Friday, March 27, 2015

APHIS: USDA Confirms 2nd H5N2 Outbreak In Minnesota Turkey Flock

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# 9876

 

Three weeks after we first learned from APHIS: HPAI H5N2 In A Minnesota Turkey Farm in Pope County, we are informed that a second farm has been affected in Lac Qui Parle County.  

 

Given that these counties are not immediately adjacent, and a  CIDRAP News report (see Wild birds nixed as Minnesota H5N2 source) from earlier this week quoted the Minnesota Department of Natural Resources (DNR) saying they found no evidence of the H5N2 strain in samples from wild birds in the area, it will be very interesting to see if there are any epidemiological links between the two poultry operations.

 

Here is the statement, posted on the APHIS website, this afternoon.

 

 

USDA Confirms Highly Pathogenic H5N2 Avian Influenza in Commercial Turkey Flock in Minnesota

Last Modified: Mar 27, 2015

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CDC considers the risk to people from these HPAI H5 infections in wild birds, backyard flocks and commercial poultry, to be low

WASHINGTON, March 27, 2015 -- The United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) has confirmed the presence of highly pathogenic H5N2 avian influenza (HPAI) in a commercial turkey flock in Lac Qui Parle County, Minnesota.  This is the second confirmation in a commercial flock in Minnesota.  The flock of 66,000 turkeys is located within the Mississippi flyway where this strain of avian influenza was previously identified. CDC considers the risk to people from these HPAI H5 infections in wild birds, backyard flocks and commercial poultry, to be low.  No human infections with the virus have been detected at this time.

Samples from the turkey flock, which experienced increased mortality, were tested at the University of Minnesota Veterinary Diagnostic Laboratory and the APHIS National Veterinary Services Laboratories in Ames, Iowa confirmed the findings. APHIS is working closely with the Minnesota Board of Animal Health on a joint incident response. State officials quarantined the affected premises and birds on the property will be depopulated to prevent the spread of the disease. Birds from the flock will not enter the food system.

The Minnesota Department of Health is working directly with poultry workers at the affected facility to ensure that they are taking the proper precautions. As a reminder, the proper handling and cooking of poultry and eggs to an internal temperature of 165 ˚F kills bacteria and viruses.

As part of existing avian influenza response plans, Federal and State partners are working jointly on additional surveillance and testing in the nearby area. The United States has the strongest AI surveillance program in the world, and USDA is working with its partners to actively look for the disease in commercial poultry operations, live bird markets and in migratory wild bird populations.

USDA will be informing the World Organization for Animal Health (OIE) as well as international trading partners of this finding. USDA also continues to communicate with trading partners to encourage adherence to OIE standards and minimize trade impacts. OIE trade guidelines call on countries to base trade restrictions on sound science and, whenever possible, limit restrictions to those animals and animal products within a defined region that pose a risk of spreading disease of concern.

These virus strains can travel in wild birds without them appearing sick. People should avoid contact with sick/dead poultry or wildlife. If contact occurs, wash your hands with soap and water and change clothing before having any contact with healthy domestic poultry and birds.

All bird owners, whether commercial producers or backyard enthusiasts, should continue to practice good biosecurity, prevent contact between their birds and wild birds, and report sick birds or unusual bird deaths to State/Federal officials, either through their state veterinarian or through USDA’s toll-free number at 1-866-536-7593.  Additional information on biosecurity for backyard flocks can be found at http://healthybirds.aphis.usda.gov.

(Continue . . . )

Media Reports: Bird Flu Detected In Romania & Italy

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# 9875

 

Yesterday’s bird flu report from Bulgaria, very near the Romanian border (see OIE: H5N1 Kills 21 Pelicans In Bulgaria) has been quickly followed by multiple media reports that dead pelicans found this week in Romania have now tested positive for the H5N1 virus as well.

 

If confirmed by the OIE, this would be the first appearance of H5N1 in Romania since 2010.

