Coronaviruses in poultry: an ongoing pandemic?

Summary

The recent Covid pandemic has introduced the global population to coronaviruses like never before. However, veterinarians and scientific researchers in the veterinary field have been working for decades to both control and better understand various coronaviruses. In the early 1930s, a respiratory disease was identified in chickens in Massachusetts, USA, which, with improved research methods, was retrospectively attributed to a coronavirus. This disease, called infectious bronchitis (IB), thus became the first proven disease caused by a coronavirus. Today, the infectious bronchitis virus (IBV) is present worldwide in domesticated chicken populations, causing problems in both the egg-laying and broiler sectors.

IBV primarily causes a respiratory infection. It infects the epithelium, a layer of specialized covering and protective cells, mainly in the upper respiratory tract (nose, trachea) of chickens. This leads to a temporary partial loss of function in this cell layer, triggering an inflammatory response and causing symptoms such as nasal discharge and watery nasal secretions. If no further complications arise, the chicken can recover within a few days, depending on its age. However, during this infection, growth and egg production decrease, leading to significant economic impact.

IBV has different variants, some of which can penetrate deeper into the chicken than just the upper respiratory tract. The better a specific IBV variant can do this, the sicker the infected chickens generally become, and for young birds, this can lead to permanent tissue damage or death. IBV can penetrate deeper into the respiratory system, causing damage to the air sacs, which are extensions of the respiratory tract found specifically in birds. Birds rely heavily on these anatomical structures for proper respiratory function. Some IBVs can penetrate even deeper, reaching the bloodstream and eventually infecting the kidneys and reproductive system. Infection of the reproductive system in young laying hens can later lead to reduced egg production, misshapen eggs, and even total loss of egg-laying ability.

Another usually severe complication arises when an IBV-infected chicken subsequently gets infected with a bacterium. Avian pathogenic Escherichia coli (APEC) is one of the most notorious bacteria in this regard, causing significant animal welfare issues and economic losses in the poultry sector. APEC causes various disease problems in different types of poultry. When it interferes with a previous IBV infection, it often leads to respiratory infections. The inflammations in these airways are then usually much more severe, often with extensive permanent tissue damage deep in the lungs and air sacs. Infected animals suffer greatly from this.

From the above, it is clear that the consequences of an IBV infection can be variable but very serious, both in terms of animal welfare and economically. The sector therefore wants to protect birds against infection through vaccination. However, because there are so many different IBV variants, this is a challenging task, as not all vaccines offer good protection against these various virus variants. There is thus a continuous need to increase knowledge about IBV, to better understand the disease, predict the impact of new virus variants, and develop better vaccines that provide broader protection. This dissertation contributes to this and describes several studies that examined certain parts of the disease process – the so-called ‘pathogenesis’ – in more detail.

For various reasons, it is common to vaccinate chickens by spraying or aerosolizing (finely spraying virus particles into the air), attempting to induce the earlier described respiratory infection in a controlled manner, thereby generating temporary immunity. To control these infections in such a manner, the virus used as a vaccine is stepwise attenuated. Chapter 2 examined the effect of attenuating a nephropathogenic IBV variant (causing kidney disease) into a so-called ‘vaccine strain’ on the virus’s spread through the chicken when exposed to it. Such detailed scale studies comparing the pathogenic, direct ‘ancestor virus’ from the poultry field one-to-one with the attenuated vaccine strain are quite rare for IBVs. It was observed that while the vaccine strain induces nearly identical tissue changes in the respiratory tract on a microscopic level, this happens in a delayed manner. The number of produced virus particles remains relatively similar at the respiratory tract level. However, a significant difference was observed at the kidney level. The vaccine strain was much less capable of reaching the kidneys, and the number of produced virus particles was noticeably lower. It was also seen that the virus infected the chicken’s gastrointestinal tract, a phenomenon that has been studied to a limited extent. The number of virus particles in the cloaca (the opening in birds where intestinal contents, uric acid from the kidneys, and eggs in females are expelled) was notably high earlier than in the kidneys. Since the ureters of the animals often contained viral protein, it seems not impossible that IBV could potentially reach the kidneys via an ascending infection, a process usually attributed mainly to spread via the blood (viremia).

