Isolation, genome analysis and comparison of a novel parainfluenza virus 5 from a Siberian tiger (Panthera tigris).

Paramyxoviruses are important pathogens affecting various animals, including mammals and humans. Parainfluenza virus 5 (PIV5)-a member of the family Paramyxoviridae-is a major threat to the health of mammals and humans. However, studies on terrestrial wild animals infected with PIV5 are scanty. In this study, we utilized reverse transcription PCR to detect PIV5 infection in the visceral organ tissues of a Siberian tiger (Panthera tigris ssp. altaica) with vomiting, diarrhea, and dyspnea before its death. A novel PIV5 (named SR strain) with a slowly progressive cytopathic effect was isolated in Vero cells and validated using a transmission electron microscope. Full-length sequencing and analysis revealed that the whole genome of the PIV5 SR strain contained 15,246 nucleotides (nt) and seven non-overlapping genes (3'-N-V/P-M-F-SH-HN-L-5') encoding eight proteins. Phylogenetic analysis of three PIV5 strains identified in the same zoo confirmed that PIV5 strains SR and ZJQ-221 shared the closest genetic relationship as they were clustered in the same branch, while the recently found Siberian tiger strain SZ2 kept a certain distance and formed a relatively unique branch. Furthermore, mutations of nt and amino acids (aa) between strains ZJQ-221, SR, and SZ2 were identified. In summary, we report the identification and genomic characterization of a novel PIV5 strain SR isolated in a Siberian tiger, which may help future research on interspecific transmission mechanisms.

Decreased SMP30 Expression Is Related With EMT in the Kidneys of Two Siberian Tigers With CKD.

BACKGROUND/AIM: Chronic kidney disease (CKD) is one of the most common causes of mortality in wild non-domestic felidae. The molecular mechanism regulating renal fibrosis in nephropathy is not fully understood especially in the felidae. This study aimed to elucidate senescence marker protein 30 (SMP30) expression patterns and its relationship with epithelial-mesenchymal transition (EMT) by immunostaining in two necropsied Siberian tigers (Panthera tigris altaica) with CKD. MATERIALS AND METHODS: Two kidney samples from male Siberian tigers were fixed and tissue sections were stained for histopathological assay. RESULTS: In CKD, renal tubular epithelial cells lost their tubular structures surrounded by severe interstitial fibrosis and were detached from the basement membrane. These damaged cells resembled the morphology of mesenchymal cells and showed much lower SMP30 expression compared with intact tubular epithelial cells. These cells also expressed vimentin, which is specifically expressed by mesenchymal cells, and through double staining, it was observed that vimentin was expressed in the tubular epithelial cells where SMP30 was not expressed. In addition, double-positive expression of pan-cytokeratin (pan-CK) and vimentin was found in damaged epithelial cells with mesenchymal features. CONCLUSION: We demonstrated possible evidence to understand the role of SMP30 as a new pivotal factor and the possibility of decreased SMP30 as a potential indicator of EMT at the end stage of CKD.

Epidemiological and Molecular Approaches for a Fatal Feline Panleukopenia Virus Infection of Captive Siberian Tigers (Panthera tigris altaica) in the Republic of Korea.

Feline panleukopenia virus (FPV), a member of the species Protoparvovirus carnivoran1, is one of the most fatal pathogens of domestic and wild carnivores. The virus endemically infects domestic carnivores worldwide and its cross-species transmission threatens endangered wild carnivores, including Siberian tigers. In this study, a fatal FPV infection in endangered Siberian tigers was investigated to trace the origin of the virus and elucidate the reason behind FPV's infection of the vaccinated tigers. Our genetic characterization and phylogenetic analysis revealed that the virus detected in the infected tigers, designated as the KTPV-2305 strain, was closely related to FPV strains circulating in Korean cats, suggesting that it might have been transmitted from stray cats wandering around the zoo. Compared with the prototype FPV reference strains, the KTPV-2305 strain carried three distinct amino acid (aa) mutations in the VP2 protein sequence (I101T, I232V, and L562V) in this study. These three mutations are commonly found in most global FPV strains, including Korean strains, indicating that these mutations are common evolutionary characteristics of currently circulating global FPVs. The reason why the vaccinated tigers were infected with FPV was most likely the insufficient protective immunity of the affected tigress or vaccine failure triggered by the interference of maternal-derived antibodies in the affected tiger cubs. These findings suggest that improved vaccination guidelines are urgently needed to save the lives of wild carnivores from this fatal virus.

