Jillian Zientek, Germany, June 19, 2012

Friedrich Loeffler Institut – Day #2

After our fascinating introduction and tour of the FLI on Monday, we returned to Insel Riems for the privilege of spending the day with a number of the FLI staff, who presented to us on various topics of interest and study at the FLI.  The amount of information we’ve received each day on this trip is truly fantastic, and it’s been incredibly humbling to be in the presence of experts who have shaped the field of veterinary medicine and public health.  Along those lines, we had the once-in-a-lifetime opportunity to attend the inaugural Loeffler Lecture, given by internationally renowned influenza specialist Dr. Ron Fouchier.  This day was full of fascinating information, stimulating discussion, and of course, great food!

Lecture #1: Transmissible Spongiform Encephalopathy (TSE)

Given by: Dr. Anna Buschmann

Dr. Buschmann is the Deputy of the FLI Institute for Novel and Emerging Infectious Diseases, a department that had its beginnings in addressing and responding to TSEs, including Scrapie and BSE (“Mad Cow” Disease).  Since these beginnings, this department has expanded to include many other new and emerging infectious diseases.

Responding to the BSE Crisis: Although the UK is perhaps the most notorious country impacted by the BSE crisis, the whole of Europe was greatly impacted by this disease.  In Germany, greater than 50% of the animals diagnosed were healthy slaughter animals, identified during mandatory screening of carcasses.  The implementation of mandatory screening of cattle above a certain age (72mo for healthy slaughter animals, 48mo for risk animals) was one of the eradication measures implemented early in the crisis, in January 2001. FLI plays an important role in facilitating this measure as the reference laboratory for BSE testing – as such, repeat positives identified at local labs by rapid testing come to FLI for confirmation.

Testing for BSE:

  • Local laboratories use a Protein Kinase K-dependent rapid test: normal prion protein is PKK-labile, but the altered prion protein in an animal with BSE is not.
  • FLI uses a variety of other confirmatory methods such as histopathology, electron microscopy, and – most commonly – immunohistochemistry or SAF-immunoblot.
  • The obex region of the brain is the optimal sample site for classical BSE – this is where the protein is most readily found.

 

The second measure in responding to the BSE crisis was removal of specified risk materials from the food supply – these include the skull, vertebrae, tonsils, and small intestine.  The only difference in these rules from the United States standards is the removal of the entire small bowel: in the US, only the ileum is removed.  These standards were built upon BSE pathogenesis studies conducted at FLI, which identified Peyer’s patches in the gut as the route of infection in calves.  Interestingly, however, the pathogenesis studies never demonstrated infectivity via the lymphoreticular system – instead, once the prions have been absorbed via Peyer’s patches, they travel via the alimentary nervous system and eventually the splanchnic and vagus nerves to infect the CNS.  These studies also found that in late stage (ie symptomatic) disease, spillover from the CNS could result in the presence of prion protein in peripheral nerves and muscles (until then, it is confined to the CNS).  Frighteningly for those of us who consume red meat in the US, infectivity studies did demonstrate an infective load in the gut outside of the distal ileum.  Infectivity of the tongue has also been called into question, as some cultures consume tongue.  While no PrPsc could be detected in the tongue, the mouse bioassay from this site was in fact positive.

The final measure in Germany’s BSE eradication effort was a ban on the feeding of animal-derived protein and fat to livestock.  Currently, this measure is in debate regarding the feeding of processed animal protein to aquaculture and fish.

Atypical BSE is a current buzz topic in US news, as several cases were recently diagnosed in California.  Dr. Buschmann touched on the pathophysiology of this disease, which differs from the typical form in several ways:

  • There are two types of atypical BSE, the H-type and the L-type, depending on the molecular weight of the protein involved
  • The anatomical distribution in the brain is different, such that the obex region is not diagnostic.  This has important implications for the rapid testing.
  • This form of disease is only seen in older cattle, and is thought to develop spontaneously.
  • During pathogenesis studies, animals exposed to infectious doses of atypical BSE prions develop disease faster than expected.

That said, typical and atypical BSE are impossible to differentiate based on clinical picture, and infectivity distribution in the tissues is the same.

In contrast to BSE in cattle, TSEs in small ruminants are detectable in peripheral nerves, lymph nodes, and muscles – this more disseminated distribution represents a greater risk to the consumer.  In small ruminants, BSE cannot be differentiated from scrapie based on clinical signs.  This lecture was the first time I’d heard about atypical Scrapie, which is much less resistant to protein kinase K digestion and may therefore go undetected by rapid tests.  This form of Scrapie heavily affects the cerebellum, and can occur in all sheep, even those of the ARR genotype, which are typically considered resistant to Scrapie.

