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.

Baseline and stress-induced corticosterone levels are higher during spring than autumn migration in European robins.

During spring and autumn migrations, birds undergo a suite of physiological and behavioral adaptations known as migratory disposition. The position of migratory seasons within the annual cycle and specifics of environmental conditions in each season could lead to formation of specific regulatory mechanisms of spring and autumn migratory disposition. However, this topic remains largely unstudied. Here we compared corticosterone (CORT) concentration (baseline and stress-induced) in European robins (Erithacus rubecula) captured during seven consecutive migratory seasons on the Courish Spit in the Baltic Sea; >650 plasma samples were analyzed in total. We found that baseline and stress-induced CORT concentrations in free-living robins during spring migration were nearly twice as high comparing to autumn passage. Moreover, the strength of relationship between these two parameters differed between the seasons. In autumn, individuals with elevated baseline CORT level invariably expressed high stress response; in spring, the stress response was more variable. These facts are in line with the hypothesis that spring and autumn migrations are separate life history stages characterized by similar physiological and behavioral adaptations but somewhat different regulatory mechanisms. Further work is needed to understand effects of seasonal differences in CORT concentrations in regulation of migratory disposition in birds.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus).

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Unusually high-pitched neonate distress calls of the open-habitat Mongolian gazelle (Procapra gutturosa) and their anatomical and hormonal predictors.

In neonate ruminants, the acoustic structure of vocalizations may depend on sex, vocal anatomy, hormonal profiles and body mass and on environmental factors. In neonate wild-living Mongolian gazelles Procapra gutturosa, hand-captured during biomedical monitoring in the Daurian steppes at the Russian-Mongolian border, we spectrographically analysed distress calls and measured body mass of 22 individuals (6 males, 16 females). For 20 (5 male, 15 female) of these individuals, serum testosterone levels were also analysed. In addition, we measured relevant dimensions of the vocal apparatus (larynx, vocal folds, vocal tract) in one stillborn male Mongolian gazelle specimen. Neonate distress calls of either sex were high in maximum fundamental frequency (800-900 Hz), but the beginning and minimum fundamental frequencies were significantly lower in males than in females. Body mass was larger in males than in females. The levels of serum testosterone were marginally higher in males. No correlations were found between either body mass or serum testosterone values and any acoustic variable for males and females analysed together or separately. We discuss that the high-frequency calls of neonate Mongolian gazelles are more typical for closed-habitat neonate ruminants, whereas other open-habitat neonate ruminants (goitred gazelle Gazella subgutturosa, saiga antelope Saiga tatarica and reindeer Rangifer tarandus) produce low-frequency (<200 Hz) distress calls. Proximate cause for the high fundamental frequency of distress calls of neonate Mongolian gazelles is their very short, atypical vocal folds (4 mm) compared to the 7-mm vocal folds of neonate goitred gazelles, producing distress calls as low as 120 Hz.

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).

Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11ß-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11ß-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11ß-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11ß-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species.

Suckling behavior in Eurasian lynx (Lynx lynx L.) cubs: characteristics and correlation with competitive interactions.

There is substantial evidence in the literature that the offspring of many mammal species prefer a particular pair of nipples. There is also a definite "nipple order" in individual litters in which each young predominantly uses one or two particular nipples. In combination with early competitive interactions, such "constancy" can play an important role in the social development of the young. In this study, we reveal an unequal use of different pairs of mothers' nipples by 42 Eurasian lynx cubs in 16 litters and investigate the relationship of this phenomenon with the early competitive interactions of the cubs and their physical development. For the lynx cubs, the most often used pair of nipples is the middle pair. There is also definite "nipple order" in each litter. We found a negative correlation between nipples use by the offspring and their competitive activity. No influence of "nipple order" on the cubs' growth rate was detected.

Relationship between acoustic traits of protesting cries of domestic kittens (Felis catus) and their individual chances for survival.