Cutaneous Amelanotic Signet-Ring Melanoma in a Siberian Tiger (Panthera tigris altaica).

A 15-year-old male white Siberian tiger (Panthera tigris altaica) was evaluated for an elevated, ulcerated, cutaneous mass on the right flank. The mass was removed by excisional biopsy and submitted for histopathology. Based on distinct histological features of intracytoplasmic globular material and positive immunohistochemical staining for Melan-A and vimentin, an amelanotic signet-ring melanoma was diagnosed. While in domestic cats this neoplasm is associated with malignancy and a short survival time, the tiger had no local recurrence or related clinical disease approximately 4 years post surgery. Cutaneous melanocytic tumours are rare in big cats, and the findings in this case suggest that amelanotic signet-ring melanoma is not as malignant as its counterpart in domestic cats.

Per- and polyfluoroalkyl substances (PFASs) in blood of captive Siberian tigers in China: Occurrence and associations with biochemical parameters.

Per- and polyfluoroalkyl substances (PFASs) have been ubiquitously detected in the environment and marine animals. However, little is known about these substances and their associations with health parameters in wild terrestrial mammals. In this study, we determined PFAS levels and distribution in the blood of captive Siberian tigers in Harbin, China, and evaluated potential exposure pathways by daily intake. In addition, for the first time, we explored the associations between serum PFAS concentrations and clinical parameters. Results showed that perfluorooctanoate (PFOA) was the dominant PFAS compound in blood (accounting for 64%), followed by perfluorooctanesulfonate (PFOS). In addition, 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) concentrations were also detected in blood and dietary food. Furthermore, significant positive age relationships were observed for levels of perfluoroheptanoate (PFHpA), PFOA, PFOS, and 6:2 Cl-PFESA in the blood of female tigers. Results showed that PFOA and PFOS in dietary food accounted for over 70% of total daily intake of PFASs, indicating that meat consumption is a predominant exposure pathway in tigers. We also found positive associations between higher exposure to PFASs (including PFOA, PFOS, and 6:2 Cl-PFESA) and elevated serum levels of alanine transaminase (ALT), a marker of liver damage. Thus, comprehensive health assessments of PFAS burdens in wildlife are needed.

Serological surveillance of avian influenza virus and canine distemper virus in captive Siberian Tigers in Northeastern China.

In order to understand infection of avian influenza A virus (AIV) and canine distemper virus (CDV) in the Siberian Tiger in Northeast China, 75 Siberian Tiger serum samples from three cap- tive facilities in northeastern China were collected. AIV and CDV antibody surveillance was test- ed by using hemagglutination inhibition and serum neutralization methods. The results showed that the seroprevalence of H5 AIV, H9 AIV and CDV was respectively 9.33% (7/75), 61.33% (46/75) and 16% (12/75). In the 1⟨years ⟨2 and > 5 year-old group, the seroprevalence of the H9 AIV was 24% and 80% (P ⟨ 0.01), and the CDV seroprevalence was 6% and 36% (P ⟨ 0.01), respectively. It was demonstrated that 3 (4%) out of 75 serum samples were AIV+CDV seropos- itive, with 2.67% (2/75) in H9+AIV and 1.33% (1/75) in H5+H9+AIV. To our knowledge, this is the first report of AIV and CDV seroprevalence in Siberian Tigers in China, which will provide base-line data for the control of AIV and CDV infection in Siberian Tigers in China.

Endoparasites of the Siberian tiger (Panthera tigris altaica).

There have been few reports on the diversity and prevalence of parasitic fauna of the endangered Siberian tiger, which inhabits the territory of the Russian Far East. The present review attempts to summarize the information about the parasitic fauna of wild Siberian tigers, which includes 15 helminths and 3 protozoan species. The most prevalent parasitic species was found to be Toxocara cati, followed by Toxascaris leonina. Another commonly recorded Platyhelminth species is Paragonimus westermani, which causes a lethal infection of the lung parenchyma in Siberian tigers. However, the information about infections by this fluke in the Siberian tigers is scarce, although P. westermani infections pose a serious health hazard to tiger populations. The nematodes Aelurostrongylus abstrusus and Thominx aerophilus are found in Siberian tigers with an occurrence rate of 2.3% and 19%, respectively. The information on the parasitic infestations of captive populations of Siberian tigers is also presented along with the sources of infection and hazards for the wild tiger populations in their natural environment.