Bird flu in Romania. Authorities found infected pelicans in the Danube

BURCHAREST  Romanian authorities have found in the Danube Delta for more than six decades of dead pelicans and laboratory tests six bodies have confirmed the presence of avian influenza virus H5N1. Informed on Friday by Agerpres, citing the report of the State Veterinary Office.

According to information from this office was 64 dead pelicans already discovered on Wednesday 25 March. Six bodies were handed over to the following day tests at the Central Veterinary Laboratory for the control of food safety and confirmed the occurrence of the virus H5N1 in all samples.

According to experts, is now in the Danube Delta are also other kinds of birds, but apart from pelicans was found dead bodies of any of them.

Any dead birds are the Dalmatian Pelican (Pelecanus crispus). It is a migratory birds in this annual time migrate from Africa to the Danube Delta.

Romanian authorities announced that in connection with the occurrence of bird flu in the Danube Delta tightened veterinary supervision of bird populations. Introduced three kilometers around the outbreak protection zone and a ten-kilometer control zone.

(Continue . . . )

A second report indicates these birds were discovered in Tulcea County, Romania.

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Credit Wikipedia.

 

March 27, 2015 3:14 p.m.

Alert avian influenza in the Danube Delta. The virus was found in corpses of pelicans

Veterinary authorities in Tulcea County are on alert after Ceaplace island in the Danube Delta has been confirmed avian influenza virus. The disease was detected in several pelicans, found dead. The case came to the attention of Tulcea, after the island Ceaplace was notified that there are more dead pelicans. Inspectors have taken six corpses DSVSA Tulcea pelicans that have undergone testing. Following laboratory tests found the presence of avian influenza virus. These samples were sent for confirmation to the National Institute for Animal Health Diagnostic, which is the National Reference Laboratory for avian influenza. Integral on adevarul.ro

 


Meanwhile, a somewhat less informative media report out of Oppeano, Italy stating that `bird flu’ has broken out at a farm in the Veneto region, but without specifying the subtype of influenza.  Italy has reported H5N8, H5N1, and H7 subtypes previously, so we’ll probably  have to wait for an FAO or OIE notification to learn the cause of this outbreak.

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Credit Wikipedia

 

Outbreak of bird flu discovered in farm

It is a farm with an adjoining farm. Recommended immediate control measures with slaughter of all susceptible animals

OPPEANO - Discovered in a herd, also home to a farm , to Oppeano, an outbreak of avian influenza .
The Section veterinary and food safety of the region Veneto , with a note sent to the mayor of the town in the province of Verona and the veterinary service of ' Ulss 21 Legnago, has recommended the adoption of immediate measures to control, with slaughter of all susceptible animals and the destruction of carcasses and materials.


The positive virological and serological virus was confirmed by the National Reference Centre for the ' AI Institute zooprofilattico delle Venezie. " The positive virological - is written in the note of the Region - testifies to the presence of avian influenza in the company and the finding of only one out of 20 seropositive taken may indicate a recent introduction of virus ', which more so requires immediate action to prevent the disease from spreading.


A second report reads:


Outbreak of bird flu discovered on a farm

Alerting Oppeano in Veronese. The Health Authority of Legnago ordered the immediate slaughter of all the heads

OPPEANO .A outbreak of bird flu was discovered in a herd, also home to a farm, to Oppeano, in the province of Verona. Section veterinary and food safety of the region Veneto, with a note sent to the mayor of Oppeano and Veterinary Service Ulss 21 Legnago (Verona) recommended the adoption of immediate measures to control, with slaughter of all susceptible animals and the destruction of carcasses and materials. The National Reference Centre for Avian Influenza Institute of zooprofilattico venezie confirmed virological and serological positivity to the virus.

 

These past few months have seen a remarkable spread of HPAI H5 viruses around the world, arguably the most impressive geographic expansion of bird flu since the great H5N1 diaspora of 2006 (see H5N8: A Case Of Deja Flu?). 

 

Europe, Asia, Africa, and North America have all see either the return, or in some cases – the very first arrival – of these highly pathogenic viruses, and the losses to the poultry industry have been substantial. 

