The natural cytotoxicity receptor genes in the family Felidae.

Natural killer (NK) cells belong to the innate immune system. The germline-encoded natural killer cell receptors represent activating and inhibitory receptors regulating multiple NK cell activities. The natural cytotoxicity receptors (NCRs) are activating natural cytotoxicity triggering receptors 1, 2, and 3 (NKp46, NKp44, and NKp30), encoded by the genes NCR1, NCR2, and NCR3, respectively. NCRs may be expressed in different cell types engaged in mechanisms of innate and adaptive immunity. The family Felidae, comprising the domestic cat and a wide variety of free-ranging species represents a well-suited model for biomedical and evolutionary studies. We characterized the NCR1, NCR2, and NCR3 genes in a panel of felid species. We confirmed the presence of potentially functional genes NCR1, NCR2, and NCR3 in all species. All three genes are conserved within the family and are similar to other phylogenetically related mammalian families. The NCR1 and NCR2 phylogenetic trees based on both nucleotide and protein sequences corresponded to the current zoological taxonomy, with some exceptions suggesting effects of different selection pressures in some species. Highly conserved NCR3 sequences did not allow a robust phylogenetic analysis. Most interspecific differences both at the nucleotide and protein level were found in NCR2. Within species, the most polymorphic CDS was detected in NCR1. Selection analyses indicated the effects of purifying selection on individual amino acid sites in all three genes. In stray cats, a rather high intraspecific diversity was observed.

Widespread anticoagulant poison exposure in predators in a rapidly growing South African city.

Anticoagulant rodenticides (ARs) are used worldwide to control rodent populations. ARs bioaccumulate across trophic levels and threaten non-target wildlife. We investigated the prevalence of AR exposure in seven predator species in the rapidly developing Greater Cape Town region of South Africa - a mosaic of natural, urban, and agricultural areas within a global biodiversity hotspot. We focused sampling on caracals (Caracal caracal, n = 28) as part of a larger caracal ecology study, but also opportunistically sampled Cape Clawless otters (Aonyx capensis, n = 9), large-spotted genets (Genetta tigrina, n = 4), honey badger (Mellivora capensis, n = 1), water mongoose (Atilax paludinosus, n = 1), small gray mongoose (Galerella pulverulenta, n = 1), and Cape Eagle owl (Bubo capensis, n = 1). We tested livers from all species, and blood from ten caracals, for eight AR compounds to assess prevalence and amount of exposure for each compound. We used generalized linear models to test spatial, demographic, and seasonal risk factors for ten measures of AR exposure in caracals. We detected at least one of the four most toxic AR compounds in six species. Exposure was high for caracals (92%) and all species combined (81%). For caracals, proximity to vineyards was the most important AR exposure risk factor. Vineyards in Cape Town do not use ARs to protect their vines but do host commercial hospitality structures where ARs are used. Vineyards may thus link caracals that forage within vineyards to the rat poisons used in and around their commercial structures. Residue levels were unexpected in large-spotted genets and Cape Clawless otters, suggesting invertebrate vectors. ARs may present a cryptic threat to populations already vulnerable to increasing habitat loss, vehicle collisions, poachers and fire. Targeted mitigation should include a mix of environmentally responsible policies that reduce AR use, particularly in areas near wildlife habitat.

Analysis of metabolic flux in felid spermatozoa using metabolomics and 13C-based fluxomics†.

Spermatozoa from three feline species-the domestic cat (Felis catus), the cheetah (Acinonyx jubatus), and the clouded leopard (Neofelis nebulosa)-were analyzed using metabolomic profiling and 13C-based fluxomics to address questions raised regarding their energy metabolism. Metabolic profiles and utilization of 13C-labeled energy substrates were detected and quantified using gas chromatography-mass spectrometry (GC-MS). Spermatozoa were collected by electroejaculation and incubated in media supplemented with 1.0 mM [U13C]-glucose, [U13C]-fructose, or [U13C]-pyruvate. Evaluation of intracellular metabolites following GC-MS analysis revealed the uptake and utilization of labeled glucose and fructose in sperm, as indicated by the presence of heavy ions in glycolytic products lactate and pyruvate. Despite evidence of substrate utilization, neither glucose nor fructose had an effect on the sperm motility index of ejaculated spermatozoa from any of the three felid species, and limited entry of pyruvate derived from these hexose substrates into mitochondria and the tricarboxylic acid cycle was detected. However, pathway utilization was species-specific for the limited number of individuals (four to seven males per species) assessed in these studies. An inhibitor of fatty acid beta-oxidation (FAO), etomoxir, altered metabolic profiles of all three felid species but decreased motility only in the cheetah. While fluxomic analysis provided direct evidence that glucose and fructose undergo catabolic metabolism, other endogenous substrates such as endogenous lipids may provide energy to fuel motility.