ACUTE NONCOMPRESSIVE NUCLEUS PULPOSUS EXTRUSION CAUSING PARAPLEGIA IN A SIBERIAN TIGER ( PANTHERA TIGRIS ALTAICA).

A neutered male Siberian tiger ( Panthera tigris altaica) presented with paraplegia of 5 days' duration. Thoracolumbar magnetic resonance imaging (MRI) revealed a focal, linear T2-W hyperintense intramedullary lesion at the level of T10-11 as well as reduced size of the nucleus pulposus of the T10-11 intervertebral disc. The animal was humanely euthanatized as a result of the severity of clinical signs and the poor prognosis. Gross necropsy revealed a dark red, firm focus on the spinal cord at the level of the T11 nerve roots. Histopathology revealed liquefactive necrosis of the ventral spinal cord at the level of T9-11, with hemorrhage and vascular changes within the cord and associated meninges. Fibrovascular tissue with hemorrhage, fibrin, and fragments of cartilage consistent with nucleus pulposus material was identified extradurally at this level. The findings were consistent with acute noncompressive nucleus pulposus extrusion.

Lead induces Siberian tiger fibroblast apoptosis by interfering with intracellular homeostasis.

Lead (Pb2+) is a poisonous heavy metal that causes many pathophysiological effects in living systems. Its toxicological effects are well known as it causes apoptosis of several cell types and tissues. This study aimed to determine the criteria required for early diagnosis of Pb2+ poisoning in the Siberian tiger using a tiger population in China, to identify a safety Pb2+ concentration threshold, and to provide suggestions for preventing Pb2+ poisoning in Siberian tigers. We investigated the apoptotic effects of Pb2+ (0, 32, 64, and 125 µM) for 12-48 h on Siberian tiger fibroblasts in vitro. Typical apoptotic effects were observed after Pb2+ exposure. Pb2+ strongly blocked DNA synthesis in the G0/G1 phase and induced cell apoptosis in a dose- and time-dependent manner. Intracellular free calcium (Ca2+) levels, reactive oxygen species levels, and efflux of extracellular Ca2+ were increased. The mitochondrial membrane potential was lowered. Caspase-3, -8, and -9 activities were increased when fibroblasts were treated with 32, 64, and 125 µM Pb2+. The gene expression levels of Bax, caspase-3, -8, Fas, and p53 were increased, while that of Bcl-2 was decreased. Calcium homeostasis and mitochondrial function were disturbed. Ca2+ efflux, oxidative damage, activation of caspases, and regulation of Bax, Bcl-2, caspase-3, -8, Fas, and p53 gene expression played an important role in the apoptotic effects. The disorder of intracellular homeostasis was the trigger for apoptosis in Siberian tiger fibroblasts.

Sterile nodular panniculitis with lung and lymph node involvement in a Siberian tiger (Panthera tigris altica).

A 2- to 4-year-old uncastrated male Siberian tiger (Panthera tigris altica) bred in a local wild animal park presented with generalized clinical signs including abdominal pain, fever, lethargy, and anorexia, along with subcutaneous nodules along the trunk. The patient subsequently died of chronic, progressive dyspnea despite 45 days of antibiotic treatment. At necropsy, mesenteric fat inflammation and multiple subcutaneous, peritoneal, and intraabdominal nodules were observed. The lungs demonstrated congestion and heavy coagulation, and necrotic foci were observed on the cut surface. Histopathologically, the nodules were identified as granulomatous fatty tissue with numerous lymphocytes, infiltration with lipid-laden macrophages, and fibrosis. These changes were also noted in the lung. The etiology of this condition remains undetermined.

Isolation and phylogenetic analysis of three feline calicivirus strains from domestic cats in Jilin Province, China.

