commercial Rapid Ag Tests Advantages 1. Rapid 2. User-friendly 3. On farm test Disadvantages 1. Very high cost 2. Poor sensitivity

Direct detection of AI viral protein or Nucleic acid Polymerase chain reaction methods used are more sensitive than virus isolation procedures

RT-PCR H7 H5 M M ctrl Ctrl- H5+ H7+ Metodo identificativo del genoma virale

3PCR-Real Time

Quantitative real time PCR 3Allows detection of the amplicon as it accumulates by measuring light emission via a specific probe. This light emission is linked to amplicon production

PCR Advantages 3Sensitivity/ specifity 3Rapid test Disadvantages High cost

Virus isolation Chicken embryos,10-11 days old,inoculated via the allantoic cavity

Virus isolation The presence of virus is demostrated by chicken erythrocyte haemagglutinating activity (HA) in the allantoic fluid

Virus isolation Plaque assay on cell monolayers (MDCK) MDCK Madin Darby Canine Kidney Cells

Virus identification 1) H.I. assay against NDV and other paramixo

Virus identification 2) Double immunodiffusion test AGP to dectect the type- specific NP and matrix proteins

Virus identification 3) ELISA with monoclonal antibodies reacts with the nucleoprotein or matrix proteins

Virus identification 4) The next spet is to determine the antigenic subtype of the surface antigens: HA and NA The HA is identified in the HI test using a panel of antisera prepared against the distinct HAs

VIRUS ISOLATION Disadvantages 3No rapid 3Cost Advantages Virus isolation is important for patogenicity studies

Serology Specific antibodies detect as earlyas seven days after infection

Serology In serological surveillance programs: A) AGP test is used for the detection of anti-NP antibodies Group specific

Serology B) ELISA assays have been developed to detect antibodies AI virus

Serology C ) Once influenza is detected by AGP or ELISA, HI test and ELISA with Mabs can be used to determine the HA subtype Subtype specific Paolo Cordioli