Study location
Samples of hunted greater cane rat were collected from Oluwo Market, Epe, Lagos State; and Odo Ona Kekere, Oluyole Local Government Area, Ibadan, Oyo State. Oluwo Market, Epe, Lagos State lies between the coordinates 6° 35′ 3 N and 3° 59′ 43′ E. During most months of the year, there is significant rainfall in Epe, interrupted by a short dry season. The average temperature is 26.3 °C and precipitation is 1990 mm per year. The climate comprises the rainy season between March to October and dry season from November to February.
Roan antelope samples were also procured on a monthly basis from hunters within the catchment area of Odo Ona Kekere in Oluyole Local Government Area Ibadan in Ibadan North, Oyo, Nigeria, with coordinates 7°14′ 1″ N, 3° 51′ 9″ E. The sampling regime spanned through the periods of May 2018 to December 2020.
Determination of trace metals in environmental media
Collection of samples
The liver and intestine of fresh greater cane rats were excised for onward tissue digestion and analysis of trace metals. For representation of the environmental media where the metals were accumulated from, the leaves that constitute the dietary intake of the animal were collected from the natural habitat and subjected to the process of digestion and analysis of heavy metals. Surface water samples and soil samples also obtained from the natural habitat of the animal where investigations proved they were sources of drinking water and shelter, respectively, to the animals. The water and soil samples were also digested using the standard procedures and analyzed for trace metals.
Analysis of samples for trace metals
Analysis of metals in water
25 mL of the preserved water sample was measured and poured into PTFE (Polytetrafluorethylene) beaker and transferred into a fume cupboard, and 10 mL of nitric acid was added to each sample in the beakers. These mixtures were then heated on a hot plate to the lowest volume possible (10 mL). They were allowed to cool and then filtered and made up with distilled water into 50 mL volumetric flask. The digested samples were then analyzed to determine the concentrations of the selected metals with the aid of a Flame Atomic Absorption Spectrometer.
Analysis of metals in sediment
Sediment samples were air-dried and sieved through 25 µm mesh. Then 1 g was weighed with the aid of a weighing balance (Model—TX3202L-V), homogenized and was transferred into a PTFE conical flask. 25 mL of ratio 3:1 hydrochloric and nitric acid (aqua regia) were added to each of the sample in a fume cupboard for digestion. It was then heated on a hot plate until the volume reduced to about 5 mL. They were filtered and made up with distilled water to 50 mL volumetric flask for the trace metal concentration analysis of zinc, cadmium, vanadium, barium, nickel, copper, lead, cobalt, chromium, and manganese, using the Flame Atomic Absorption Spectrometer (Philips model PU 9100).
Analysis of metals in liver, intestine and parasites
Frozen tissues were thawed and two (2) grams wet-weight sample of liver and intestine (from both infected and uninfected fish) were weighed, and the enteric parasites were separately pulled to obtain same weight. These samples were separately placed in a beaker and digested with 25 mL of ratio 1:1 hydrogen peroxide and nitric acid. The mixture was heated to about 5 mL and allowed to cool afterward. It was then filtered and made up with distilled water to the 50 mL. Flame Atomic Absorption Spectrometer (Philips model PU 9100) was then used in analyzing the concentrations of zinc, cadmium, vanadium, barium, nickel, copper, lead, cobalt, chromium, and manganese with detection limits of 0.5 μg g−1, 0.01 μg g−1, 0.01 μg g−1, 0.03 μg g−1, 0.1 μg g−1, 0.05 μg g−1, 0.1 μg g−1, 0.05 μg g−1, 0.01 μg g−1, and 0.5 μg g−1, respectively. All the analytical procedures adopted were strictly in compliance with the guidelines of Whiteside (1981).
