COMPARISON OF BLOOD SYMMETRIC DIMETHYLARGININE AND CREATININE AS ENDOGENOUS MARKERS OF KIDNEY FUNCTION IN CAPTIVE TIGERS (PANTHERA TIGRIS).

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in captive tigers (Panthera tigris). Blood creatinine (Cr) and blood urea nitrogen measurements are inexpensive and common biomarkers used to evaluate renal function. However, several limitations have been reported regarding their sensitivity and interindividual variability. Symmetric dimethylarginine (SDMA) has been suggested to be a more sensitive biomarker that is less affected by extrarenal factors and has a strong correlation with glomerular filtration rate and blood Cr in several species. This project aimed to identify the usefulness of SDMA as an endogenous marker of kidney function in captive tigers. The hypothesis of this study is that increased circulating SDMA is positively associated with increased blood Cr. SDMA and Cr were measured in 65 banked samples (serum and plasma) from 30 individual captive tigers. The samples were collected over a 38-y period and stored at -21°C. SDMA and Cr concentrations were determined using the commercially available SDMA test and enzymatic colorimetric methods, respectively. SDMA had a significant positive association with Cr (for every 1 unit increase of log SDMA, Cr increased by 82%, P = 0.0002). Age and subspecies influenced Cr but not SDMA concentrations. In one animal, blood SDMA increased above the ZIMS reported range. approximately 3.6 mo before Cr increased. SDMA is currently indicated for the diagnosis of CKD in domestic felids and seems also promising in nondomestic felids. Further prospective studies might improve the understanding of the performance of this biomarker.

Exposure of Wild Sumatran Tiger (Panthera tigris sumatrae) to Canine Distemper Virus.

Canine distemper virus (CDV) is recognized as a conservation threat to Amur tigers (Panthera tigris altaica) in Russia, but the risk to other subspecies remains unknown. We detected CDV neutralizing antibodies in nine of 21 wild-caught Sumatran tigers (42.9%), including one sampled on the day of capture, confirming exposure in the wild.

Microsatellite characterization and development of unified STR panel for big cats in captivity: a case study from a Seoul Grand Park Zoo, Republic of Korea.

The zoos manage small populations of endangered big cat species like tiger, lion, and leopard for display, research, and conservation breeding. Genetic management of these populations is essential to ensure long term survival and conservation utility. Here we propose a simple and cost effective microsatellite based protocol for the genetic management of captive big cats. We sampled 36 big cat individuals from Seoul Grand Park Zoo (Republic of Korea) and amplified 33 published microsatellite loci. Overall, allelic richness and gene diversity was found highest for leopards, followed by lions and tigers. Twelve of the thirty-three markers showed a high degree of polymorphism across all target species. These microsatellites provide a high degree of discrimination for tiger (1.45 × 10-8), lion (1.54 × 10-10), and leopard (1.88 × 10-12) and thus can be adopted for the genetic characterization of big cats in accredited zoos globally. During captive breeding, zoo authorities rely on pedigree records maintained in studbooks to ensure mating of genetically fit unrelated individuals. Several studies have reported errors in studbook records of big cat species. Microsatellites are simple and cost effective tool for DNA fingerprinting, estimation of genetic diversity, and paternity assessment. Our unified microsatellite panel (12-plex) for big cats is efficient and can easily be adopted by zoo authorities for regular population management.

Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard.

Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365 bp outer and a 98 bp inner marker for nested tiger species identification assay, 136 bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365 bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98 bp FAM-labelled marker amplified 89% (n = 33; 33/37) scat samples independently. The 136 bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n = 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n = 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

Haematological and Biochemical Parameters of Captive Siberian Tigers (Panthera tigris altaica) from the Heilongjiang Province, China.