Sex and species differences of stress markers in sympatric cheetahs and leopards in Namibia.

Physiological stress markers may provide valuable insight for our understanding of costs of given life-history strategies or of wildlife health condition, most importantly in case of threatened species. In the last decade, there has been growing interest in the ecological relevance of cellular oxidative stress, which would provide complimentary information to that obtained by the classic analyses of glucocorticoid hormones. In this study, we analysed the sex and species variation of five blood-based markers of oxidative status, both molecular oxidative damage and antioxidant protection, in sympatric cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) living on Namibian farmlands. Both these terrestrial carnivores are classified as vulnerable by the International Union for Conservation of Nature. We found that female cheetahs had significantly higher serum reactive oxygen metabolites of non-protein origin and lower glutathione peroxidase activity in whole blood than both male and female leopards and male cheetahs. We also found that cheetahs and leopards differed in the association between the two antioxidant enzymes glutathione peroxidase and superoxide dismutase. Correlations among oxidative status markers were stronger in female cheetahs than leopards or male cheetahs. Our results suggest that female cheetahs are more sensitive to local sources of stress. Our work did not corroborate the assumption that two species with different life histories consistently differ in key physiological traits.

What makes a feline fatal in Toxoplasma gondii's fatal feline attraction? Infected rats choose wild cats.

Toxoplasma gondii is an indirectly transmitted protozoan parasite, of which members of the cat family (Felidae) are the only definitive hosts and small mammals such as rats serve as intermediate hosts. The innate aversion of rodents to cat odor provides an obstacle for the parasite against successful predation by the feline definitive host. Previous research has demonstrated that T. gondii appears to alter a rat's perception of the risk of being preyed upon by cats. Although uninfected rats display normal aversion to cat odor, infected rats show no avoidance and in some cases even show attraction to cat odor, which we originally termed the "Fatal Feline Attraction." In this study, we tested for the first time whether the "Fatal Feline Attraction" of T. gondii-infected rats differed according to the type of feline odor used, specifically whether it came from domestic cats (Felis silvestris catus) or wild cats-cheetahs (Acinonyx jubatus) or pumas (Felis concolor). In two-choice odor trials, where wild and domestic cat odors were competed against one another, consistent with previous findings we demonstrated that infected rats spent more time in feline odor zones compared with uninfected rats. However, we further demonstrated that all cat odors are not equal: infected rats had a stronger preference for wild cat odor over that of domestic cats, an effect that did not differ significantly according to the type of wild cat odor used (cheetah or puma). We discuss these results in terms of the potential mechanism of action and their implications for the current and evolutionary role of wild, in addition to domestic, cats in transmission of T. gondii.

Fecal endocrine monitoring of reproduction in female snow leopards (Uncia uncia).

Although the snow leopard (Uncia uncia) is a common endangered felid species in zoos, little is known about the complex endocrine interactions controlling ovarian function and conception in this species. The goal of this work was to characterize ovarian activity throughout the estrous cycle, nonpregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. This goal was accomplished using an enzyme immunoassay to measure fecal concentrations of estrogen metabolites (E) and progesterone metabolites (P). Fecal samples were collected from 12 female snow leopards (ages 18 months to 18 years) during one to three breeding seasons. In each breeding season, the majority of females (78%, 88%, and 100%, respectively) began to exhibit ovarian activity in December or January. The estrous cycle, defined by the first day of estrus (E ≥ 2 × basal concentration) to the first day of the subsequent estrus, was 12.7 ± 0.6 days (n = 145 cycles). Estrus lasted 4.3 ± 0.4 days with mean concentrations of fecal E during the follicular phase (1661 ± 139 ng/g feces) increasing 3.2-fold above basal concentrations (515 ± 32 ng/g feces). No spontaneous ovulations were observed in any of the cycling females. Nonpregnant luteal phases were observed in eight females that bred but did not become pregnant. The length of the nonpregnant luteal phase ranged from 11 to 72 days (45.7 ± 5.7 days; n = 10) with mean concentrations of fecal P during the luteal phase (12.46 ± 1.7 µg/g feces) increasing 6.2-fold above basal concentrations of P (2.01 ± 0.2 µg/g feces). Three of the females in the study became pregnant and gave birth after a gestation of 93 (n = 2) and 95 (n = 1) days. Fecal P concentrations during pregnancy increased to 11.64 ± 1.3 µg/g feces, or 5.8-fold above basal concentrations. The results of this study provide a comprehensive characterization of reproductive endocrinology in snow leopards, and confirm that fecal hormone monitoring is an effective way to monitor female snow leopards throughout the breeding season.