Domestic cat (Felis catus) mothers may rely on offspring cries to allocate resources in use of individuals with greater chances for survival and sacrifice the weak ones in case of impossibility to raise the entire large litter. Potential victims of this maternal strategy can enhance their chances of survival, by producing vocalizations with traits mimicking those of higher-quality offspring. We compared acoustic traits of 4990 cries produced during blood sampling by 57 two-week-old captive feral kittens (28 males, 29 females); 47 of them survived to 90 days of age and 10 died by reasons not related to traumas or aggression. No relationship was found between acoustic parameters and kitten survival, however, positive relationship was found between survival and body weight. The cries had moderate cues to individuality and lacked cues to sex. Body weight correlated positively with fundamental frequency and negatively with call rate, duration, peak frequency and power quartiles. We discuss that dishonesty of acoustic traits of kitten quality could develop as adaptation for misleading a mother from allocation resources between the weaker and stronger individuals, thus enhancing individual chances for survival for the weaker littermates. Physical constraint, as body weight, may prevent extensive developing the deceptive vocal traits.

Meeting with a male can lead to stress: Female reaction to a pairing with a partner in Felidae.

The aim of this study was to test the hypothesis that the level of sexual dimorphism modulates the effects of male-female social interaction on the female physiological condition in felids. We predicted that: 1 - contacts of females with males in species with low level of sexual dimorphism in body size will not lead to significant changes in the activity of the hypothalamus-pituitary-adrenal axis (female stress); 2 - contacts of females with males in species with a high level of sexual dimorphism can result in significant increase in the cortisol level in females. Our study did not support these hypotheses. Despite the fact that sexual dimorphism influenced partner relationships, changes of the activity of HPA in response to social interaction with a partner appeared to be determined by the biology of the species rather than by the degree of sexual dimorphism. In species with unexpressed sexual dimorphism in body size, females determined the character of relationships in the pair. In species with pronounced sexual dimorphism biased towards males, the pattern of relationships was determined by males. However, meeting a partner led to increased cortisol levels in females not in pairs with pronounced sexual dimorphism, but in pairs with a high frequency of partner interactions. This frequency was determined by the species life history and was probably related to the seasonality of reproduction and the degree of monopolization of the home range.

Inbreeding Coefficient and Distance in MHC Genes of Parents as Predictors of Reproductive Success in Domestic Cat.

Inbreeding and low diversity in MHC genes are considered to have a negative effect on reproductive success in animals. This study presents an analysis of the number and body mass of offspring in domestic cat, depending on the inbreeding coefficient and the degree of similarity in MHC genes of class I and II in parents. Inbred partners had a lower number of live kittens at birth than outbred ones. At the same time, the inbreeding coefficient did not affect the litter size and the number of offspring who survived until the period of transition to solid food. The most significant predictor for the number of surviving offspring was the degree of parental similarity in MHC genes: the parents with the maximum distance in MHC genes had more survived kittens. Moreover, this effect was most pronounced immediately after birth. A significant percentage of kittens from parents with a minimum distance in MHC genes were either stillborn or died on the first day after birth. By the age of transition to solid food, this effect is no longer so pronounced. Furthermore, neither the inbreeding coefficient nor the distance in MHC genes of parents had any effect on the body mass of kittens.

Application of Felid Hair for Non-Invasive Tracking of Animal Reproductive Status and Adrenal Activity.

Hair can be a useful matrix to examine the hormonal status of an animal, although it is difficult to correlate the results to a specific time point. The aim of this study was to evaluate seasonal changes in cortisol and testosterone levels in the hair of four feline species (lynx, Lynx lynx, n = 8; Amur wildcat, Prionailurus bengalensis euptilurus, n = 8; caracal, Caracal caracal, n = 6 and domestic cat, Felis catus, n = 17) with different breeding strategies. Animals of both sexes were sampled over the year, once per season (every three months), and the concentrations of hair testosterone and cortisol were measured by EIA. Both hormones showed annual dynamic changes, which coincided with the reproductive seasonality of the studied species. Sexual differences in testosterone level were found only during the mating season (spring for lynx and Amur wildcats, spring-summer for domestic cats), when testosterone levels were higher in males than in females. Cortisol levels were higher in males than in females in domestic cats and Amur wildcat, but also only during the mating season. Seasonal increases in testosterone were observed in three seasonal breeders (lynx, Amur wildcat and domestic cat) but not in caracal, which had high testosterone levels over the whole year. In lynx and Amur wildcat, it decreased sharply in the summer. Cortisol levels increased during the mating period in domestic cat males and lynx of both sexes; in caracal, an increase in cortisol was related to the transfer of animals to smaller winter cages. Measurements of steroids in hair can provide a reliable method for evaluating the reproductive status and the activity of the hypothalamus-pituitary-adrenal axis in several felid species.