Feline calicivirus (FCV) is a highly prevalent pathogen that can cause infectious felid upper respiratory tract disease. The majority of complete genome sequences of FCV strains reported to date are from the USA. In this study, three FCV strains, CH-JL1, CH-JL2 and CH-JL3, were isolated from domestic cats in Jilin Province, China. Sequence analysis revealed that except for strains HRB-SS, WZ-1, XH, 12Q087-1 and 12Q087-5, the 3' untranslated regions (UTRs) of CH-JL2 and CH-JL3 are more than 20 nucleotides longer than those of all other reference isolates. The complete sequences of the three CH-JLs were compared with other reference strains, with nucleotide sequence identity values in the range of 76.2%-82.2%, 76.8%-96.4 and 76.8%-96.4%. Phylogenetic analysis showed that CH-JL1 forms a branch with FB-NJ-13, GD, 12Q087-1 and 12Q087-5. CH-JL2 was found to be most closely related to CH-JL3, forming another branch together with the other isolates. CH-JL1 shares a long nucleotide span with CH-JL2 and CH-JL3. It can be inferred that many FCV strains are co-circulating in Jilin Province. The availability of complete genome sequences will serve as a reference for future epidemiological studies of FCV.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis.

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd2+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd2+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

De novo transcriptomic analysis and development of EST-SSR markers in the Siberian tiger (Panthera tigris altaica).

The Siberian tiger, Panthera tigris altaica, is an endangered species, and much more work is needed to protect this species, which is still vulnerable to extinction. Conservation efforts may be supported by the genetic assessment of wild populations, for which highly specific microsatellite markers are required. However, only a limited amount of genetic sequence data is available for this species. To identify the genes involved in the lung transcriptome and to develop additional simple sequence repeat (SSR) markers for the Siberian tiger, we used high-throughput RNA-Seq to characterize the Siberian tiger transcriptome in lung tissue (designated 'PTA-lung') and a pooled tissue sample (designated 'PTA'). Approximately 47.5 % (33,187/69,836) of the lung transcriptome was annotated in four public databases (Nr, Swiss-Prot, KEGG, and COG). The annotated genes formed a potential pool for gene identification in the tiger. An analysis of the genes differentially expressed in the PTA lung, and PTA samples revealed that the tiger may have suffered a series of diseases before death. In total, 1062 non-redundant SSRs were identified in the Siberian tiger transcriptome. Forty-three primer pairs were randomly selected for amplification reactions, and 26 of the 43 pairs were also used to evaluate the levels of genetic polymorphism. Fourteen primer pairs (32.56 %) amplified products that were polymorphic in size in P. tigris altaica. In conclusion, the transcriptome sequences will provide a valuable genomic resource for genetic research, and these new SSR markers comprise a reasonable number of loci for the genetic analysis of wild and captive populations of P. tigris altaica.

Molecular characterization of a feline calicivirus isolated from tiger and its pathogenesis in cats.

Feline calicivirus (FCV) is a virus that causes respiratory disease in cats. In this study, the FCV TIG-1 was isolated from Siberian tiger feces collected in 2014 in Heilongjiang Province, China. Phylogenetic analysis among TIG-1 and other FCVs showed that TIG-1 does not share the same lineage with other FCV isolates from Heilongjiang or other regions in China but is located in the same cluster with the FCV strain Urbana, which was isolated from the United States. The growth kinetics in vitro and the pathogenicity in cats between TIG-1 and the domestic cat-origin FCV strain F9 (vaccine strain) and strain 2280 were compared. We found that the growth kinetics of strains TIG-1 and 2280 were faster than that of strain F9 from 12h to 36h post-infection, indicating that strains TIG-1 and 2280 produce infectious virions and reach peak yields earlier. Challenge experiments in cats showed that TIG-1 grew faster than the other two strains in the lungs of cats and that TIG-1 is a virulent FCV with 100% morbidity and lethality. In addition, the histopathological results showed that the virulent TIG-1 strain directly led to severe lung tissue damage and indirectly led to intestinal damage. The results presented here show that a tiger-origin FCV exhibits high virulence in cats.

Ascarid infestation in captive Siberian tigers in China.