Comparative metabolism of PGFM (13,14-dihydro-15-keto-PGF2α) in feces of felids.

Methods for monitoring endocrine activities are useful tools for reproduction management. In particular, captive breeding of endangered felid species is considered to be an important part of the species conservation efforts. Within breeding programs, reliable methods for pregnancy diagnosis are highly demanded to prevent peri- and postpartal losses, but pregnancy diagnosis based on gestagen metabolites in felids is hampered by pseudopregnancies. Recently, we described fecal PGFM as an indicator for pregnancy in several feline species, but peak levels of PGFM secretion differed dramatically between species. It is believed that prostaglandin composition and metabolism pathways may differ as well. Therefore, a study was devised to both compare various fecal immunoreactive PGFM metabolites and to identify prostaglandins in fecal extracts by liquid chromatography-mass spectrometry (LCMS). Our results confirmed that fecal metabolite patterns differ between feline species. The identity of PGFM was confirmed in six of eight felids. In Iberian lynx and the Sumatran tiger, PGFM did not exceed 5% of all immunoreactivities. The total number of immunoreactivities varied between two (e.g., domestic cat) and four (e.g., oncilla). Several prostaglandins were identified by LCMS; apart from PGFM, all LCMS-identified prostaglandins, including tetranor-PGFM, did not show any cross-reactivity with our PGFM-specific antibody. This indicates the existence of still unknown eicosanoids and further studies are needed to clarify the origin of the different metabolites. Although differing stages of pregnancy did not reveal significant differences in the composition of metabolites, we could not exclude the possibility that metabolites from other prostaglandins (e.g. PGE2) contributed to the fecal metabolite patterns.

Specifying and sustaining pigmentation patterns in domestic and wild cats.

Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.

High proportion of male faeces in jaguar populations.

Faeces provide relevant biological information which includes, with the application of genetic techniques, the sex and identity of individuals that defecated, thus providing potentially useful data on the behaviour and ecology of individuals, as well as the dynamics and structure of populations. This paper presents estimates of the sex ratio of different felid species (jaguar, Panthera onca; puma, Puma concolor; and ocelot/margay, Leopardus pardalis/Leopardus wiedi) as observed in field collected faeces, and proposes several hypotheses that could explain the strikingly high proportion of faeces from male jaguars. The proportion of male and female faeces was estimated using a non-invasive faecal sampling method in 14 study areas in Mexico and Brazil. Faecal samples were genetically analysed to identify the species, the sex and the individual (the latter only for samples identified as belonging to jaguars). Considering the three species, 72.6% of faeces (n = 493) were from males; however, there were significant differences among them, with the proportion from males being higher for jaguars than for pumas and ocelots/margays. A male-bias was consistently observed in all study areas for jaguar faeces, but not for the other species. For jaguars the trend was the same when considering the number of individuals identified (n = 68), with an average of 4.2±0.56 faeces per male and 2.0±0.36 per female. The observed faecal marking patterns might be related to the behaviour of female jaguars directed toward protecting litters from males, and in both male and female pumas, to prevent interspecific aggressions from male jaguars. The hypothesis that there are effectively more males than females in jaguar populations cannot be discarded, which could be due to the fact that females are territorial and males are not, or a tendency for males to disperse into suboptimal areas for the species.

Using PGFM (13,14-dihydro-15-keto-prostaglandin F2α) as a non-invasive pregnancy marker for felids.

