Front or back – which end of the duck should you swab?

By Jonas Waldenström

Simple questions can have simple or advanced answers. The question in the title – which end of a bird you should sample for flu – will be answered in due time, but first I need to take you on an odyssey of duck flu history. Let’s start in Italy:

A Blue rock thrush view of Colloseum (Photo from Flickr, T. van Ardenne under a CC BY-NC-ND 2.0 license)

A Blue rock thrush view of Colloseum (Photo from Flickr, T. van Ardenne under a CC BY-NC-ND 2.0 license)

It was a beautiful day in Rome. The sun poured down on Colloseum and the hordes of tourists that meandered through the old remnants of the Roman empire. A Blue rock thrush fluted melodiously from the ruins, ignorant of the people below.

Meanwhile, in a concrete slab on the other side of Forum Romanum, the world’s leading experts on influenza A virus had gathered at the OIE headquarters. The acronym stands for Office International des Epizooties, but as no one could remember that name it has since been renamed the World Organization for Animal Health, a UN body devoted to fight disease in food production animals.

This was back in 2006, and the reason we were all there was the looming threat of highly pathogenic avian influenza H5N1 – or the bird flu as it was called in the media. This particular virus had crossed species barriers from the wild waterfowl reservoir into poultry and mutated into a highly pathogenic form that spread rapidly in southern China. This is in itself wasn’t too worrying – as outbreaks of highly pathogenic avian influenza emerge from time to time in poultry – but the unprecedented events were that H5N1 also spilled back into wild birds and that it infected and killed humans. Thus, it was a virus of substantial concern for the poultry industry and one which also had the potential to become a new human pandemic virus. The spread had been slow at start, with outbreaks mainly regionally in Asia but then the virus took a sudden leap across Russia into Turkey, and then further into Europe and Africa. People were scared, authorities had no proper preparedness plans, and research on avian flu was still limited.

Inside the big hearing room sat a mix of researchers, health care professionals, vets, and officials from various governments (and yours truly, in the back row, perplexed and overwhelmed of it all). The atmosphere was tense. What did we know? How could we learn more? What did we need to know to stop it? Questions without answers at the time.

A particular problem was the elusive nature of the virus. If it infected a poultry farm we would see it – it was easily detected due to its high mortality in chickens. If your chickens were dying in heaps, chances were that H5N1 had made a visit. But unlike most other previous poultry outbreaks of highly pathogenic avian influenza it also popped up in wild birds. The most covered outbreak in Europe was the dead swans on the shores of Rügen in Germany, but swans and diving ducks were found dead in several other places too, including Sweden during the winter 2005/2006. At the same time, no one found any infected dabbling ducks, despite thousands of samples being tested. Where was this bloody virus?

And it was then Ron Fouchier, a virologist from the Erasmus Medical Center in Rotterdam, dropped the bomb. In his talk he showed data from infection experiments showing that H5N1 was not primarily a gastrointestinal virus in ducks (as they usually are). Rather, the highest titers of virus were found in samples from the upper airways, suggesting an all-together different epidemiology. He had a punch line in his PowerPoint presentation that said (if I remember correctly) “Are we sampling the wrong end of the bird?” It was a deafening silence while it sank in, and then a forest of hands was raised for questions. These results, later repeated by other laboratories, changed the recommendations for wild bird H5N1 sampling more or less over night.

Let’s get back to 2014 again. We know more today than we did in 2006, but still the influenza A virus research field is full of surprises. Adding the sampling of the respiratory tract (or rather the oropharyngeal cavity or the pharynx) did not yield many H5N1 positive samples from wild birds in the EU – most found cases were still from dead birds, especially swans. The virus disappeared from Europe, and the interest from European authorities and media vaned with time. However, the question regarding front or rear remained among researchers. Not so for highly pathogenic forms of influenza A virus – which at least for H5N1 is clearly different in pathogenesis – but for the more normal, waterfowl-adapted low pathogenic precursor viruses.

The old dogma was that the low pathogenic viruses primarily infected the lower gastrointestinal tract, but that it could be found at lower frequency also in the respiratory tract. Indeed, this is often what you find in field studies; at our sampling location oropharyngeal swabs normally peak at 2-5% prevalence, compared to up to 30-40% in fecal/cloacal samples. But detection is not the same as infection, as the viruses detected there may be contaminants from feces or water. In infection experiments with low pathogenic viruses, replicating virus is sometimes found in the respiratory tract, but this may be an aberrant result due to large infection doses and mode of inoculation, where some virus may be washed down where it normally would not go.

