Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach.

BACKGROUND: Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests. RESULTS: The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively. CONCLUSION: To the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.

CARNIVORE PROTOPARVOVIRUS 1 (PARVOVIRUSES) AT THE DOMESTIC-WILD CARNIVORE INTERFACE IN INDIA.

Carnivore protoparvovirus 1 (CP1, earlier called Feline panleukopenia virus) variants such as canine parvovirus (CPV) and feline parvovirus (FPV) are significant, emerging, multihost pathogens of domestic and wild carnivores. The diversity of CP1 variants was studied between 2008 and 2014 in Wayanad, India, where flagship wildlife species such as tigers (Panthera tigris) and leopards (Panthera pardus) coexist alongside domestic carnivores, including dogs (Canis lupus familiaris) and cats (Felis catus). Using polymerase chain reaction, FPV and CPV sequences were obtained from the heart blood of a necropsied leopard individual for the first time in the world and from rectal swabs of three sympatric and clinically ill domestic dogs. CP1 amplicons were also detected in a tiger. Cross-species transmission possibilities were identified, as the closest relatives to the leopard FPV sequence were found in domestic cats from a neighboring state.

Faecal cortisol metabolites in Bengal (Panthera tigris tigris) and Sumatran tigers (Panthera tigris sumatrae).

The tiger (Panthera tigris) faces a great risk of extinction as its wild numbers have plummeted due to poaching and habitat destruction so ex-situ conservation programs are becoming ever more necessary. Reliable non-invasive biomarkers of the stress hormone (cortisol) are necessary for assessing the health and welfare of tigers in captivity. To our knowledge, non-invasive stress endocrinology methods have not been tested as widely in tigers. The first aim of this study was to describe and validate a faecal cortisol metabolite enzyme-immmunoassay (FCM EIA) for two tiger sub-species, the Bengal tiger (Panthera tigris tigris) and the Sumatran tiger (Panthera tigris sumatrae). Individual tigers (n=22) were studied in two large Zoos in Queensland, Australia (Dreamworld Theme Park and Australia Zoo). Fresh faecal samples (<12 h old) were collected each morning from both Zoos over a study period of 21 days. Biological validation was conducted separately by collecting feces 5 days before and 5 days after blood was taken from four male and five female tigers. Results showed that mean FCM levels increased by 138% and 285% in the male and female tigers within 1 day after bloods were taken, returning to baseline in 5 days. Laboratory validations of the FCM EIA were done using an extraction efficiency test and parallelism. Results showed >89% recovery of the cortisol standard that was added to tiger faecal extract. We also obtained parallel displacement of the serially diluted cortisol standard against serially diluted tiger faecal extract. Our second aim was to determine whether the FCM levels were significantly different between tiger sub-species and sex. Results showed no significant difference in mean FCM levels between the Bengal and Sumatran tiger sub-species. Mean levels of FCMs were significantly higher in females than in male tigers. Those male and female tigers with reported health issues during the study period expressed higher FCM levels than the reportedly healthy tigers. Interestingly, those tigers that took part in some activity (such as walks, photos, presentations and guest feeds) expressed moderately higher FCM levels at Dreamworld and lower FCM levels at Australia Zoo in comparison to those tigers that did not take part in such activities. These results indicate potential habituation in some tigers for routine activity through specialized training and pre-conditioning. In conclusion, the FCM EIA described in this study provides a reliable non-invasive method for evaluating the stress status of tigers in Zoos.

Anatomic Study of the Elbow Joint in a Bengal Tiger (Panthera tigris tigris) Using Magnetic Resonance Imaging and Gross Dissections.

The objective of our research was to describe the normal appearance of the bony and soft tissue structures of the elbow joint in a cadaver of a male mature Bengal tiger (Panthera tigris tigris) scanned via MRI. Using a 0.2 Tesla magnet, Spin-echo (SE) T1-weighting, and Gradient-echo short tau inversion recovery (GE-STIR), T2-weighting pulse sequences were selected to generate sagittal, transverse, and dorsal planes. In addition, gross dissections of the forelimb and its elbow joint were made. On anatomic dissections, all bony, articular, and muscular structures could be identified. The MRI images allowed us to observe the bony and many soft tissues of the tiger elbow joint. The SE T1-weighted MR images provided good anatomic detail of this joint, whereas the GE-STIR T2-weighted MR pulse sequence was best for synovial cavities. Detailed information is provided that may be used as initial anatomic reference for interpretation of MR images of the Bengal tiger (Panthera tigris tigris) elbow joint and in the diagnosis of disorders of this region.

Maximizing the reliability of non-invasive endocrine sampling in the tiger (Panthera tigris): environmental decay and intra-sample variation in faecal glucocorticoid metabolites.

Evaluation of physiological stress in the tiger (Panthera tigris) using faecal cortisol metabolite (FCM) enzyme immunoassays (EIAs) provides a powerful conservation physiology tool for the species. However, it is important to validate non-invasive endocrine sampling techniques in field conditions to ensure that the method provides a reliable parameter of physiological stress in the species. This is because endocrine measurements are highly species specific and FCM concentrations can be influenced by environmental factors. Here, we studied the impact of the decay rate of FCMs and intra-sample variation of FCMs using a previously validated EIA. To determine the decay rate of FCMs, we measured FCMs in freshly deposited tiger faeces (n = 8 tigers and 48 scats) that were randomly exposed to the natural environment (dry conditions with no rainfall) for up to 192 h. To determine intra-sample variation in FCMs, we used 10 scats from 10 tigers, divided each sample into four sections and each section into four sub-sections and measured FCMs in each section and sub-section. The results of this decay-rate experiment showed that FCMs in tiger faeces began to decay after 48 h exposure to the environmental conditions available. Thus, FCMs within freshly deposited tiger faeces are influenced by available environmental conditions. Changes in weather conditions (e.g. increased rainfall and humidity) could influence the stability of FCMs. The results of the intra-sample variation study showed that inter-variation among scats accounted for 52% of the variations in FCMs, while intra-sample variation between sections (32%) was greater than the sub-sample variation (16%). Intra-sample variation can be reduced by homogenizing the entire lyophilized faecal sample prior to the EIA. In conclusion, careful evaluation of decay rate and complete homogenization of faeces prior to EIA analysis will increase the reliability of FCMs as a non-invasive index of physiological stress in the tiger.