 

With the exception of Egypt’s record-setting H5N1 outbreak and occasional human cases in China, the impact on human health this winter been negligible.  Some of these viruses (H5N8 and H5N2) have no record of infecting humans, but as they are related to far less benign strains, they are viewed with understandable caution.

 

As these viruses spread through wild and migratory birds, and among commercial poultry, they have the potential to come in contact with other influenza viruses, and possibly reassort into new hybrids.  A process that could yield new subtypes with unpredictable results.

A Genetic Predisposition To Severe Flu Infection

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Photo Credit – CDC PHIL

 

 

# 9874

 

Influenza can produce a wide spectrum of illness, ranging from mild or even asymptomatic presentation to severe and/or life threatening disease.  

 

Often we talk about `high risk’ patients – where their age and/or co-morbidities (COPD, Asthma, pregnancy, etc. ) can sometimes lead to a greater chance of complications, but there may be other factors at work as well. 

 

As we saw in the 2009 H1N1 pandemic, with seasonal flu, and with several strains of avian flu (H5N1, H7N9), young, otherwise healthy adults can be quickly overwhelmed by their infections.

 

For a number of years researchers have been looking for a hematological or genetic marker that would help predict which patients would be most likely to experience severe influenza.

 

In 2008, in the Journal of Infectious Diseases, we saw a study that suggested there might be a heritable susceptibility to death from the influenza virus:

Evidence for a heritable predisposition to death due to influenza.

Albright FS, Orlando P, Pavia AT, Jackson GG, Cannon Albright LA.  

 

While interesting, this study didn’t provide us with a smoking gene.

 

In 2009 (see The Best Defense) we inched a bit closer, with research from Harvard Medical School and the Howard Hughes Medical Institute, that identified the IFITM3 protein as capable of inhibiting the replication of influenza, and other viruses, such as West Nile and Dengue. 

 

We revisited the IFITM3 story again in early 2012, in Luck Of The Draw, when we looked at research from the Wellcome Trust Sanger Institute, that found that people who carried a particular variant of the IFITM3 gene - (SNP rs12252-C) - were more likely to be hospitalized with severe influenza

 

In 2013, a study by Professor Peter Doherty (see PNAS: Genetic Marker & Cytokine Levels Linked To Severity Of Human H7N9 Infection) linked IFITM3 CC gene variant (aka C/C Genotype)  to hypercytokinemia (aka a `Cytokine Storm’), and severe outcomes in H7N9 infections.

 

This genetic marker– while comparatively rare in Caucasians - is far more common in Han Chinese, and may (partially) account for some of the particularly high mortality rates we’ve seen with novel influenza’s in Asia. 

 

Fast forward to yesterday, and we get a study published in Science Express that identifies yet another (rare) genetic marker -  a mutation of the IRF7 gene -  linked to a lack of interferon production which can lead to a more severe influenza infection. 

 

Published Online March 26 2015

Science DOI: 10.1126/science.aaa1578

Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency

Michael J. Ciancanelli1, Sarah X. L. Huang2,3,*, Priya Luthra4,*, Hannah Garner5,*, Yuval Itan1, Stefano Volpi6,7, Fabien G. Lafaille1, Céline Trouillet5, Mirco Schmolke4, Randy A. Albrecht4,8, Elisabeth Israelsson9, Hye Kyung Lim1, Melina Casadio1, Tamar Hermesh1, Lazaro Lorenzo10,11, Lawrence W. Leung4, Vincent Pedergnana10,11, Bertrand Boisson1, Satoshi Okada1,12, Capucine Picard1,10,11,13, Benedicte Ringuier14, Françoise Troussier15, Damien Chaussabel9,16,†, Laurent Abel1,10,11,†, Isabelle Pellier17,†, Luigi D. Notarangelo6,†, Adolfo García-Sastre4,8,18,†, Christopher F. Basler4,†, Frédéric Geissmann5,†, Shen-Ying Zhang1,10,11,†, Hans-Willem Snoeck2,3,†, Jean-Laurent Casanova1,10,11,19,20,‡ 

Abstract

Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient’s leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient’s dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

Some excerpts from the Rockefeller University press release follow:

 

Genetic mutation helps explain why, in rare cases, flu can kill

March 26, 2015 | Science News

(Excerpts)

The researchers scrutinized blood and tissue samples from a young girl who, at the age of two-and-a-half, developed acute respiratory distress syndrome after catching the flu, and ended up fighting for her life in the hospital. Years after her ordeal, which she survived, scientists led by Jean-Laurent Casanova discovered that it could be explained by a rare mutation she carries that prevented her from producing a protein, interferon, that helps fight off the virus.

