After our success in isolating influenza viruses from seabirds on the Great Barrier Reef, Graeme Laver and I decided to test whether influenza viruses related to those in humans could be found in other species of birds in other parts of the world. One of the world’s largest populations of pelagic (open ocean) birds is found off the coast of Peru and Chile, on the Guano Islands, which had built up from the droppings of millions of seabirds over time and were mined for agricultural fertiliser. Smaller populations of many other gull species and noddy terns also inhabit the Dry Tortugas, islands off the western end of the Florida Keys in the United States. It was these latter populations that we targeted in 1974, using the techniques honed on the Great Barrier Reef. Over a thousand samples were collected from the Dry Tortugas for serological and virological study.
Four years later we mounted an expedition to the Guano Islands. It was supported by the WHO in conjunction with the Peruvian government, which supplied its research ship for transport to the islands. We collected thousands of serum samples, throat swabs and faecal samples from these huge populations of birds, which spend their entire lives at sea except when they nest and raise their young. Both studies came up empty in that we detected no antibodies or influenza viruses in any of the samples. Clearly not all populations of pelagic birds were infected with influenza. Finding those that were would require being at the right place at the right time. We needed to change our approach.
I decided to focus on my own back yard. Publications from colleagues had reported the detection of antibodies to avian influenza viruses in migratory Canadian geese (Branta canadensis),30 and influenza viruses had been found in free-flying ducks in California.31 Since Memphis lies on the Mississippi Flyway, the route taken by migratory waterfowl flying from Canada to their overwintering grounds in South America, it is an excellent site for studying these birds.
Each year millions of wild ducks and geese fly south, and each year hunters harvest a portion of them. The numbers of ducks and geese that hunters can shoot and the length of the hunting season in different areas are set by the Canadian and American wildlife authorities, who monitor the bird populations. Hunting is usually permitted in two periods: one in mid- to late November and the other in early December.
Obtaining samples from waterfowl shot by hunters turned out to be easy. Many American hunters don’t like to dress (clean) their own ducks, so they take them to a dressing station, where they pay to have them gutted and plucked. With the permission of the owner of the Minnow Bucket, a dressing station in West Memphis, Arkansas, we joined two very cheerful women in the back room to collect samples from the ducks before they were dressed. The employees held the dead ducks against a rotating drum with long rubber fingers that removed all of the down and feathers. They then eviscerated and washed the ducks and gave them back to the hunters beautifully wrapped and ready for cooking. The feathers were bagged for marketing to down and feather bedding and clothing manufacturers. Meanwhile we collected throat swabs from the ducks and stored them in a chilly bin (cooler) containing ice.
Back in the laboratory, a small volume of the solution surrounding the swab was injected into 10-day-old developing chicken eggs, as described in Chapter 2. The eggs were kept for two days at 35°C, and then a small sample of fluid was removed to test for the presence of virus by adding a few drops of chicken red blood cells. If an influenza virus was present, the cells would clump or haemagglutinate.
In the very first batch of samples from the ducks we found influenza virus, which was designated A/Duck/Memphis/546/74 (H11N9). Like the influenza virus from birds on the Great Barrier Reef, this virus caused no disease when put into the throat and eyes of young ducks.32
In that first season of sampling wild ducks, approximately 2 per cent of the birds yielded influenza viruses of different subtypes. We wanted to know if the virus had increased to high levels in the ducks and whether it was present anywhere besides the respiratory tract. A visiting Russian scientist, Maya Yakhno, was given the job of separating all of the organs from the infected ducks and determining the level of virus in each. This simple study resulted in another of those eureka moments. Yakhno found that the virus was present in all parts of the intestinal tract she examined, and that the highest amount of virus was in the faeces. This finding eventually led to the significant realisation that in aquatic birds, the influenza virus caused an intestinal infection.33 Virus counts in the faeces were as high as 100 million virus units per gram of faeces. The virus was probably spread from bird to bird through contaminated water. This meant that hunters could easily tramp influenza viruses on their boots back to their homes – and, if they were farmers, to their poultry houses. It also meant that wild ducks could contaminate smaller ponds with enough viruses to infect other animals.
Since Memphis is well along in the birds’ southern migration route, we wondered if the incidence we detected was low because we were detecting the end of an outbreak. Perhaps the best place to look for influenza in wild ducks would be in Canada during the summer, when the wildlife authorities banded birds for identification before their migration south. I sent out about 20 letters to Canadian wildlife officials explaining the proposed study. I received a reply from Bruce Turner of the Edmonton office of the Canadian Wildlife Service (now part of Environment Canada), who agreed to let me join his banding team in July 1976. I flew to Edmonton with the same array of equipment that we had used on the Great Barrier Reef – Dacron throat swabs, collection vials, vials for serum and a Dewar flask of liquid nitrogen for cold storage of the samples (Figure 4.1).
Turner’s banding team was an extremely cooperative group of young men who made me very welcome. Their trucks were loaded with duck traps and bags of grain. These traps were large wire cages that birds could swim into but not get out of. The traps were baited with grain and set on floating polystyrene blocks near the edges of the small lakes in southern Alberta, near Vermillion. A hessian grain bag over the trap protected the captive ducks from getting too much sun.