Paraffin-embedded Mallard tissue samples waiting to be analyzed. Photo by Michelle Wille

Paraffin-embedded Mallard tissue samples waiting to be analyzed. Photo by Michelle Wille

A week ago we published a paper in Veterinary Research that aimed to test the ‘to be, or not to be’-hypothesis of natural respiratory infections. During November, the peak the flu season in ducks, we trapped and sampled roughly 125 Mallards at our field site. Instead of letting the birds go, we kept them in the trap a couple of hours until the test results were back from the lab. Four birds with positive RRT-PCR results from the oropharyngeal swab, and one which was negative, were sacrificed for assessing presence, or absence of virus infection in different body tissues; all other birds were released. The decision to kill a bird for science isn’t one to take lightly, but in this case we believed the benefit of mapping the pathogenesis of the virus justified the study (and procedures, sample size etc. were assessed by an ethical committee and approved by different authorities).

The method we used to look for replicating virus particles is called immunohistochemistry. Fresh tissue specimens are preserved in formalin, and then embedded in paraffin. Very thin slides of tissue (3 micrometer thick) are then mounted on glass slides and treated in such a way that cells infected with influenza A virus will appear red under the microscope. We (Michelle Wille from my lab, and Peter van Run and Thijs Kuiken from the Erasmus Medical Centre in Rotterdam) screened a large number of slides from the full lengths of the respiratory and gastrointestinal tracts. None of the slides from respiratory tract were positive, in contrast to several from the intestinal tract.

Figure 1 from the paper. Selected tissues of the respiratory and gastrointestinal tracts of Mallard following immunohistochemical staining to detect nucleoprotein of influenza A virus. Tissues from the respiratory tract did not show virus antigen expression, such as (A) the respiratory epithelium of the nasal cavity and (B) air sac epithelium. In contrast, some tissues from the gastrointestinal tract such as (C) the epithelium lining the jejunal villi of the gastrointestinal tract, and (D) surface epithelium of the cloacal bursa did show virus antigen expression. Virus antigen expression is visible as diffuse to granular red staining, which is usually darker in the nucleus than in the cytoplasm. The tissues are counterstained blue with hematoxylin. Arrows have been included to illustrate positive cells.  [Used under a CC 2.0 license]

Figure 1 from the paper. Selected tissues of the respiratory and gastrointestinal tracts of Mallard following immunohistochemical staining to detect nucleoprotein of influenza A virus. Tissues from the respiratory tract did not show virus antigen expression, such as (A) the respiratory epithelium of the nasal cavity and (B) air sac epithelium. In contrast, some tissues from the gastrointestinal tract such as (C) the epithelium lining the jejunal villi of the gastrointestinal tract, and (D) surface epithelium of the cloacal bursa did show virus antigen expression. Virus antigen expression is visible as diffuse to granular red staining, which is usually darker in the nucleus than in the cytoplasm. The tissues are counterstained blue with hematoxylin. Arrows have been included to illustrate positive cells. [Used under a CC 2.0 license]

Immunohistochemistry is a very sensitive method for assessing infections. While PCR-based methods are extremely sensitive for detecting presence of virus RNA, they do not say which cells that are infected, and whether they are associated with pathological changes. In our study, the Mallards were positive for influenza A virus in the oropharyngeal cavity by RRT-PCR, and in some cases we were even able to culture the virus, but none of them had any infected cells in the respiratory tract. If we cant find it in these birds (which belong to the main reservoir species), during the peak of infection in autumn, I think it is unlikely that respiratory infections are commonplace. The fact that you may find PCR-positive samples from the front end of the duck is then not due to infection in this site, but from virus received through feeding, drinking or preening.

And this is important, as the pathogenesis of a pathogen is the basis for transmission. Without knowing which parts of an animal that are infected we cannot fully understand the epidemiology and make informed decisions. Thus, if you sample for low pathogenic avian influenza in wild waterfowl go for the rear end of the bird.

And in Colloseum, the Blue rock thrush continues to sing, regardless.

Link to the paper:

Wille, M., van Run, P., Waldenström, J. & Kuiken, T. 2014. Infected or not: are PCR-positive oropharyngeal swabs indicative of low pathogenic influenza A virus infection in the respiratory tract of Mallard Anas platyrhynchos? Veterinary Research 45: 53.

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