Resolving the structure of crocodilian secondary lymphoid organs: A morphological and immunohistochemical study

Introduction
The adaptive immune response is initiated when reactive lymphocytes meet their specific antigen. To optimize the chance of this serendipitous encounter, secondary lymphatic organs (SLO) evolved as anatomic locations for the concentration of antigen-specific lymphocytes and their cognate antigen. For this, SLO complexity evolved in parallel with the increasing functionality of adaptive immunity. Paradigms of lymphoid organ function and development are derived from studies on mammals, particularly mice and humans, and less frequently on avian, amphibians and piscine species.
Knowledge of the reptilian immune system is limited, and the characterization of secondary lymphoid organs of crocodilians is needed.

Aim of Investigation
To characterize the immune-cell populations forming secondary lymphatic organs of crocodilians.

Methods
Tissues obtained postmortem from crocodiles and alligators were assessed using standard histopathology stains and immunohistochemistry (anti-CD3, anti-PAX5, anti-IBA1, anti-MHCII).

Results
Our study, for the first time, determines the architecture of secondary lymphoid organs of crocodilians, showing the characteristic compartmentalization of B and T cell zones similar to warm-blooded species. In contrast to the current literature, we show that germinal centres can form in the lymphatic follicles of gut associated lymphatic tissue (GALT) of Nile crocodiles (Crocodylus niloticus) and American Alligators (Alligator mississippiensis).

Conclusions
This discovery suggests that the evolution of germinal centers, are as ancient as the common ancestor of birds and crocodilians.

Acknowledgements
The authors would like to thank Lynn Stevenson and Frazer Bell, Diagnostic Services, MVLS, School of Veterinary Medicine for their technical contribution on immunohistochemistry.