Haematological and biochemical parameters play important roles in safeguarding animal health and preventing disease, but the blood reference values of many wild animals are still unknown. Recently, few descriptions of the blood parameters of Siberian tigers (Panthera tigris altaica) have been reported because these tigers comprise an endangered species; however, it is extremely difficult to obtain blood samples necessary for these analyses. This study presents 14 haematological and 16 biochemical parameters of 133 Siberian tigers, of which 112 and 21 were from Heilongjiang Siberian Tiger Park (HB) and Hailin Siberian Tiger Park (HD), China, respectively. Our study is the first to determine the following parameters in Siberian tigers: red blood cell volume distribution width, platelet count, mean platelet volume, amylase (AMY), sodium/potassium, globulin and albumin/globulin levels. As the data for total bilirubin and AMY were not statistically significant, no statistical analysis was conducted for these parameters. Few parameters were significantly different according to sex and region (p < 0.05). The concentration of alkaline phosphatase decreased with age, whereas the creatinine (CREA) increased with age. The CREA concentration of tigers raised in HB was much lower than that of tigers raised in HD. The data obtained in this study provide a reference for monitoring the health of wild and captive Siberian tigers and will add important information to the standards for haematological and biochemical parameters of wild felines.

Distemper, extinction, and vaccination of the Amur tiger.

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species.

Insights in the regulation of trimetylamine N-oxide production using a comparative biomimetic approach suggest a metabolic switch in hibernating bears.

Experimental studies suggest involvement of trimethylamine N-oxide (TMAO) in the aetiology of cardiometabolic diseases and chronic kidney disease (CKD), in part via metabolism of ingested food. Using a comparative biomimetic approach, we have investigated circulating levels of the gut metabolites betaine, choline, and TMAO in human CKD, across animal species as well as during hibernation in two animal species. Betaine, choline, and TMAO levels were associated with renal function in humans and differed significantly across animal species. Free-ranging brown bears showed a distinct regulation pattern with an increase in betaine (422%) and choline (18%) levels during hibernation, but exhibited undetectable levels of TMAO. Free-ranging brown bears had higher betaine, lower choline, and undetectable TMAO levels compared to captive brown bears. Endogenously produced betaine may protect bears and garden dormice during the vulnerable hibernating period. Carnivorous eating habits are linked to TMAO levels in the animal kingdom. Captivity may alter the microbiota and cause a subsequent increase of TMAO production. Since free-ranging bears seems to turn on a metabolic switch that shunts choline to generate betaine instead of TMAO, characterisation and understanding of such an adaptive switch could hold clues for novel treatment options in burden of lifestyle diseases, such as CKD.

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.

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.

PHARMACOKINETIC PARAMETERS OF CEFOVECIN SODIUM (CONVENIA) IN CAPTIVE TIGERS ( PANTHERA TIGRIS).

A single 8 mg/kg dose of Cefovecin (Convenia®) was administered intramuscularly in the hindlimb of eight anesthetized captive tigers ( Panthera tigris) and serial blood samples were collected over the next 56 days to determine pharmacokinetic characteristics. High-performance liquid chromatography was utilized to determine plasma levels, and demonstrated a mean terminal half-life of 227.8 ± 29.3 hr. Cefovecin, >1 µg/ml, was detected in plasma levels up to 56 days postinjection. Protein binding of tiger plasma for cefovecin was 98%. The long duration of therapeutic plasma concentrations and lack of adverse effects make cefovecin a useful antimicrobial drug for tigers.

Selected clinical, biochemical, and electrolyte alterations in anesthetized captive tigers (Panthera tigris) and lions (Panthera leo).

A prospective study to assess changes in selected plasma biochemistry and electrolyte values, plasma insulin and aldosterone concentrations, and electrocardiography (ECG) was performed on eight female captive tigers (Panthera tigris) and three lions (Panthera leo) undergoing general anesthesia for elective laparoscopic ovariectomy. Each animal was sedated with medetomidine (18-25 microg/kg) and midazolam (0.06-0.1 mg/kg) intramuscularly, and anesthesia was induced with ketamine (1.9-3.5 mg/kg) intramuscularly and maintained with isoflurane. Venous blood samples were collected and analyzed for plasma biochemistry parameters and insulin and aldosterone concentrations. An ECG was recorded at the time of each blood sample collection. Mean plasma potassium, glucose, phosphorus, and aldosterone concentrations increased during anesthesia (P < or = 0.05). One tiger developed hyperkalemia (6.5 mmol/L) 2.5 hr after anesthetic induction. Plasma insulin concentrations were initially below the low end of the domestic cat reference interval (72-583 pmol/L), but mean insulin concentration increased (P < or = 0.05) over time compared with the baseline values. Three tigers and two lions had ECG changes that were representative of myocardial hypoxemia. Based on these results, continuous monitoring of clinical and biochemical alterations during general anesthesia in large nondomestic felids is warranted, and consideration should be given to reversal of medetomidine in these animals should significant changes in electrolytes or ECG occur.

