Development and validation of selected biomarkers for the health status of the Nile crocodile (Crocodylus niloticus) and sharptooth catfish (Clarias gariepinus)

Abstract

Aquatic species, such as the African sharptooth catfish (Clarias gariepinus) and Nile crocodile (Crocodylus niloticus), are considered to be valuable sentinels of ecosystem health. Although the Nile crocodile is still widely distributed in Africa, viable populations of wild crocodiles are nowadays only found in protected areas in South Africa, like the Kruger National Park and iSimangaliso Wetland Park. The sharptooth catfish, on the other hand, also has a wide distribution in Africa, but is still commonly found in nearly all waterbodies in South Africa. Biomarkers to determine the health status of the sharptooth catfish and Nile crocodile are, unfortunately, lacking. This PhD project can be divided into three focus areas, namely: (1) the development of blood and urine sampling techniques for the Nile crocodile; (2) determining of blood biochemical parameters in the African sharptooth catfish if a standard veterinary clinical pathology profile is used; and (3) the establishment of two in vitro methods (primary hepatocyte system and gill filament assay) for the African sharptooth catfish. Although the Nile crocodile blood sample collection technique has been established, the clinical anatomy of the post-occipital spinal venous sinus collection site has not been thoroughly investigated. The anatomy of the cranial neck region was investigated macro- and microscopically, radiographically and by means of computed tomography. The spinal vein runs within the vertebral canal, dorsal to, and closely associated with the spinal cord and it changes into a venous sinus, cranially, in the post-occipital region. For blood collection the spinal venous sinus is accessed through the interarcuate space between the atlas and axis (cervical vertebrae: C1 and C2) by inserting a needle angled just off the perpendicular, in the midline through the craniodorsal cervical skin, just cranial to the cranial borders of the first cervical osteoderms. Hypodermic and spinal needles with short bevels are the most suitable to prevent complications. Crocodilian urine is not often used as a diagnostic sample - most likely due to the fact that a practical urine collection technique has not been described before. However, urine is a very useful diagnostic sample in human and veterinary medicine, and can most probably be used for the same purpose in crocodilians. A simple urine benzo[a]pyrene (B[a]P). The primary catfish hepatocyte cell culture system, expressing CYP1A when exposed to B[a]P, could in future be used as a biomarker for aromatic hydrocarbon pollutants in aquatic ecosystems. A gill filament-based EROD assay was also developed for the African sharptooth catfish as an ecosystem health monitoring tool. The ability of C. gariepinus in inducing cytochrome P-450 class 1A (CYP1A) and glutathione S-transferase (GST) biomarkers was determined in gill filaments and liver cells after four days of waterborne exposure to the polycyclic aromatic hydrocarbon, (B[a]P). The CYP1A activity was measured in hepatic microsomes and gill filaments. Benzo[a]pyrene strongly induced CYP1A activities in gill filaments. These findings suggest that the sharptooth catfish gill filament-based CYP1A assay can be used to monitor AhR agonist pollutants in aquatic ecosystems. In conclusion, the focus of the Nile crocodile projects was on the establishment of sample collection techniques. However, the development of the blood and urine sample collection techniques not only confirmed the normal anatomy of the sample collection sites, but also created the potential for screening crocodile blood and urine samples for an array of chemicals and laboratory parameters. Screening of blood samples, collected from apparently healthy African sharptooth catfish, contributed to our database of reference ranges, as well as evaluating the potential diagnostic value of standard veterinary clinical pathology tests. The establishment of specific in vitro assays for the African sharptooth catfish contributed to the implementation of innovative methods that can be used to effectively monitor aquatic animal health.