<SNIP>

Turning their attention to influenza, Michael J. Ciancanelli, a research associate and senior member of Casanova’s lab, and his colleagues sequenced all genes in the genomes of the young girl who survived her dangerous bout of the flu and her parents, looking for mutations that might explain her vulnerability. Knowing how rare her reaction to the flu was, they narrowed their search to mutations that were unique to her, then focused only on those that affected the immune system.

What emerged from their work was the finding that the girl had inherited two differently mutated copies of the gene IRF7, which encodes a protein that amplifies the production of interferon, a critical part of the body’s response to viral infections. “No other mutations could have explained her reaction to the influenza virus,” says Ciancanelli. “Each mutation is very uncommon and thus the likelihood of carrying two damaged copies of the gene is extremely rare.”

(Continue . . . )

 

In a separate press release from HHMI, in view of the likelihood that others care this or similar genetic factors, Dr. Casanova recommends that clinicians may want to consider including interferon alpha in the treatment of severe unexplained flu.

 

[ March 26, 2015 ]

Rare Genetic Mutations May Make Influenza Life-Threatening for Some

Summary

HHMI researchers identify a rare genetic mutation that tamps down immune response to influenza.

Highlights

  • Why did the flu develop into a life-threatening infection in a two-year-old girl?
  • HHMI researchers tried to identify the cause of the girl's severe response to influenza infection.
  • They found that the girl carries a genetic mutation that affects production of natural antiviral molecules.

(Continue . . . )

NIH: Ebola Virus More Stable Than Previous Study Suggested

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Credit CDC PHIL

# 9873

 

Last August, while Ebola was still accelerating in West Africa, we saw a study warning that the Ebola Virus Is Rapidly Evolving.  Specifically, that 99 Ebola viruses taken from 78 people from Sierra Leone during the month of June, and found that the virus is showing a marked propensity to accumulate `interhost and intrahost genetic variation’ as it passages through the population.


This led to media reports such as Ebola virus mutating rapidly as it spreads from Nature News, and  Ebola Is Rapidly Mutating As It Spreads Across West Africa via NPR’s Goats & Soda Blog.


As I noted at the time,  while scientists have the ability to sequence and compare these variant viruses, they don’t necessarily know what these individual mutations (or their aggregate) means to the virus, or how it might change its behavior.

 

Still, this study raised the level of concern over how this virus was evolving, and what changes in its behavior that might eventually bring.

 

Yesterday the NIH announced a far more reassuring survey of the Ebola virus’s evolution, which found a far less aggressive rate of change in the virus than previously announced.  First the NIH announcement, followed by a link to the study, then I’ll be back with a bit more.

 

NIH study finds no evidence of accelerated Ebola virus evolution in West Africa

 

The Ebola virus circulating in humans in West Africa is undergoing relatively few mutations, none of which suggest that it is becoming more severe or transmissible, according to a National Institutes of Health study in Science. The study compares virus sequencing data from samples taken from patients in Guinea (March 2014), Sierra Leone (June 2014) and Mali (November 2014).

 

“The Ebola virus in the ongoing West African outbreak appears to be stable—that is, it does not appear to be mutating more rapidly than viruses in previous Ebola outbreaks, and that is reassuring,” said Anthony S. Fauci, director of the National Institute of Allergy and Infectious Diseases (NIAID), part of NIH. “We look forward to additional information to validate this finding, because understanding and tracking Ebola virus evolution are critical to ensuring that our scientific and public health response keeps pace.”

