Study area and animals’ enrolment
Twenty tigers housed at a Safari Park (Apulia region, Brindisi Province, southern Italy) (40°50’ N, 17°20’ E) were involved in the study. Those animals have been previously enrolled in an epidemiological investigation aimed at evaluate the prevalence of L. infantum infection after the identification of an index case in the tiger population, using both serological and molecular methods. Nine (45%) out of 20 tigers were positive to L. infantum by immunofluorescence antibody test (IFAT) (9/20, 45%) and/or by real time-PCR (5/20, 25%), being five of them (5/20, 25%) positive by both methods.
Out of 9 serologically positive tigers, three animals presented an antibody titre of 1:40, four of 1:80, one of 1:160 and one of 1:640 each, respectively. Therefore, according to these results (Iatta et al., under review) in the overall tiger population two groups were set up: L. infantum-positive animals (n=9; group A) and L. infantum-negative animals (n=11; group B).
Food and water were withheld for 12 hr prior to anaesthesia. Upon arrival at the zoological park, each animal was anesthetised, via remote injection, with a combination of 250 mg of zolazepam/tiletamine (Zoletil 50/50, Virbac) plus 3 mg of detomidine (Mesden 10mg/ml, FM Italia) (Laricchiuta et al., 2015). Then, a complete physical exam was performed, and for each animal body condition score was assessed (AZA Tiger Species Survival Plan ®, 2016). The clinical visit included a whole-body visual examination for cutaneous lesions and lymph nodes evaluation. After verifying sex and reproductive status, external genitalia and perineum were inspected. Mucous membrane colour and moisture were observed. Other visual observations included evaluation for normal breathing patterns and rate.
At the end of the procedures (i.e. 30-45 min after darting), immobilization of the tigers was reversed with intramuscular administration of 15 mg of atipamezole (Antisedan 0.5%, Pfizer, Milan, Italy) (Laricchiuta et al., 2015). After recovery (i.e. ≃10 min), each tiger was closely monitored for 8 h and, thereafter, reintroduced into the colony of the zoo.
Blood samples and analysis
Blood (12 mL) was collected from the cephalic vein and placed in tubes with EDTA (2 mL) to perform routine haematology, and in plain tubes (10 mL) to obtain serum. Serum was obtained within 4 hours from collection, by centrifugation (15 minutes at 1500 x g).
Results from complete blood count (CBC) (Siemens, ADVIA 2120), serum biochemical analysis (Beckman Coulter, Clinical Chemistry Analyzer AU680) and electrophoresis (SEBIA, Capillarys 2 Flex Piercing) were achieved with the same methods in all samples.
Urine collection and urinalysis
All urine samples were collected by catheterization, using a 20 mL syringe connected to a 14 Ch catheter. Urine samples were placed in 10 mL, sterile, evacuated collection tubes, stored at room temperature (approximately 20°C [676°F]), and analysed within 4 hours from collection. Urine sediment was obtained by centrifugation (10 minutes at 900 x g) of 5 mL of urine, followed by removal of 4.5 mL of supernatant, and resuspension of the remaining 0.5 mL of urine. A sample of 12 µL of the resuspended urine was microscopically assessed. Red blood cells and white blood cells were expressed as mean number of cells/10 hpf (40 × magnification). Urine sediment with bacteriuria, and/or >5 red blood cells or white blood cells/hpf, was considered indicative of active inflammation and excluded from the UPC ratio evaluation. The supernatant was transferred into separate tubes to determine UPC ratio. To calculate the UPC ratio, protein concentration (mg/dL) was measured with pyrogallol red, and creatinine (mg/dL) was measured using the Jaffé method on undiluted urine supernatant that was thawed before analysis. Results were achieved with the same method in all samples (Beckman Coulter, Clinical Chemistry Analyzer AU680).
The Kolmogorov-Smirnov test was used to assess the normality of the data. Statistical differences between group A and group B were tested for significance by Student’s t-test for normally distributed data and with the Mann-Whitney test for not normally distributed data. A value of P < 0.05 was considered statistically significant. The statistical analyses were performed using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego California, USA).