Understanding the complex endocrine interactions that control reproduction in felids is essential for captive breeding management. The most important demand is a quick and reliable pregnancy diagnosis. However, the occurrence of pseudopregnancies in felids complicates matters. We investigated whether the fecal prostaglandin metabolite (PGFM) recently suggested for pregnancy diagnosis in the lynx is suitable for all felid species. We found that increased levels of PGFM during the last trimester indicate pregnancy in seven of the eight main lineages of the carnivore family Felidae. PGFM levels in a sand cat (domestic cat lineage) were basal at mating and remained so until Day 40 post-mating. Day 41 marked the beginning of a distinct increase culminating in peak levels of 6.5 µg/g before parturition and decreasing again to baseline thereafter. Similar pregnancy profiles were obtained from the domestic cat, the leopard cat, the lynx, the ocelot and the caracal lineage, whereas in pseudopregnant individuals (sand cat, Iberian and Eurasian lynx) fecal PGFM remained at basal levels. In pregnant cheetahs (puma lineage) PGFM increased above basal following day ∼48 peaking before pregnancy but remained at baseline in pseudopregnant females. Discrepancies existed in the Panthera lineage. While Chinese leopard, Sumatran tiger, and the black panther showed marked increases of PGFM during the last weeks of pregnancy, only moderate increases in PGFM levels were found in the Indochinese tiger and the Persian leopard. Altogether, PGFM as tool for pregnancy diagnosis has been proven to be useful in breeding management of felids.

Quantification and molecular characterization of the feline leukemia virus A receptor.

Virus receptors and their expression patterns on the cell surface determine the cell tropism of the virus, host susceptibility and the pathogenesis of the infection. Feline thiamine transport protein 1 (fTHTR1) has been identified as the receptor for feline leukemia virus (FeLV) A. The goal of the present study was to develop a quantitative, TaqMan real-time PCR assay to investigate fTHTR1 mRNA expression in tissues of uninfected and FeLV-infected cats, cats of different ages, in tumor tissues and leukocyte subsets. Moreover, the receptor was molecularly characterized in different feline species. fTHTR1 mRNA expression was detected in all 30 feline tissues investigated, oral mucosa scrapings and blood. Importantly, identification of significant differences in fTHTR1 expression relied on normalization with an appropriate reference gene. The lowest levels were found in the blood, whereas high levels were measured in the oral mucosa, salivary glands and the musculature. In the blood, T lymphocytes showed significantly higher fTHTR1 mRNA expression levels than neutrophil granulocytes. In vitro activation of peripheral blood mononuclear cells with concanavalin A alone or followed by interleukin-2 led to a transient increase of fTHTR1 mRNA expression. In the blood, but not in the examined tissues, FeLV-infected cats tended to have lower fTHTR1 mRNA levels than uninfected cats. The fTHTR1 mRNA levels were not significantly different between tissues with lymphomas and the corresponding non-neoplastic tissues. fTHTR1 was highly conserved among different feline species (Iberian lynx, Asiatic and Indian lion, European wildcat, jaguarundi, domestic cat). In conclusion, while ubiquitous fTHTR1 mRNA expression corresponded to the broad target tissue range of FeLV, particularly high fTHTR1 levels were found at sites of virus entry and shedding. The differential susceptibility of different species to FeLV could not be attributed to variations in the fTHTR1 sequence.

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus).

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17ß and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3ß-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17ß concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.

Vif of feline immunodeficiency virus from domestic cats protects against APOBEC3 restriction factors from many felids.

To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

Stress in wildlife species: noninvasive monitoring of glucocorticoids.

Depression and stress are related pathologies extensively studied in humans. However, this relationship is not well known in animals kept in zoos and even less known in wild animals. In zoo animals, acute and chronic stress caused by difficulties in coping with stressors such as public presence and noise, among others, can induce the appearance of repetitive pathological behaviors such as stereotypies, many times associated with organic pathologies that deeply affect their health and welfare. In the wild, factors such as deforestation, habitat fragmentation, lack of food and water, and human disturbances are potential causes of acute and chronic stress for the resident fauna. Glucocorticoids (GC) have been extensively used as stress indicators in many species including humans. Since chase and handling of wild animals immediately raise their GC serum levels, noninvasive methods have been developed to assess stress without interference caused by sample collection. The hormones and their metabolites can be measured in various body fluids and excreta and detect basal feedback free hormone concentrations as well as the response to ACTH and handling. In order to study the influence of disturbing factors we have measured GC as stress indicators by noninvasive techniques in dolphins and felids (ocelots, jaguarundis and margays) and cortisol and testosterone in spider monkeys.

