Factsheet #119

Air Sac Proteins and their Role in Defence Against Infection with E.Coli in Poultry
Drs. Wineeta Weebadda, Tony Hayes and Bruce Hunter
University of Guelph
(based on research funded in part by the Poultry Industry Council)

Respiratory diseases cause significant economic losses to all segments of the poultry industry. Confinement housing systems
utilized in Canada require efficient, properly maintained mechanical ventilation systems to vent dust, gases and humidity,
and maintain acceptable air and litter quality. Substandard barn air and litter quality are well documented risk factors for
respiratory disease in poultry.
Very little is known about the basic protective mechanisms of the avian respiratory system, despite research on respiratory
pathogens of poultry and the development of massive vaccination protocols. We do know that poultry have quite efficient
mechanisms for clearing inhaled bacteria and dust from the upper respiratory tract (nostrils, turbinates, sinuses and trachea).
However, once bacteria penetrate to the lungs and air sacs, the bird's respiratory defense is sluggish and inefficient. In closed
barn situations where birds are commonly exposed to high bacterial loads, dusty environments and sometimes high levels of
noxious gases like ammonia, it is surprising that so few actually develop respiratory disease. This is particularly true in
commercial heavy tom turkeys that often mouth breathe, effectively bypassing the efficient upper respiratory clearance
mechanisms. Clearly, the lower airways of birds must have protective mechanisms against inhaled pathogens.
Our past studies have shown that the air sacs of birds are lined by cells that secrete a lipid-rich substance. In order to
determine the characteristics of this secretory product, we obtained and examined air sac fluid using a variety of techniques
including affinity chromatography. We were looking for certain protective proteins that might bind to the surface of
pathogenic E. coli bacteria.
Several protein bands were isolated from turkey air sac fluids by binding to chromatographic columns to which surface
polysaccharides of E. coli were attached. These proteins included immunoglobulins (both light and heavy chains) and
albumin that were expected. In addition, a distinct major protein of approximately 40 kDa was consistently isolated from
turkey air sac fluid. We have called this novel protein Air Sac Lectin-40 (ASL-40). Through a series of studies, this protein
was shown to bind to the surface polysaccharides of pathogenic E. coli 02 (isolated from an acute field out break of
colibacillosis in chickens). The N-terminal amino acid sequence of this protein was identified as
DINGGAZTLPQHLYLTPD. By sequence comparisons, this showed that ASL-40 is a previously unknown protein that is
related to a partially characterized human protein processing T cell stimulation activity in the joints of rheumatoid arthritis
patients.
Sera from chickens and turkeys were examined to determine if this new protein was also in the blood. A similar protein was
isolated in sera, but it had binding functional differences with the air sac form, and its concentrations in blood were much
lower than in the air sac fluids. To determine the origin of these proteins, we produced antibodies to the air sac form, and to
a synthetic peptide sequence corresponding to the N-terminal sequence above, and used these antibodies to examine tissue
samples by Western blotting of tissue homogenates and immunohistochemistry of frozen tissue sections. Western blots
confirmed the presence of a 40 kDa immmunoreactive band in the air sac membranes, but we did not detect this band in
tissue homogenates from trachea, liver, spleen and synovial membranes. Immunohistochemistry revealed weak positive
staining only in the epithelial cells of the air sacs. Liver, lung, spleen and synovial lining cells did not positively stain for
ASL-40. In addition, we examined egg yolk, known to be a rich source of a variety of innate proteins such as lysozymes. The
results were negative.
In summary, this research has identified a novel protein in the air sac fluid of turkeys. Tissue distribution studies indicate
that the protein is most likely produced by the type II epithelial cells that line turkey air sacs. A similar related protein, but
with some biochemical differences, was identified in the blood (serum) of both chickens and turkeys. Both the air sac form

and the serum form of the protein have the capability of binding to the polysaccharide outer coating of pathogenic strains of
E. coli (serogroup 02).
The functions of these proteins are not yet understood. Their demonstrated ability to bind to sugars and to the surface of
intact E. coli (serogroup 02 isolated from diseased chickens) suggest that these proteins may play a role in protecting the air
sac from infection with pathogenic bacteria. In previous studies, electron microscopic examination of turkey air sacs
revealed that birds raised in dusty commercial settings had a higher percentage of air sac lining cells actively secreting this
product compared to turkeys raised in low dust isolation facilities. Further, the percentage of actively secreting air sac lining
cells was higher in very young turkey poults compared to birds older than 6 weeks. Both of the findings support the theory
that this newly identified protein is involved in the defence of air sacs against bacterial pathogens. Clearly, more work is
required to determine the biological functions of this protein, and to better understand the defence mechanisms of bird air
sacs.