Age estimation using methylation-sensitive high-resolution melting (MS-HRM) in both healthy felines and those with chronic kidney disease.

Age is an important ecological tool in wildlife conservation. However, it is difficult to estimate in most animals, including felines-most of whom are endangered. Here, we developed the first DNA methylation-based age-estimation technique-as an alternative to current age-estimation methods-for two feline species that share a relatively long genetic distance with each other: domestic cat (Felis catus; 79 blood samples) and an endangered Panthera, the snow leopard (Panthera uncia; 11 blood samples). We measured the methylation rates of two gene regions using methylation-sensitive high-resolution melting (MS-HRM). Domestic cat age was estimated with a mean absolute deviation (MAD) of 3.83 years. Health conditions influenced accuracy of the model. Specifically, the models built on cats with chronic kidney disease (CKD) had lower accuracy than those built on healthy cats. The snow leopard-specific model (i.e. the model that resets the model settings for snow leopards) had a better accuracy (MAD = 2.10 years) than that obtained on using the domestic cat model directly. This implies that our markers could be utilised across species, although changing the model settings when targeting different species could lead to better estimation accuracy. The snow leopard-specific model also successfully distinguished between sexually immature and mature individuals.

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.

Partial replacement of dietary buffalo meat on the bone with chicken carcass improves serum antioxidant profile of zoo-housed Indian leopards (Panthera pardus fusca).

This experiment was conducted to study the effect of gradual replacement of dietary buffalo meat on the bone (BMB) with chicken carcass (CC) on nutrient utilization, serum cortisol, and total serum antioxidant profile of zoo-housed Indian leopard. Twelve adult leopards were randomly distributed into a replicated Latin square design comprising three treatments, three periods, four animals, and three sequences. Leopards in group T1 were fed normal zoo diet of BMB. On the basis of dry matter, 10% and 20% of BMB was replaced with CC in groups T2 and T3 , respectively. Each experimental period comprised 21 days. During each period, a digestion trial of 4-day collection period was conducted after an adaptation period of 17 days. On Day 21 of each experimental period, blood was collected from all the animals by puncturing the ventral coccygial vein. Intake and apparent digestibility of major nutrients were similar among the groups. Replacement of 20% BMB with addition of CC increased (p < 0.001) the calculated supply of I, niacin, and vitamin A. Carotenoid intake increased (p < 0.01) with increased level of CC in the diet. Serum concentration of cortisol decreased (p < 0.01) whereas serum concentration of total carotenoids increased (p < 0.001) with increased level of CC in the diet. Serum concentration of antioxidant enzymes increased (p < 0.001) with increased level of CC in the diet. It was concluded that replacement of 20% of BMB with CC increased antioxidant profile. This may reduce oxidative stress in zoo-housed Indian leopards without any adverse effect on nutrient utilization.

Blood meal identification in the cryptic species Anopheles gambiae and Anopheles coluzzii using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged in entomology as a technique to identify arthropods and their blood meal source. In this study, female Anopheles gambiae were fed on five host blood sources: ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) and Hamadryas baboon (Papio hamadryas), while Anopheles coluzzii were fed on three hosts: dromedary (Camelus dromedarius), Barbary sheep (Ammotragus lervia) and pig (Sus scrofa). We obtained the MS spectra from 240 engorged mosquito abdomens and selected high quality ones from 72 mosquito abdomens to upgrade our home-made database. We excluded from the analysis any spectra of low quality (n = 80), and the remaining 88 specimens were subjected to a blind test analysis against the home-made database. We obtained 100% correct identification of the blood meal source for the specimens collected, 1, 12 and 24 h post-feeding, whereas for the specimens collected 36 h post-feeding, the correct identification rate decreased dramatically. We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes, which opens new perspectives for further studies, including the impact of the mosquito species on blood meal identification.

TITLE: Identification du repas sanguin des espèces cryptiques Anopheles gambiae et Anopheles coluzzii par l'utilisation de MALDI-TOF MS. ABSTRACT: L'identification par spectrométrie de masse à temps de vol par désorption/ionisation assistée par matrice (MALDI-TOF MS) a récemment émergé en entomologie pour l'identification des arthropodes et de leur source de sang. Des femelles d'Anopheles gambiae ont été nourries de sang de cinq hôtes, ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) et babouin Hamadryas (Papio hamadryas), et des femelles d'Anopheles coluzzii ont été nourries sur trois hôtes, dromadaire (Camelus dromedarius), mouflon à manchettes (Ammotragus lervia) et porc (Sus scrofa). Nous avons obtenu les spectres MS à partir de 240 abdomens de moustiques engorgés et avons sélectionné ceux de 72 abdomens de moustiques de haute qualité pour améliorer notre base de données maison. Nous avons exclu de l'analyse les spectres de faible qualité (n = 80) et les 88 échantillons restants ont été soumis à une analyse de test en aveugle contre la base de données maison. Nous avons obtenu 100 % d'identification correcte de la source de sang pour les échantillons collectés, 1, 12 et 24 heures après l'alimentation, mais le taux d'identification correct a diminué de façon spectaculaire pour les échantillons collectés 36 heures après l'alimentation. Nous confirmons ici que la MALDI-TOF MS peut être utilisée pour identifier l'origine des repas sanguins des moustiques fraîchement engorgés et ouvre de nouvelles perspectives pour d'autres études, y compris l'impact des espèces de moustiques sur l'identification des repas sanguins.

Effect of feeding Jerusalem artichoke (Helianthus tuberosus) root as prebiotic on nutrient utilization, fecal characteristics and serum metabolite profile of captive Indian leopard (Panthera pardus fusca) fed a meat-on-bone diet.

An experiment was conducted to determine the effect of incorporating Jerusalem artichoke (JA) as a prebiotic in the diet of Indian leopards (n = 11 adults) fed a meat-on-bone diet. The trial consisted of three periods (A1 , B, and A2 ). Each period comprised 17 days of adaptation and four days of collection. During the control periods (A1 and A2 ), the leopards were fed their normal zoo diets of 2.5-3 kg of buffalo meat-on-bone six days a week without any supplement. During trial B, meat-on-bone diets of the leopards were supplemented with JA at 2% of dietary dry matter (DM). Meat consumption was similar among the treatments. Supplementation of JA decreased the digestibility of crude protein (P < 0.01). Digestibilities of organic matter and ether extract were similar among the treatments. Serum concentrations of urea and triglycerides were lower (P < 0.05) when JA was added to the diet. Incorporation of JA to the basal diet increased fecal concentrations of acetate (P < 0.01), butyrate (P < 0.01), lactate (P < 0.01), Lactobacillus spp., and Bifidobacterium spp. (P < 0.01) with a simultaneous decrease in the concentration of ammonia (P < 0.01), Clostridia spp. (P < 0.01), and fecal pH (P < 0.01). Fecal microbial profiles and hind gut fermentation were improved, without any adverse effects on feed consumption, nutrient utilization, and serum metabolite profiles. Results of this experiment showed that feeding JA at 2% DM in the whole diet could be potentially beneficial for captive Indian leopards fed meat-on-bone diets.

Hematology and serum chemistry of free-ranging jaguars (Panthera onca).

We collected and analyzed blood samples from 12 free-ranging jaguars (Panthera onca). Clinical examinations, hematology, and serum chemistry indicate the jaguars were in good overall health. Results may help as values for free-ranging jaguars under the same handling conditions.

Tick-borne bacteria in free-living jaguars (Panthera onca) in Pantanal, Brazil.

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer ≥ 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.