Frustrated appetitive foraging behavior, stereotypic pacing, and fecal glucocorticoid levels in snow leopards (Uncia uncia) in the Zurich Zoo.

This study hypothesized that permanently frustrated, appetitive-foraging behavior caused the stereotypic pacing regularly observed in captive carnivores. Using 2 adult female snow leopards (Uncia uncia), solitarily housed in the Zurich Zoo, the study tested this hypothesis experimentally with a novel feeding method: electronically controlled, time-regulated feeding boxes. The expected result of employing this active foraging device as a successful coping strategy was reduced behavioral and physiological measures of stress, compared with a control-feeding regime without feeding boxes. The study assessed this through behavioral observations and by evaluating glucocorticoid levels noninvasively from feces. Results indicated that the 2 snow leopards did not perform successful coping behavior through exercising active foraging behavior or through displaying the stereotypic pacing. The data support a possible explanation: The box-feeding method did not provide the 2 snow leopards with the external stimuli to satisfy their appetitive behavioral needs. Moreover, numerous other factors not necessarily or exclusively related to appetitive behavior could have caused and influenced the stereotypic pacing.

Species-, sex-, and age-dependent urinary excretion of cauxin, a mammalian carboxylesterase.

Domestic cats exhibit physiological proteinuria due to the excretion of cauxin, a carboxylesterase, into the urine. In the present report, we demonstrate that cauxin is excreted in a species-, sex-, and age-dependent manner. Although the cauxin gene is conserved in mammals, including human, mouse, and dog, urinary cauxin was found only in member of the genus Felis and lynx (bobcat, and lynx) and not in other Felidae (genus: Panthera and puma) tested. In mature cats, cauxin excretion was higher in intact males than in castrated males or in intact or spayed females. Daily cauxin excretion decreased immediately after castration. Immunohistochemistry confirmed that cauxin expression in the kidney proximal straight tubules was higher in intact males than in castrated males. Urinary cauxin was detectable by Western blotting in cats older than about 3 months, and its excretion increased with age. In a zymographic esterase assay, urine contained a major cauxin band; by contrast, kidney homogenates contained three major bands, comprising two carboxylesterases and an unidentified esterase, and one minor cauxin band. These results suggest that 1. cauxin excretion is regulated by sex hormones, such as testosterone, 2. cauxin functions as an esterase in the urine rather than in kidney cells, and 3. the decomposition products by cauxin are excreted in a species-, sex-, and age-dependent manner, as is cauxin itself.

Transitional cell carcinoma in fishing cats (Prionailurus viverrinus): pathology and expression of cyclooxygenase-1, -2, and p53.

A high prevalence of urinary bladder transitional-cell carcinoma (TCC) has been noted in captive fishing cats (Prionailurus viverrinus). Of the 91 adult deaths between 1995 and 2004, 12 (13%) were attributed to TCC. To help elucidate mechanisms of carcinogenesis, archival sections of urinary bladder from 14 fishing cats were examined histologically and immunohistochemically for p53, cyclooxygenase (COX)-1, and COX-2 expression. Ten cats had TCC, and 4 were unaffected. The average age at death was 10.8 years in affected individuals and 10.5 years in unaffected individuals. There was no sex predilection. Fishing cat TCCs were characterized histologically as papillary and infiltrating (n = 6), nonpapillary and infiltrating (n = 3), or carcinoma in situ (n = 1). Glandular and squamous metaplasia, necrosis, and lymphatic invasion were prominent histologic features. Two individuals had documented metastasis. p53 nuclear immunolabeling was detected in 4/10 (40%) TCCs. In two cases, immunolabeling was limited to less than 10% of the neoplastic cellular population and was comparable to staining of normal fishing cat bladder. Therefore, p53 gene mutation did not appear to be an essential component of TCC carcinogenesis in examined fishing cats. COX-1 immunohistochemistry was negative in all cases. All TCCs had some degree of COX-2 cytoplasmic immunolabeling, which was exclusively within the invasive portions of the neoplasms. Papillary portions were uniformly negative. COX-2 overexpression was a prominent feature in the majority of the examined fishing cat TCCs, suggesting that COX-2-mediated mechanisms of carcinogenesis are important in this species and that COX-inhibiting drugs may be of therapeutic benefit.