The next morning each trap contained five to ten birds. The species of each bird was recorded (mallard, pintail, etc.) before the bird was given a leg band with a unique number and its age was assessed. Young ducks grow so quickly that it took an expert to decide whether they were juveniles or adults. Very occasionally a bird already had a band; this information was recorded, and the bird was given a second band.
I took swab samples initially just from the throat, but by the second year, as at the Great Barrier Reef, I sampled both ends of the bird, as well as collecting a blood sample from the wing vein. The men bringing the ducks ashore were very patient – the swabbing and bleeding slowed down their normal banding work and made some days very long. All ducks, of all the different species, were fat and healthy, preparing for their southern migration.
Turner and his crew were very sceptical that any of these healthy birds could possibly carry the influenza virus. We discussed the matter many evenings over dinner, since they were all biologists and not easily put off their food! I was left with the distinct impression that they were tolerating a rather nutty professor determined to take butt swabs from robust birds.
But analysis of the samples in the Memphis laboratory yielded amazing results: up to 18.5 per cent of juvenile birds (hatched that year) and 5 per cent of adults were excreting influenza viruses. While one strain of influenza was dominant, small numbers of many different subtypes were isolated.34 When the results were sent back to Turner and his crew, I hope they decided the professor wasn’t so nutty after all (Figure 4.2).
This sampling of wild ducks in Canada has been going on for nearly 40 years now, and not all the outcomes are published in the scientific literature or followed up. One sampling study attempted to determine whether the fish in the lakes are infected with influenza from the ducks or contain detectable levels of virus. In the second year of sampling (1977), I had driven from Memphis to Alberta with my family with the usual sampling supplies but also had a gill net for catching fish, specially made by the North American department store Sears Roebuck.
At the first sampling site on the first morning. I rolled out the gill net from my car and asked Turner where I should put it. Turner was a Newfoundlander and unfazed by most things. On this occasion, however, he went bright purple and told me exactly where I should put that net. I didn’t know that gill nets were illegal in Canada or that for wildlife personnel to be seen with one would be scandalous. No fish were sampled for influenza on that trip.
That night my youngest son, James, surprised everyone by catching his limit of very large northern pike (Esox lucius) on a 10-cent white woolly lure that he had bought in Memphis at a variety store. The locals, who were not getting bites, came to inspect his lure. In our eagerness to eat the catch, we forgot to sample the fish before cooking them, so despite over 40 years of collaborative study on influenza with Canadian wildlife personnel, I have still never taken scientific samples from the fish.
Figure 4.1 A duck trap on a lake near Vermillion, Alberta, Canada, with Bruce Turner (red shirt) of the Canadian Wildlife Services (now part of Environment Canada) showing the different influenza viruses isolated from the ducks in the trap and the water, on 22 August 1977.
On the return road trip to Memphis after that sampling trip, my wife Marjorie was the driver, taking the family and the samples back to Memphis, the latter in the insulated liquid nitrogen container. I had flown to Australia to join Laver. As my family headed south that morning, the children wanted to know why that car behind had all those flashing lights. Marjorie’s first worry was the samples – and whether she had copies of the right import permits. The police officer was very gracious about her travelling over 80 miles per hour when he saw that the car was registered in Tennessee. He asked if she was hurrying back for Elvis Presley’s funeral, which was to take place the next day. He then asked her to slow down, for at the time the speed limit on United States highways was 55 miles per hour.
Figure 4.2 Results of surveillance for influenza viruses among wild ducks in Alberta, Canada, 1976–2016, showing the different combinations of haemagglutinin and neuraminidase. The dominant viruses change from year to year, with H1N1, H3N8, H4N6, H6N2, H6N5 and H6N6 the most frequently isolated viruses. Counterparts of human H1N1 and H3N2 were isolated, but H2N2 was isolated only once. No isolates of H13, H14, H15 and H16 were detected in these migratory ducks. Several different duck species were sampled, including mallard, pintail, gadwall and teal, and the dominant influenza virus was isolated from all species. Most duck species migrate to the southern states of United States to overwinter, but the teals migrate as far as northern South America. Table courtesy of Scott Krauss, St Jude Children’s Research Hospital
The other question that remained unanswered was whether influenza viruses overwinter in the frozen lakes and re-infect the ducks when they return in the spring. In midwinter 1978 Turner and his team drilled holes in the ice and sampled the water. We never did succeed in isolating influenza viruses from the dozens of water samples, but other studies showed that in the laboratory these viruses remain viable for months at low temperatures. Later, in the collaborative studies, our Canadian colleagues collected cloacal and throat swabs from ducks on their northern spring migration returning to their breeding grounds in Canada. Approximately 0.2 per cent of the apparently healthy returning ducks were ‘shedding’ influenza viruses of different subtypes. Thus it is possible that the influenza viruses remain present in a flock over winter, albeit at low levels, but we cannot rule out the possibility that some influenza viruses come from the lakes after thawing.
***
Initial scepticism towards our findings gave way to acceptance. Our pioneering studies of Canadian ducks in many ways triggered the global interest in influenza viruses in aquatic birds and established many of the now-accepted ecological principles of influenza viruses, including the fact that wild aquatic birds are indeed a major reservoir of the influenza viruses that evolve into pandemic viruses for humans. The studies continue to the present day. In 1975 the WHO invited St Jude Children’s Research Hospital to become a collaborating centre on the Ecology of Influenza Viruses at the human–animal interface, a role it continues to fulfil.