Bacterial and fungal penetration of the Nile crocodile (Crocodylus niloticus) egg in relation to the eggshell and eggshell membrane anatomy and microstructure

Abstract

Commercial farming with the Nile crocodile (Crocodylus niloticus) for both leather and meat is an economically significant industry in South Africa. Due to the economic value of these products, effective captive breeding and hatchling rearing management programmes are of critical importance. An increase of knowledge and the investigation of specific crocodile industry related questions (identified interventions) in South Africa are of utmost importance to standardize and optimize Good Operating Practices for Nile crocodile husbandry, as well as to increase productivity on commercial farms. Low hatching rates have been identified as a management problem by most South African farms. Although many factors may contribute to the low hatching percentage, the role of bacterial and fungal contamination of the embryo inside the egg, has not been investigated before. For these microorganisms to clinically affect the healthy embryo, they must be able to gain access through the eggshell and the eggshell membrane. The role of the anatomy and microstructures of the eggshell in the prevention of intra-egg infections, and its changes during the incubation period is also not well known. The Objectives of this project were to: (1) test, optimize and determine the most suitable preparation technique / set of techniques to comprehensively describe the microstructure of the Nile crocodile eggshell and eggshell membrane; (2) investigate and describe the normal anatomy of the Nile crocodile’s eggshell and eggshell membranes and its changes during the incubation period; (3) identify the bacterial and fungal species prevalent on commercial crocodile farms and associated with the Nile crocodile egg after oviposition and during the incubation period; (4) investigate the penetration of bacterial and fungal species through the eggshell and membrane; and (5) determine if there is a correlation between the changing microstructure of the egg during incubation and the ability of microbes to penetrate the eggshell and eggshell membranes. Fifty unbanded Crocodylus niloticus eggs from 22 clutches, 12 nesting sites on three different farms were used for Objective 1. A variety of sample preparation and analysis methodologies and techniques was tested and optimized to study the micro- and ultrastructure of the inorganic eggshell and its associated organic eggshell membrane. For Objectives 2 and 3, Crocodylus niloticus eggs (n = 48) from 12 clutches, each from different nesting sites on three separate farms were collected. Four eggs from different incubation stages (one day -, one week -, five weeks – and nine weeks after oviposition) were collected from each of the 12 clutches. During the first egg collection relevant environmental samples and samples from areas associated with the egg during incubation (nesting material, water, incubator boxes, vermiculite, and egg handlers’ hands) were also collected for microorganism identification. For Objective 4, additional eggs from any clutch produced on the farms (n = 17) which showed external signs of microbial infection (discoloration, fine cracking, slimy to the touch, abnormal odour) or embryo death (egg cool to the touch) during the incubation period were identified and collected. Bacterial and fungal species were identified on all the collected samples and material using MALDI-TOF biomolecule analysis and ITS sequencing, respectively.