The First Case of Cytauxzoon spp. in Russia: The Parasite Conquers Eurasia.

Over the last two decades, Cytauxzoon spp. has been conquering Eurasia, although this fact has only been brought to light through recent more intensive research after the discovery of C. manul in Pallas' cat. In Europe, Cytauxzoon was detected mainly in southern countries and later in central Europe. This pathogen has now been found in Russia for the first time (50 km from Moscow), this being the most northern sighting in Eurasia. A captive serval (Leptailurus serval) was found to be infected. Hematological analysis showed a crucial decrease in the number of leukocytes and erythrocytes, as well as in hemoglobin concentration. Genetic analysis confirmed the presence of Cytauxzoon spp. in serval blood at the beginning of the disease period. The identical pathogen was found in one bobcat at the same breeding center. Two other haplotypes of Cytauxzoon spp. were obtained from domestic cats at the same location, identical to the samples from Italy. One new haplotype, which was sequenced for the first time, was found in 7/7 investigated Amur wildcats (100%). The high occurrence and diversity of these pathogens suggest that they are present in free-ranging domestic cats and wild felids in Russia, and may be considered a potential threat to the endangered species. Current research shows that the genetic diversity of this pathogen may be even higher than it was suggested previously. Further genetic research is necessary to describe the diversity and phylogeny of this pathogen in Eurasia.

Leukocytes count and profile during early postnatal ontogenesis in domestic cat: Effect of litter size and multiple paternity.

Since blood cells count is the most important indicator of animals' physiological status, we investigated the effects of age, litter size, and multiple paternity on the total number of white blood cells, the number of their individual types (lymphocytes, neutrophils, eosinophils, monocytes), and the ratio of neutrophils to lymphocytes in domestic cat during early postnatal ontogenesis. The study was conducted on kittens living in outdoor conditions, aged from birth to 6 months. The number of white blood cells was evaluated using a hemoanalyzer, the leukocyte formula and the proportion of cell types were determined manually from blood smears. Age significantly affected the number of leukocytes in the first 3 months of kittens' age. The number and proportion of neutrophils were the highest after birth and gradually decreased during the first month. Lymphocytes number, on the contrary, increased during this period. Monocytes and eosinophils increased in number in the first 2 months. The litter size affected the number of leukocytes and neutrophils in the first 2 months of life, their number being significantly higher in kittens from the small litters than from the large ones. In kittens from the litters with multiple paternity, the number of leukocytes and the proportion of neutrophils was higher than in litters from a single male. Thus, age, litter size and type of paternity may affect the hematological indices in domestic cats, which must be taken into account during the estimation of the health status of kittens in domestic and wild cats.

Size Matters: Zoo Data Analysis Shows that the White Blood Cell Ratio Differs between Large and Small Felids.

The total number of white blood cells (WBCs) is related the immune system. In mammals, it is affected by the body mass, but it is unclear how the numbers of different WBC types correlate with this parameter. We analyzed the effect of body mass on WBC number and ratio in felids, where species are similar in diet (warm-blood vertebrates) and reproductive strategy (promiscuity). Based on zoo veterinary data (ZIMS database) we analyzed the effect of body mass on WBC number and neutrophils/lymphocytes ratio in 26 species of felids. The number of WBCs correlated with the body masses of animals: large cats had more WBC, which may be due to greater risks of infection associated with larger body surface, lifespan and home range size. For the first time we found obvious differences in the number of WBC types. Large cats also had more neutrophils and monocytes but fewer lymphocytes than smaller cats. The ratio of neutrophils to lymphocytes is greater in large felids. This phenomenon may be related to diet (relative prey size and kill utilization time), which suggests regular contact of large cats with bacterial and protozoal pathogens in contrast to the small cats.