Lecture #2: Foot and Mouth Disease (FMD)

Given by: Dr. Bernd Haas, Institute of Diagnostic Virology

Given the intimate role of FMD in the founding and history of FLI and Insel Riems, it seems only appropriate that we learn about the current research addressing this disease.  FMD is a very challenging virus in that the seven serotypes, 46 topotypes, and hundreds of sublineages and strains make a universal vaccine impossible.  It is dangerous due to its characteristics such as low infectious dose, high virus excretion and aerosolization, environmental stability, creation of subclinical carriers, and tropisim in a variety species.  In addition, in both pigs and sheep, the peak of viral excretion precedes clinical signs.  FLI studies have determined that pigs excrete about 1000X more virus than cattle.  Other studies at FLI demonstrated that esophageal samples from cattle and sheep showed intermittent shedding of the virus for months to years following infection.

Perhaps the most unique research conducted by FLI regarding FMD is that done in wild boars.  FLI maintains a herd of wild boars for research purposes (pictured below), and has demonstrated that while these animals are highly susceptible to infection with FMD, they remain quite ambulatory despite the presence of lesions on their feet.  Their behavior is so unaffected that diseased animals are difficult to detect until closely examined.  This finding suggests that these wild animals could play an important role in spreading the disease should an outbreak occur in Europe.

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Epidemiology of FMD is very different between the developing world and the developed world.  The “red belt” of FMD encompassing North Africa and the Middle East describes regions where FMD is endemic.  In the Eurozone, the current fear of incursion is focused on spread from Turkey or introduction by smuggled feedstuff belonging to refugees from endemic areas.  An outbreak in a developing nation generally follows a typical epidemic curve and control revolves around culling, with the greatest concern being overwhelming of rendering capacity.  The decision regarding whether or not to vaccinate is guided by population density, the availability of a suitable vaccine, and the distribution of the outbreak. FMD must be handled in BSL3+ laboratories, which makes studying this agent quite cumbersome and expensive.

As such, the study and regulations surrounding BSE are a hotbutton political issue.  Testing of vaccines costs millions of Euro, and EU trade restrictions are very strict.  Dr. Haas was refreshingly frank and candid regarding the external factors governing the study of this disease.  This open discussion of political issues and how they impact scientific activities and other aspects of the field is something that seems common to many German academics and professionals – in stark contrast to American classrooms, where elaboration of such issues is often regarded as inappropriate or taboo.  As such, Dr. Haas’ willingness to openly discuss these factors prompted many questions from the students, and a lively discussion of sociological factors governing vaccine use, trade restrictions, and other intersectional issues ensued.

Lecture #3: Schmallenberg Virus

Given by: Dr. Martin Beer, Head of the Institute of Diagnostic Virology

Dr. Beer shared with us the fascinating story behind the discovery of this emerging disease.  Schmallenberg is a disease of ruminants similar to bluetongue, causing fever, decreased milk production, and diarrhea.  Milking robots, which are gaining popularity in Germany, were instrumental in detecting this cluster of clinical signs.  Concerns at the beginning of the outbreak included BHV and Rift Valley Fever, but exhaustive diagnostic evaluation turned up completely negative for known agents.  As such, the hunt for a new agent began.

Metagenomics.  It was a word I had vaguely heard before, but probably couldn’t have defined before this lecture.  Metagenomics refers to the process of analyzing a host sample with new sequencing technology, which utilizes software to distinguish host nucleic acids from pathogen.  This software than uses a server to compare to a database.  Within hours of beginning the metagenomic analysis, a novel Akabane virus had been identified & sequenced, and PCR primers could be developed.  Utilizing this technology, only 2-3 weeks elapsed between first detection and virus isolation – astounding speed when compared with “old” methods of virology.  Interestingly, this virus was only able to replicate on Culicoides cell lines, not any of the mammalian cell lines such as bovine or hamster.  This pointed towards an insect vector.

Meanwhile, the saga of the new disease took a dramatic twist and reared its ugly head in a new way: horrendously malformed lambs were being born to sheep in the affected area.  Up to 50% of lambs on some farms were born with the classical clinical signs: arthrogryphosis, torticollis, and hydrancephaly.   Although the virus has been isolated, the pathogenesis of both the diarrhea and disease in cattle and the malformation in sheep remains unknown.

 

Thanks to metagenomics, diagnostics such as RT-PCR and a serological assay (neutralization) were rapidly developed.  The virus has spread quickly despite the short viremia, implicating replication within the Culicoides vector.

This lecture was fascinating not only because it provided a real-world introduction to metagenomics, but because it detailed an emerging disease issue in such recent history.  In many of our classes, we are used to hearing about notable outbreaks from the distant past, and seeing the dates from 2011 and 2012 on Dr. Haas’ slides was an unsettling reminder than emerging diseases are a reality of the present and future, not just the past.

Lecture #4: Avian Influenza     

Given by: Dr. Timm Harder, Institute of Diagnostic Virology

FLI boasts the OIE/FAE reference laboratory for Avian Influenza.  As its’ director, Dr. Harder gave us a great overview of the pathobiology and clinical picture of Type A influenza, and proceeded to discuss the issue as it pertains to Germany.  All infections of AI in Germany are the result of introduction by wild birds.  As such, surveillance efforts for HPAI revolve around passive surveillance of wild birds.