Identification of gastrointestinal parasites
Gastrointestinal contents of T. swinderianus were collected from the bushmeat processing sections of the market. The gut was partitioned into stomach, caecum, and small and large intestines; the small and large intestines were unfolded by detaching them from the mesentery. The gastrointestinal tract (GIT) was dissected, and contents emptied in sterile dishes. The linings of each region of the GIT were scraped, washed in saline solution (9 g salt dissolved in 1 L of water), and examined for any helminth attaching to it. A hand lens was used to examine the intestinal content of the mammals for adult parasites. Helminth parasites were recovered with a pair of forceps and fixed in 70% alcohol for parasite identification (Opara and Fagbemi 2008). The parasites were identified based on standard morphological characteristics and the representative images of the parasites Identification of intestinal parasites was undertaken at the pathology laboratory of the Department of Veterinary Pathology, University of Ibadan, Nigeria.
Live parasite specimens collected from the dissected host guts were kept in a deionized water until parasite proboscis were everted. Afterward, they were fixed in 70% ethanol. The specimens were stained in Mayer’s acid carmine, distained in 4% hydrochloric acid in 70% ethanol, then dehydrated in ascending concentrations of ethanol (70%, 80%, 90%, 90%, 100%), and cleared in 100% xylene, then in 50% Canada balsam and 50% xylene. Each step was at the interval of 24 h. Whole worms were then mounted on slide and analyzed. All measurements were recorded in micrometer. The width was measured as the maximum width, while the trunk length was measured without including the proboscis, neck, or bursa.
Specimens were fixed in 70% ethanol, then placed in critical-point drying baskets and dehydrated using ethanol series of 95% and 100% for at least 10 min per soak followed by critical-point drying Lee (1992). Samples were gold coated and observed under a scanning electron microscope XL30 ESEMFEG (FEI, Hillsboro, Oregon, USA). Digital images of the structures were obtained with the aid of Olympus BH2 compound light microscope (Olympus Optical Co., Tokyo, Japan), equipped with an AmScope camera MU900 (United Scope, Irvine, California), in conjunction with digital imaging software. Detailed studies were then carried out on the para-receptacle structure by sectioning the specimens using plastic and diamond knives under the scanning electron microscope.
Using Omar et al. (2016) as the parasite identification manual, some of the taxonomic identification keys used include possession of proboscis which is spineless at the anterior and apical ends. The posterior end of the proboscis and conical neck exhibited depressions like sensory structures. Furthermore, the apical end of the proboscis possesses an apical epidermis cone, while the posterior of the proboscis had thin latero-dorsal and massive ventral hooks as described by Omar et al. (2016).
A coprological survey was also conducted where a total of eighty fecal samples were put in sample bottles with saline solution added to it and twenty fecal samples were collected. Aliquots were taken and examined under the light microscope to observe eggs or parasites larvae; intensity was estimated by the number of parasites per mL.
The predominant parasites identified in the host was Strongyloides spp., which recruited into the study for further analysis.
Determination of biochemical biomarkers
Determination of cholesterol
Total cholesterol in the liver, intestine and parasites of the rats was determined using enzymatic end point method described by Roeschlau et al. (1974).
Determination of high-density lipoprotein-associated cholesterol (HDL)
The high-density lipoprotein-associated cholesterol was spectrophotometrically measured using a series of coupled reactions as described by Burstein et al. (1980).
Low-density lipoprotein-associated cholesterol (LDL)
All reagents used in the analysis were provided as ready to use. The method of Assman et al. (1984) was adopted in analysis of low-density lipoprotein-associated cholesterol, which is a combination of polyvinyl sulfate precipitation and enzymatic method.
Determination of protein (PRO)
The protein content of the liver and intestine of 16 uninfected and 49 infected fish was estimated using Biuret method as described by Umemoto (1966).
Triglycerides
Triglycerides were analyzed in the 65 fish samples using the enzymatic method described by Tietz (1990).
Glucose
The glucose concentrations in the liver and intestine of the 16 uninfected and 49 infected fish were determined within 30 min of collection using the method of Wedermeyer and Yasutake (1977).
Catalase (CAT)
Catalase (CAT) was assayed calorimetrically at 620 nm and expressed as moles of hydrogen peroxide (H2O2) consumed/min mg−1 protein as described by Quinlan et al. (1994). The reaction mixture (1.5 mL) contained 1.0 mL of 0.01 M pH7.0 phosphate buffer, 0.1 mL of Plasma and 0.4 mL of 2 M H2O2. The reaction was stopped by the addition of 2.0 mL of dichromate–acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). The specific activity of catalase was expressed as moles of reduced per minute per mg protein.