The Siberian tiger is endangered and is listed by the International Union for the Conservation of Nature; the captive environment is utilized to maintain Siberian tiger numbers. Little information regarding the prevalence of parasites in Siberian tigers is available. A total of 277 fecal samples of Siberian tigers were analyzed in this study. The microscopic analysis indicated the presence of ascarid eggs of Toxascaris leonina and Toxocara cati. The ascarid infection rate was 67.5% in Siberian tigers. The internal transcribed spacer-1 (ITS-1) phylogenetic analysis indicated that T. leonina belonged to Toxascaris and that Toxo. cati belonged to Toxocara. The infestation rate and intensity of T. leonina were higher than those of Toxo. cati. One-way analysis of variance showed that the presence of T. leonina was significantly associated with age (P<0.05). Temperature changes also influenced T. leonina and Toxo. cati infestation, and a rise in temperature caused an increase in the number of T. leonina and Toxo. cati eggs. This study provides a better understanding of ascarid infestation among the captive Siberian tigers and is helpful for the prevention of the spread of infectious parasitic diseases among other tigers in the zoo.

Analysis of Odorants in Marking Fluid of Siberian Tiger (Panthera tigris altaica) Using Simultaneous Sensory and Chemical Analysis with Headspace Solid-Phase Microextraction and Multidimensional Gas Chromatography-Mass Spectrometry-Olfactometry.

Scent-marking is the most effective method of communication in the presence or absence of a signaler. These complex mixtures result in a multifaceted interaction triggered by the sense of smell. The objective was to identify volatile organic compound (VOC) composition and odors emitted by total marking fluid (MF) associated with Siberian tigers (Panthera tigris altaica). Siberian tiger, an endangered species, was chosen because its MF had never been analyzed. Solid phase microextraction (SPME) for headspace volatile collection combined with multidimensional gas chromatography-mass spectrometry-olfactometry for simultaneous chemical and sensory analyses were used. Thirty-two VOCs emitted from MF were identified. 2-acetyl-1-pyrroline, the sole previously identified compound responsible for the "characteristic" odor of P. tigris MF, was identified along with two additional compounds confirmed with standards (urea, furfural) and four tentatively identified compounds (3-methylbutanamine, (R)-3-methylcyclopentanone, propanedioic acid, and 3-hydroxybutanal) as being responsible for the characteristic aroma of Siberian tiger MF. Simultaneous chemical and sensory analyses improved characterization of scent-markings and identified compounds not previously reported in MF of other tiger species. This research will assist animal ecologists, behaviorists, and zookeepers in understanding how scents from specific MF compounds impact tiger and wildlife communication and improve management practices related to animal behavior. Simultaneous chemical and sensory analyses is applicable to unlocking scent-marking information for other species.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd²+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd²+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

Simultaneous Occurrence of Pancreatic Adenocarcinoma and Brunner's Gland Adenoma in a Siberian Tiger (Panthera tigris altaica).

We describe a case of pancreatic adenocarcinoma and Brunner's gland adenoma in an 18-year-old male Siberian tiger (Panthera tigris altaica) from the Ljubljana Zoo. The tiger was humanely destroyed due to weakness and progressive weight loss. Necropsy examination revealed a large, grey, predominantly necrotic mass replacing the major part of the pancreatic body. Microscopically, the mass was unencapsulated, poorly demarcated, highly cellular and composed of highly pleomorphic, cuboidal to tall columnar cells with basal, round or oval, moderately anisokaryotic nuclei with prominent nucleoli and moderate to large amounts of eosinophilic cytoplasm. The tumour was diagnosed as pancreatic tubular adenocarcinoma with infiltration into the duodenum and mesentery. There were tumour emboli in mesenteric blood vessels and hepatic metastases. The non-affected part of the pancreas exhibited severe chronic pancreatitis. In addition, one firm white neoplastic nodule was observed in the duodenal wall. The nodule was set in the tunica muscularis and was unencapsulated, well demarcated and highly cellular, and consisted of a closely packed layer of normal Brunner's glands and a centrally positioned group of irregularly branched tubules with small amounts of debris in the lumen. The neoplastic nodule was diagnosed as Brunner's gland adenoma. The present case is, to the best of our knowledge, the first report of concurrent pancreatic adenocarcinoma and Brunner's gland adenoma, most probably induced by chronic pancreatitis, either in man or animals.

Complete mitochondrial genome of a wild Siberian tiger.

In this study, the complete mitochondrial genome of Siberian tiger (Panthera tigris altaica) was sequenced, using muscle tissue obtained from a male wild tiger. The total length of the mitochondrial genome is 16,996 bp. The genome structure of this tiger is in accordance with other Siberian tigers and it contains 12S rRNA gene, 16S rRNA gene, 22 tRNA genes, 13 protein-coding genes, and 1 control region.

Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future.