Stressed by Maternity: Changes of Cortisol Level in Lactating Domestic Cats.

Lactation is the most energetically expensive component of maternal care in mammals. Increased reproductive investment can lead to physiological stress for the mothers, based on the exhaustion of energy resources and increase in glucocorticoids level. This study aimed to estimate the changes in cortisol concentrations during lactation in domestic cats and compared the differences among litter sizes. Eleven females gave birth to 27 litters, which were divided in two groups-small (1-3 kittens) and large (4-7 kittens) litters. Blood samples were collected from each female before mating, after parturition, at 4 and 8 weeks of lactation. We showed that the cortisol level in females changed significantly during lactation-the highest concentrations were observed at the peak of lactation at 4 weeks. Cortisol levels varied significantly among females but did not depend on their maternal experience. We also revealed that there were no differences in cortisol levels between females with small and large litters, but at 4 weeks of lactation, the hormone concentrations were higher in females with small litters. It is likely that these females initially invested less in reproduction, giving birth to fewer offspring.

Assessing the health risks of reintroduction: The example of the Amur leopard, Panthera pardus orientalis.

Translocation of wildlife as a means of reintroducing or reinforcing threatened populations is an important conservation tool but carries health risks for the translocated animals and their progeny, as well as wildlife, domestic animals and humans in the release area. Disease risk analyses (DRA) are used to identify, prioritize and design mitigation strategies to address these threats. Here, we use a DRA undertaken for Amur leopards (Panthera pardus orientalis) to illustrate how specific methodology can optimize mitigation strategy design. A literature review identified a total of 98 infectious hazards and 28 non-infectious hazards. Separate analyses were undertaken for disease risks in leopards from hazards of source origin (captive zoo collections and the transit pathway to the Russian Far East), or of destination origin (in breeding enclosures and wider release areas); and for disease risks in other wildlife, domesticated species or humans, similarly from hazards of source or destination origin. Hazards were assessed and ranked as priority 1, priority 2, priority 3 or low priority in each of the defined scenarios. In addition, we undertook a generic assessment of stress on individual leopards. We use three examples to illustrate the process: Chlamydophila felis, canine distemper virus (CDV) and feline immunodeficiency virus (FIV). We found that many potentially expensive screening procedures could be performed prior to export of leopards, putting the onus of responsibility onto the zoo sector, for which access to diagnostic testing facilities is likely to be optimal. We discuss how our methods highlighted significant data gaps relating to pathogen prevalence in the Russian Far East and likely future unpredictability, in particular with respect to CDV. There was emphasis at all stages on record keeping, meticulous planning, design, staff training and enclosure management, which are relatively financially inexpensive. Actions to minimize stress featured at all time points in the strategy and also focussed on planning, design and management.

Comparison of tigers' fecal glucocorticoids level in two extreme habitats.

Application of different antibodies and extraction methods results in a wide range of steroid metabolite concentrations obtained during noninvasive hormones monitoring. It makes regional comparisons of steroid concentration very difficult. We compared three methods for extraction of glucocorticoids metabolites in tiger feces to examine correct stress level in Bengal and Amur tigers in India and Russia respectively. The results obtained with three different extraction methods correlate with each other positively and significantly. The highest concentration of fecal glucocorticoids metabolites (FGCM) was found after the extraction of wet feces samples with 90% methanol. The level of FGCM was significantly higher in Bengal tigers in India than in Amur tigers in Russian Far East. The reasons might be related to tigers' density or anthropogenic pressure.

Physiological stress responses of tigers due to anthropogenic disturbance especially tourism in two central Indian tiger reserves.