Another virus causing significant problems worldwide in the poultry sector (and beyond) is the avian influenza virus (AIV). It was mentioned above that IBV infections can lead to additional problems when a secondary bacterial infection such as APEC occurs. Such double infections have been studied to some extent in chickens. However, much less research has been done on the effects of an infection with two viruses and what the consequences of such an infection are on the pathogenesis. In Chapter 3, using a model of cultured chicken tracheal organ cultures (TOCs), the effect of a first IBV infection on a subsequent AIV infection and a second IBV infection was investigated. While AIV seemed able to infect and multiply in the already IBV-infected trachea with some delay but relatively unimpeded, it was much more difficult for a second IBV virus to re-enter the epithelial layer. Microscopic studies of the binding proteins of AIV and IBV to the IBV-infected trachea suggested that these differences partly resulted from a reduced ability of the second IBV variant to bind to the trachea. This seems to be related to changes on the surface of the epithelial layer, where certain molecules (sialic acids) needed specifically by IBV for attachment largely disappear under the influence of the initial IBV infection. Such interactions could occur in the poultry field when vaccinating for IBV in the presence of AIV and could influence the course of the disease and possibly immunity buildup.

If a secondary bacterial infection occurs after an IBV infection, the consequences for the animal and ultimately the poultry farmer can be disastrous. Since antibiotic use due to bacterial resistance ideally should be applied with renewed critical insight, it is desirable to gain more knowledge about these double infections with virus and bacteria. Chapters 4 and 5 studied several aspects of such infections. While the interactions between chicken, IBV, and APEC have been studied previously, comparing IBV with other respiratory viruses in this context is novel. Thus, in Chapter 4, animals were also infected with a Newcastle Disease Virus (NDV) and an avian metapneumovirus (aMPV) (both also major respiratory disease-causing agents in chickens) before being exposed to APEC. The virus seems largely determinant of where in the respiratory tract after APEC infection damage occurs and the extent and severity of it. After aMPV infection, a virus known to often not infect beyond the nose and trachea in chickens, barely any APEC-correlated tissue changes were seen, while after IBV and most after NDV infection (viruses known to variably also infect deeper airways), much more extensive damage was discovered in the lungs and air sacs. Compared to animals that had only received APEC, the inflammatory changes were much more extensive, especially after IBV and NDV infection. Interestingly, the bacterium itself was rarely found in the persistent tissue damage. These changes were correlated with variations in the presence of types of immune cells in different infection groups. The presence of APEC in lung tissue was mainly associated with the presence of phagocytic cell types (granulocytes and macrophages that can ingest and neutralize the bacterium), while the presence of the various viruses influenced different populations of T cells (some of which can specifically neutralize virus-infected cells). Notably, when IBV and NDV preceded APEC infection, these cells remained in the tissue longer, and higher levels of cytokines (molecules used for signal transmission between immune cells) were measured over time. In Chapter 5, the effects of IBV and APEC in the air sacs were specifically examined. Signals from this study suggest that the persistent tissue damage after exposure to APEC might largely be due to virus-induced and bacteria-aggravated damage. This is a subtly different view from the usual one, where it is generally thought that the virus hinders the antibacterial immune response, allowing the bacterium to cause damage unhindered for a longer time.

It was mentioned above that, depending on the virus variant, IBV can also infect the gastrointestinal tract. Over the years, it has been somewhat debatable how much animals suffer from this. However, a closely related virus in turkeys, the turkey coronavirus (TCoV), is known for its effects on the gastrointestinal tract, causing variable degrees of intestinal inflammation (enteritis) and associated diarrhea. TCoV was initially discovered in the United States (TCoV-US variant), but more recently, a closely related yet genetically distinct TCoV was discovered in Europe (France) (TCoV-Fr variant). In Chapter 6, this TCoV-Fr variant was examined for viral transmission between animals, viral particle shedding during infection, and microscopic effects at the intestinal level. This study shows that infected animals shed virus particles with their intestinal contents over several weeks of infection, soon after infection and then for a long time. Notably, these virus particles sometimes seemed infectious and sometimes not (or less) infectious to a healthy animal that ingested them. Animals successfully infected by these virus particles had a lot of viral protein in their hindgut segments a few days after infection, but intestinal damage was very limited. This consisted of slight, difficult

-to-observe changes in the length and thickness of the epithelial cells that form the microscopic villi of the intestinal mucosa. Infected animals did lose weight compared to uninfected animals. With the long-term viral particle shedding seen in this study, a role for carrier animals (which become infected without themselves falling ill) seems not impossible in the epidemiology of this TCoV-Fr variant.

A common theme in the studies described above is that IBV – and related coronaviruses – vary greatly in their effects on infected animals. By better understanding how and why these differences arise, it will be possible to develop better strategies for combating IBV infections in the poultry industry.

Full text