Interferon-beta, interferon-gamma and their fusion interferon of Siberian tigers (Panthera tigris altaica) in China are involved in positive-feedback regulation of interferon production.

As a group of cytokines, interferons are the first line of defense in the antiviral immunity. In this study, Siberian tiger IFN-ß (PtIFN-ß) and IFN-γ (PtIFN-γ) were successfully amplified, and the two were fused (PtIFN-γ) by overlap extension polymerase chain reaction (SOE-PCR). Bioinformatics analysis disclosed that PtIFN-ß and PtIFN-γ have species-specificity and conservation in the course of evolution. After being expressed in prokaryotes, the antiviral activities and physicochemical properties of PtIFN-ß, PtIFN-γ and PtIFNß-γ were analyzed. In Feline kidney cells (F81), PtIFNß-γ showed more active antiviral activity than PtIFN-ß and PtIFN-γ, which has more stable physicochemical properties (acid and alkali resistance, high temperature resistance). In addition, PtIFN-ß, PtIFN-γ and PtIFN-γ activated the JAK-STAT pathway and induced the transcription and expression of interferon-stimulated genes (ISGs). Janus kinase (JAK) 1 inhibitor inhibited ISGs expression induced by PtIFN-ß, PtIFN-γ and PtIFN-γ. Overall, this research clarified that PtIFN-ß, PtIFN-γ and PtIFNß-γ have the ability to inhibit viral replication and send signals through the JAK-STAT pathway. These findings may facilitate further study on the role of PtIFN in the antiviral immune response, and help to develop approaches for the prophylactic and therapeutic of viral diseases based on fusion interferon.

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.

Detection of Lion Podoplanin Using the Antitiger Podoplanin Monoclonal Antibody PMab-231.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.

TIGER (PANTHERA TIGRIS) AND DOMESTIC CAT (FELIS CATUS) IMMUNE RESPONSES TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINATION.

Two methods for delivering a canarypox-vectored canine distemper vaccine to tigers (Panthera tigris) and domestic cats (Felis catus) were investigated. Eight tigers were divided randomly into two vaccination groups: subcutaneous injection or topical tonsillar application. Each tiger received 2 ml of canine distemper virus (CDV) vaccine (Merial Ferret Distemper Vaccine). Blood was collected from tigers on days 0, 21, 35 or 37, and 112 post-initial vaccination (PIV). Domestic cats were divided randomly into four treatment groups: saline injection (negative controls), low- and high-dose oral, and subcutaneous vaccinates. Blood was collected from domestic cats on days 0, 7, 21, and 28 and 165 or 208 PIV. Sera were tested for CDV antibodies by virus neutralization. All individuals were seronegative at the beginning of the study. One tiger vaccinated subcutaneously developed a titer of 32 by day 35, which reduced to 16 by day 112. Another tiger vaccinated by tonsillar application developed a titer of 8 on day 112. All other tigers remained seronegative. Cats that received saline injection or oral vaccination remained seronegative at each sampling time. Domestic cats vaccinated subcutaneously developed titers ranging from 4 to >128 by day 28, and those re-bled at day 166 had titers of 16 or 64. The disparity in response between domestic cats and tigers may be due to species differences or it may represent a dose-dependent effect. Subcutaneous vaccination with canarypox-vectored Purevax Ferret Distemper® is safe and elicits persistent antibody titers in domestic cats vaccinated parenterally.

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231.

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.

Detection of Tiger Podoplanin Using the Anti-Cat Podoplanin Monoclonal Antibody PMab-52.

Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies (mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, and cat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab-52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These results indicate that PMab-52 may be useful for the detection of tiger PDPN.

Molecular cloning and functional characterization of eleven subtypes of interferon-α in Amur tigers (Panthera tigris altaica).

Interferon has a broad-spectrum of antiviral effects and represents an ideal choice for the development of antiviral drugs. Nonetheless, information about alpha interferon (IFN-α) is vacant in Amur tiger (Panthera tigris altaica), an endangered species and indigenous to northeast Asia. Herein, 11 PtIFN-αs genes, which encoded proteins of 164-165 amino acids, were amplified. Afterwards, expression and purification were conducted in Escherichia coli. In physicochemical analysis, PtIFN-αs were shown to be highly sensitive to trypsin and remained stable despite changes in pH and temperature. In feline kidney cells (F81)/vesicular stomatitis virus (VSV)/canine distemper virus (CDV)/avian influenza virus (AIV) systems, PtIFN-αs were demonstrated to have distinct antiviral activities, some of them (PtIFN-α and PtIFN-α9) inhibited viral transcription levels more effectively than the other subtypes including Felis catus IFN-α, an effective therapeutic agent used for viral infections clinically. Additionally, PtIFN-α and PtIFN-α9 can up-regulate the transcription and expression of p53, a tumor suppressor factor, which could promote apoptosis of virus-infected cells. In conclusion, we cloned and expressed 11 subtypes of PtIFN-α for the first time. Furthermore, PtIFN-α and PtIFN-α9 were likely to be more efficient against both chronic viral infections and neoplastic diseases that affect the Amur tiger population. It will be of significant importance for further studies to protect this endangered species.

Comparative serological investigation between cat and tiger blood for transfusion.

Evidence suggests that non-domesticated felids inherited the same AB-erythrocyte antigens as domestic cats. To study the possible compatibility of tiger blood with that of other endangered felidae, blood samples from captive tigers and domestic cats were subjected to an in vitro study. The objectives of this study were to (1) identify whether the captive tigers had blood type AB and (2) determine the compatibility between the blood of captive tigers and that of domestic cats with a similar blood type. The anti-coagulated blood with ethylenediaminetetraacetic acid of 30 tigers was examined to determine blood type, and a crossmatching test was performed between tiger and cat blood. All 30 tigers had blood type A. Tube agglutination tests using tiger plasma with cat erythrocytes resulted in 100% agglutination (n=30) with type B cat erythrocytes and 76.7% agglutination (n=23) with type A cat erythrocytes. The 80% of major and 60% of minor compatibilities between blood from 10 tigers and 10 domestic cats with blood type A were found to pass compatibility tests. Interestingly, 3/10 of the tigers' red blood cell samples were fully compatible with all cat plasmas, and 1/10 of the tiger plasma samples were fully compatible with the type A red cells of domestic cats. Although the result of present findings revealed type-A blood group in the surveyed tigers, the reaction of tiger plasma with Type-A red cell from cats suggested a possibility of other blood type in tigers.

MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.