Superoxide dismutase (SOD)
Superoxide dismutase activity in liver homogenates was determined using the procedure described by Marklund and Marklund (1974). The method is based on the ability of SOD to inhibit the autoxidation of pyrogallol. In 970 µL of buffer (100 mM Tris—HCl, 1 mM EDTA, pH 8.2), 10 µL of homogenates and 20 µL pyrogallol 13 mM were mixed. Assay was performed in thermostated cuvettes at 25 °C and changes in absorption were recorded by a spectrophotometer (Spectronic 20D) at 480 nm. SOD activity was determined estimating the amount of enzyme that inhibited the auto-oxidation of 50% the total pyrogallol in the reaction.
Reduced glutathione (GSH)
Reduced glutathione (GSH) was determined by the method of Ellman (1959). To the liver homogenate 10% TCA was added and centrifuged. 1.0 mL of supernatant was treated with 0.5 mL of Ellmans reagent (19.8 mg of 5,5′-dithiobis nitro benzoic acid (DTNB) in 100 mL of 0.1% sodium nitrate) and 3.0 mL of phosphate buffer (0.2 M, pH8.0). The absorbance was read at 412 nm.
Malonaldehyde (MDA)
Malondialdehyde (MDA) an index of lipid peroxidation was determined by adding 1.0 mL of the supernatant was added to 2 mL of (1:1:1) TCA-TBA HCL reagent (thioarbituric acid 0.37%, 0.24 n HCL and 15% TCA) tricarboxylic acid-thioarbituric acid-hydrochloric acid reagent boiled at 1000C for 15 min, and allowed to cool. Flocculent materials were removed by centrifuging at 3000 rpm for 10 min. The supernatant was removed and the absorbance read at 532 against a blank. MDA was calculated using the molar extinction coefficient for MDATBA-complex of 1.5 × 105 M cm−1.
Quality assurance/quality control
All standards, replicates, and blanks were prepared at the same time and used immediately to prevent contamination or compromise of quality. The standards were calibrated, and the calibration curves were verified with ICV standard. The ICV standard was prepared from an independent (second source) material at or near the mid-range of the calibration curve. The acceptance criteria for the ICV standard were ± 20% of its true value. The analysis data for the ICV was kept on file with the sample analysis data. The calibration curve was verified at the end of each analysis batch and after every 20 samples using continuing calibration verification (CCV) standard and a continuing calibration blank.
A certified Standard Reference Material (SRM) was prepared with each analytical batch of samples using the same preparation method as that employed for the samples with the frequency of 1 in 20 samples per matrix. The SRM results for each analyte was validated to be within the specifications supplied by the vendor or within 75–125% of the true value. Samples that exceeded the linear calibration were diluted and reanalyzed to sensitive line for which quality control data was already established.
All reagents used were analar grade which were permissible and standard reagent for laboratory analysis as obtained from the vendor with their certificate of analysis. Each batch of sample analysis was run with certified reagent from same Lot/Batch with Lot number properly documented. Gases purchased from the gas vendor were of high purity as shown in the certificate of analysis. Standard regents, of high purity with certificate of analysis were obtained from certified manufacturers.
All glassware were treated with chromic acid before washed with detergent. They were then cleaned all glassware by detergent washing with hot water, and rinse with tap water, distilled water and acetone and oven-dried at 150 to 200 °C for 30 min. The volumetric flask was rinsed with dichloromethane only. After drying and cooling, they were sealed and stored in a clean environment to prevent post-cleaning contamination.
Ethical permission
Ethical approval was obtained from the University of Lagos College of Medicine health research ethics committee with reference number CMUL/HREC/05/20/724.
Statistical analysis
The descriptive statistics of the trace metals, lipid profile and antioxidants of the live and intestine of the greater cane rat were subject to analysis of variance (ANOVA) using Microsoft Excel 2020 to test for the significant differences among the infected and uninfected greater cane rat with regards to the concentration of trace metals, lipid profile, and antioxidant biomarkers. The Bonferroni post hoc test was employed in determining the actual locations of the significant difference at the probability level of 0.05.