Lecture #5: Rabies

Given by: Dr. Thomas Müller

Dr. Müller gave us an informative overview of Rabies with a focus on how the disease burden differs between Europe and the developed world as compared to the developing world.  In the early 1980s, rabies remained widely distributed in Europe, whereas today it has basically disappeared from western Europe.  This eradication success is largely attributable to legislation such as sanitary policy, dog registration, and movement restrictions.

The unfortunate caveat to this success story is that fox rabies has arisen as dog rabies declined.  Conventional “elimination” efforts resulted in decimation of the fox population but were ineffective at controlling the disease.  The problem was most effectively addressed in the 1970s by oral vaccination campaigns.  Switzerland was the first to show field viability of the oral vaccinations.  For an oral vaccination campaign to be successful, large-scale efforts must take place, requiring strong political support.  In the case of fox rabies, Switzerland, Germany, and France became the driving force behind the oral vaccination campaign.  In the current moment, EU support for the ongoing effort remains crucial, and legislation governing pet travel and financing have been integral to its success.

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In the developing world, human rabies remains a major problem.  Greater than 90% of human rabies deaths occur in Asia and Africa, and more than half of these occur in children less than 15 years old.  Dog bites account for the overwhelming majority of human rabies deaths, and the great bulk of these occur in rural areas where access to post-exposure prophylaxis is essentially nonexistent.  Dr. Müller attributes the ongoing problem with rabies in certain countries to a “circle of neglect”, and suggested that the key to human rabies control is canine rabies control.

 

Lecture #6: Pathology Overview and Insel Riems Trivia

Given by: Dr. Jens Peter Teifke

Dr. Teifke capped off our day of lectures at FLI with a whirlwind overview of various animal diseases, pathology, and Insel Riems history.  We learned that the quaint city of Greifswald where we’d been staying boasts one of the oldest universities in Germany and even in Europe, the Ernst Moritz Arndt Universität Greifswald.  We discussed the pathological and clinical findings of Rabbit Hemorrhagic Disease, which provides a good potential model for human hemorrhagic diseases.  We heard about the 2006 H5N1 outbreak in swans near Insel Rügen, which sparked rumors of a biosecurity breach at FLI (these were false, of course).  This grab-bag of applied pathology was an ideal capstone to a day of learning in the Loeffler house!

The First Annual Loeffler Lecture

Topic: The Influenza Virus Riddle – Pandemic Potential or Not?

Presenter: Dr. Ron Fouchier, Erasmus University, Rotterdam

After a brief break to spend some time in downtown Greifswald, a quaint and cobblestoned little square, we attended the First Annual Loeffler Lecture at the Alfried Krupp Wissenschaftskolleg. Dr. Fouchier was named one of Time Magazines 100 Most Influential People this past year for his work studying the evolution of pandemics in humans.  His research team investigates species jumps and tries to predict disease spread, and their recent focus is H5N1 influenza.

Since its first recognition in Hong Kong in199, H5N1 has been documented in numerous countries on three continents.  This virus circulates primarily in poultry but transmission events to mammals, including humans, have been highly lethal.  This begs the question: Could H5N1 cause the next pandemic?

To answer this question, Dr. Fouchier’s research team first determined what the virus would need to acquire for human-to-human transmission:

  1. Replication in the upper respiratory tract
  2. Replication at high titers
  3. Shedding of single particles (to facilitate aerosolization)

His lab demonstrated that when the virus is serially passed between ferrets (the current model for human infection), it naturally acquires adaptation to the upper respiratory tract, easily meeting the first criteria.  When these adapted viruses were sequenced, the researchers discovered that the newly transmissible virus had only undergone five mutations from the wildtype virus to gain this adaptation – all of these occurred in the hemagglutinin protein.  Frighteningly, sequence comparison to circulating strains identified viruses in Japan and Mongolia that only needed three more mutations to acquire this ability.  This points strongly to a pandemic potential for the virus.

Dr. Fouchier’s lab become somewhat notorious when Science imposed a moratorium on the publication of their research, due to fears that in the wrong hands, his research could contribute to manipulation of these viruses by bioterrorists.  When addressing these concerns, Dr. Fouchier pointed out that any one with the tools to study and synthesize these viruses already has the tools to do the work that he did.  Moreover, he made the strong point that the conduction and communication of this work is the only way to advance scientific understanding of this important disease threat.

This talk sparked a lively discussion of research ethics and the principles behind Dr. Fouchier’s work.  Afterwards, Laura, Elizabeth, and I got a great picture with Dr. Fouchier, who is incredibly tall in addition to being incredibly intelligent!

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We capped off a brilliant day of lecture and discussion with a fantastic dinner at the Hotel Utkiek.  Everyone enjoyed wonderful seafood dishes while watching the sunset over the Baltic.  Thus ended another truly fantastic day on our journey!

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