Obtaining virus samples for analysis was challenging for researchers during the outbreak. The NIAID study published today relies on data from the Guinea and Sierra Leone cases as well as samples from two case clusters in Mali obtained from the International Center for Excellence in Research (ICER) located in Bamako. NIAID and the Malian government have been partners in the ICER since 2002. The Mali case clusters originated from people who became infected in Guinea and traveled to Mali, where they were diagnosed.

Today’s study, from NIAID’s Rocky Mountain Laboratories, finds that there appear to be no genetic changes that would increase the virulence or change the transmissibility of the circulating Ebola virus, and that despite extensive human-to-human transmission during the outbreak, the virus is not mutating at a rate beyond what is expected. Further, they say, based on their data it is unlikely that the types of genetic changes thus far observed would impair diagnostic measures, or affect the efficacy of candidate vaccines or potential virus-specific treatments.

As of March 11, the World Health Organization listed more than 24,000 confirmed, suspected or probable cases of Ebola virus disease in West Africa, with about 10,000 deaths.

 

Mutation rate and genotype variation of Ebola virus from Mali case sequences

T. Hoenen1,*, D. Safronetz1,*, A. Groseth1,*,  K. R. Wollenberg2,*, O. A. Koita3,  B. Diarra3, I. S. Fall4, F. C. Haidara5, F. Diallo5, M. Sanogo3, Y. S. Sarro3, A. Kone3, A. C. G. Togo3, A. Traore5, M. Kodio5, A. Dosseh6, K. Rosenke1, E. de Wit1, F. Feldmann7, H. Ebihara1, V. J. Munster1, K. C. Zoon8, H. Feldmann1,†,‡, S. Sow5,†,‡

 

The occurrence of Ebola virus (EBOV) in West Africa during 2013–2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (9.6 × 10–4 substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.

 

 

Last night Lisa Schnirring of CIDRAP News reviewed these findings, and looked at reports of two new Ebola cases in Liberia, an update on the American being treated at the NIH, and news on one of the candidate vaccines in:

 

Gene study finds no worrisome changes in Ebola virus

Lisa Schnirring | Staff Writer | CIDRAP News

A genetic analysis published today of Ebola virus samples over the course of the outbreak found few changes and noted that the virus is apparently more stable than a study back in August had suggested.

(Continue . .. )

Thursday, March 26, 2015

APHIS Statement: HPAI H5N2 Confirmed In Wyoming Goose

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# 9872

 

 

Although the story has been meandering through the local press for a number of hours (see Crofsblog US: H5N2 avian influenza discovered in Wyoming), we now have an official statement from the USDA’s APHIS  website:

 

 

 

USDA Confirms Highly Pathogenic H5N2 Avian Influenza in Wild Bird in Wyoming

Last Modified: Mar 26, 2015

Print

CDC considers the risk to people from these HPAI H5 infections in wild birds, backyard flocks and commercial poultry, to be low

WASHINGTON, March 26, 2015 -- The United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) has confirmed the presence of highly pathogenic H5N2 avian influenza (HPAI) in a wild Canada goose in Laramie County, Wyoming.  This is the first finding of the Eurasian lineage avian influenza viruses in wild birds in the Central flyway. CDC considers the risk to people from these HPAI H5 infections in wild birds, backyard flocks and commercial poultry, to be low.  No human infections with the virus have been detected at this time.

The sample, taken from a sick bird, was tested by the Wyoming State Veterinary Laboratory and confirmed by USDA’s National Veterinary Services Laboratories in Ames, Iowa.  The United States has the strongest AI surveillance program in the world, and USDA is working with its partners to actively look for the disease in commercial poultry operations, live bird markets and in migratory wild bird populations.

USDA will be informing the World Organisation for Animal Health (OIE) as well as international trading partners of this finding. USDA also continues to communicate with trading partners to encourage adherence to OIE standards and minimize trade impacts. OIE trade guidelines call on countries to base trade restrictions on sound science and, whenever possible, limit restrictions to those animals and animal products within a defined region that pose a risk of spreading disease of concern.