Tigers continue to face unprecedented threats to their existence due to poaching, habitat loss, habitat fragmentation and anthropogenic disturbances. The present study examines the physiological stress response of tigers due to anthropogenic activities including wildlife tourism in Bandhavgarh Tiger Reserve and Kanha Tiger Reserve using faecal glucocorticoid metabolite (fGCM) measurement. We collected a total of 341 faecal samples from both reserves during tourism and non-tourism periods. Data on various anthropogenic disturbances including tourism activities like number of vehicles and visitors were also collected. We ascertained the species identity and sex of all the samples collected using genetic markers. fGCMs were extracted using a previously reported procedure, and fGCM concentrations were subsequently determined using an established enzyme immunoassay. There was no significant difference in overall mean fGCM concentrations between the two tiger reserves, but within each reserve, concentrations were significantly higher in tigers during the tourism period as compared to the non-tourism period. We also found that the number of tourist vehicles and disturbance level significantly correlated with fGCM concentrations. This study further supports the assumption that unbridled tourism associated with high anthropogenic disturbance can be related to perceived stress and consequently may have an impact on the reproductive fitness of tigers and long-term survival of isolated populations.

The method matters: The effect of handling time on cortisol level and blood parameters in wild cats.

Blood analysis has recently become a popular tool to assess the welfare of the wild cats. However, the estimates of blood parameters may depend on the sampling method. We have tested (1) if the sampling procedure influences blood parameters and (2) what parameters are the most efficient in assessing the physiological status in wild cat species. We assessed the effect of handling time on red blood cells (RBC) and white blood cells (WBC) counts, the ratio of neutrophils to lymphocytes (N/L ratio), and serum cortisol level within 1 hr after the capture of the animal in six far-east wild cats (Prionailurus bengalensis euptilura). Also, we analyzed literature data in 17 cat species to assess the effect of place of study, type of immobilization, and handling time on WBC count and N/L ratio. Serum cortisol level varied significantly with the handling time. RBC and WBC counts were strongly affected by the handling time. N/L ratio was very robust and did not depend on the handling time. However, the analysis of literature data has shown that the prolonged handling time (over 1 hr) and the type of immobilization significantly influence the N/L ratio, whereas the WBC count does not depend on any of considered factors. We conclude that while most blood parameters of cats are affected by routine handling time, the N/L ratio does not vary if the samples are collected within 1 hr after the capture of the animal. All other tested parameters should be treated with caution.

Investigations on reproductive physiology in the male Eurasian lynx (Lynx lynx).

This study characterized (in vivo) morphological and functional parameters of reproductive organs of adult male lynx (n = 3) prior to, during, and after the breeding season (n = 3). Size and morphology of the reproductive tract were monitored by transcutaneous (testes) and transrectal (accessory sex glands) ultrasonography. Semen was collected by electroejaculation. Ejaculate volume, sperm number, motility, and morphology of spermatozoa as well as testosterone concentrations in blood serum and feces were evaluated. The testes and prostate had seasonal changes in size and echotexture. The mean (+/- S.D.) maximum and minimum testicular volume were 2.8 +/- 0.8 cm3 and 1.5 +/- 0.3 cm3, respectively. Fecal testosterone concentrations were highest in February (1240 +/- 393 ng/g feces), with a second increase in May (971 +/- 202 ng/g feces), but concentrations were lowest in January (481 +/- 52.9 ng/g feces). Ejaculate volume, total sperm number and percentage of motile, and intact spermatozoa were maximal in March (the middle of the breeding season). In one of the eight litters, multiple paternity was proven; however, in the remaining seven litters, all 16 cubs were sired by the same male. This particular male had the most developed and active testes and best semen quality, which may be important for sperm competition.

Genetic structure of the Amur tiger (Panthera tigris altaica) population: Are tigers in Sikhote-Alin and southwest Primorye truly isolated?

We used molecular genetic analyses to noninvasively identify individual Amur tigers and define subpopulations of tigers in the Russian Far East. We identified 63 individuals after genotyping 256 feces, 7 hair and 11 blood samples collected within southern, central and northern Sikhote-Alin, as well as Southwest Primorye. Analysis of nuclear DNA at 9 microsatellite loci demonstrated greater genetic similarity between animals from southern and northern Sikhote-Alin (some 500 km apart) than between animals from Ussuriskii State Nature Reserve and Southwest Primorye (less than 10 km apart at their nearest point), suggesting that a true barrier exists preventing movements of tigers between Southwest Primorye and the southern Sikhote-Alin Mountains.