Serosurvey of free-ranging Amur tigers in the Russian Far East.

Wild Amur tigers (Panthera tigris altaica, n=44) from the Russian Far East were tested for antibodies to feline leukemia virus, feline corona virus (FCoV), feline immunodeficiency virus, feline parvovirus (FPV), canine distemper virus (CDV), Toxoplasma gondii, and Bartonella henselae. Antibodies to FCoV, CDV, FPV, and T. gondii were detected in 43, 15, 68, and 42% of tigers, respectively. No differences were detected in antibody prevalence estimates between tigers captured as part of a research program and those captured to mitigate human-tiger conflicts. Domestic dogs (Canis familiaris) were tested as a potential source for CDV; 16% were vaccinated against CDV and 58% of unvaccinated dogs were antibody positive for CDV. A high percentage of tigers were exposed to potential pathogens that could affect the survival of this species. We recommend continued monitoring of wild tigers throughout Asia, development of standardized sampling and postmortem examination procedures, and additional research to better understand potential domestic and wild animal sources for these pathogens.

Perfluorooctanesulfonate and related fluorochemicals in the Amur tiger (Panthera tigris altaica) from China.

Perfluorinated compounds (PFCs) are used in a variety of industrial applications. We tested the hypothesis that, in Amur tigers (Panthera tigris altaica), captivity in industrialized areas increases PFC levels, potentially presenting a health risk to these animals. Serum samples were collected from 100 tigers from industrialized or nonindustrialized regions in China with nonpoint sources of PFCs. Mean concentrations of PFCs in these samples ranged from 1.57 +/- 0.83 ng/mL in nonindustrial Hailin to 4.31 +/- 2.90 ng/mL in industrial Beijing. PFC concentrations were significantly higher in tigers from the industrial city of Harbin than those from Hailin (p < 0.05). Perfluorooctanesulfonate (PFOS) was the most abundant PFC in all tigers and increased with age, regardless of industrial/ nonindustrial background (p < 0.01). However, PFOS concentrations were 2-4 orders of magnitude less than the current no-observed-effect level. In addition, overall PFC levels in Amur tigers were low compared with various species living in other countries, consistent with the relatively short history of PFC use in China. These results are consistent with the hypothesis that captivity in industrialized areas increases PFC levels in Amurtigers. They also suggestthat PFC accumulation will persist, and even increase, with continued use of PFCs in China.

Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal steroid analyses.

Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated with natural ovarian activity with those after chorionic gonadotropin ovulation induction and artificial insemination (AI). Baseline fecal estradiol concentrations were 65.77 +/- 3.61 ng/g with estrual peaks of 167.39 +/- 9.92 ng/g and an anovulatory cycle length of 17.96 +/- 0.70 days. Peak fecal estradiol was higher when females were housed with a male for breeding (262.30 +/- 41.43 vs. 165.30 +/- 3.67 ng/g; P < 0.05). The majority of animals showed some seasonal differences in fecal estradiol however, patterns were inconsistent. Fecal progestagens increased only after breeding confirming tigers are primarily induced ovulators. The non-pregnant luteal phase was 34.50 +/- 1.85 days in duration. In pregnant tigers, fecal progestagens remained elevated for 108 days until parturition and the diagnosis of pregnancy was possible based on the elevated fecal progestagens after 35 days of gestation. Tigers were administered equine chorionic gonadotropin (eCG) to stimulate follicular growth and human chorionic gonadotropin (hCG) to induce ovulation prior to AI [200 IU eCG/100 IU hCG (n = 5); 400 IU eCG/200 IU hCG (n = 2); 500 IU eCG/100 IU hCG (n = 2); 1000 IU eCG/750 IU hCG (n = 11); 1000 IU eCG/1000 IU hCG (n = 4)]. None of the tigers subjected to AI became pregnant (n = 9). Fecal endocrine patterns in gonadotropin-stimulated tigers were considerably different from those observed in naturally bred tigers. In particular, fecal estradiol concentrations were higher than those observed during natural estrus and remained elevated for longer periods of time in tigers administered the higher doses of gonadotropins typically used in conjunction with AI in this species. These abnormal endocrine patterns may help explain the poor success rate of AI in this species.