These virus strains can travel in wild birds without them appearing sick. People should avoid contact with sick/dead poultry or wildlife. If contact occurs, wash your hands with soap and water and change clothing before having any contact with healthy domestic poultry and birds.

All bird owners, whether commercial producers or backyard enthusiasts, should continue to practice good biosecurity, prevent contact between their birds and wild birds, and report sick birds or unusual bird deaths to State/Federal officials, either through their state veterinarian or through USDA’s toll-free number at 1-866-536-7593.  Additional information on biosecurity for backyard flocks can be found at http://healthybirds.aphis.usda.gov.

(Continue . . . )

 

Two days ago, in  CDC: HPAI H5 Viruses In The United States,  we looked at the CDC’s advice regarding these avian viruses.

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While the USDA offers the following biosecurity advice for those who may come in contact with wild birds:

Bird Enthusiasts:

Do not pick up deceased or obviously sick birds. Contact your State, tribal, or Federal natural resources agency if you find sick or dead birds.

  • Wear rubber gloves when cleaning your bird feeders.
  • Wash hands with soap and water immediately after cleaning feeders.
  • Do not eat, drink, or smoke while cleaning bird feeders.

Hunters:

Follow routine precautions when handling wild birds.

  • Do not handle or consume game animals that are obviously sick or found dead.
  • Do not eat, drink, or smoke while cleaning game.
  • Wear rubber gloves when cleaning game.
  • Wash hands with soap and water, or alcohol wipes, immediately after handling game.
  • Wash tools and working surfaces with soap and water and then disinfect.
  • Keep uncooked game in a separate container, away from cooked or ready-to-eat foods.
  • Cook game meat thoroughly; poultry should reach an internal temperature of 165 degree Fahrenheit to kill disease organisms and parasites.
  • To report unusual signs in birds you have seen in the wild, call 1-866-4-USDA-WS. To learn more about how you can help, visit usda.gov/birdflu.

 

 

For additional background on the arrival and spread of HPAI H5 and other avian viruses via America’s Flyways you may wish to revisit:

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Credit FAO

J. Virol: Spread & Persistence Of Avian Flu Viruses In The Mississippi Flyway

OIE: H5N1 Kills 21 Pelicans In Bulgaria

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Bulgaria Lies beneath Black Sea / Mediterranean Flyway

 

# 9871

 

Two months ago we learned of the first appearance of H5N1 in Bulgaria since 2010, in OIE Notification Of H5N1 In Bulgaria, which was followed by Bulgaria: Additional H5N1 Outbreak Reported a few days later. 

 

Since then Bulgaria has reported two additional outbreaks, including this latest one involving 21 dead pelicans.


First a media report, followed by the OIE notification.

Bird Flu Kills 21 Pelicans
in Srebarna
Nature Reserve

Silistra, on the Danube, March 26 (BTA) - Laboratory tests  confirmed that bird flu was the cause of death for 21 pelicans in the Srebarna Nature Reserve (Northeast Bulgaria), said on Thursday the Director of the Silistra Food Safety Regional Directorate, Dr. Sebahtin Halid.


The birds were removed from the pelican colony on Tuesday by a team of the reserve's security guards and experts of the Regional Food Safety Directorate.


Two protection zones have been designated. The first one covers a three-km long perimeter surrounding the outbreak area. The second zone covers a perimeter of ten km and is a surveillance area, which includes Silistra and the villages of Vetren, Aydemir, Lambrinovo, Sitovo, Popina and Polyana. Restrictions have been introduced for transporting birds and hunting wild birds in the area. The authorities will monitor for other cases of sick or dead birds and any infected domestic birds will be destroyed. PK/MY

 

 

The OIE Notification follows:

 

 

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This year, in addition to seeing the robust spread of HPAI H5N8 (and H5N2) via migratory birds, we’ve also seen a strong resurgence of H5N1 in the Middle East, India, and Africa.

 

Last November, in The North Atlantic Flyway Revisited, we looked at the ability of seabirds to carry avian flu viruses, along with the potential for lateral creep of flu viruses across overlapping flyways.