Evolution of the germline mutation rate across vertebrates.

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself1. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies2. Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent-offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis3. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates.

Polar bear-adapted Ursidibacter maritimus are remarkably conserved after generations in captivity.

Most species in the bacterial family of Pasteurellaceae colonize one specific host species. Vertebrates of very different evolutionary descent including fish, turtles, marsupials, eutherians and birds are colonized by different members of Pasteurellaceae. This one-to-one microbial-host species partnership makes Pasteurellaceae species valuable candidates to study biodiversity, bacterial-host co-evolution and host adaptation, and their widespread distribution across vertebrates provide the possibility to collect a wide array of data, where wildlife species are essential. However, obtaining samples from wild animals comes with logistic, technical and ethical challenges, and previous microbiota studies have led to the presumption that captive animals are poor models for microbial studies in wildlife. Here, we show that colonization of polar bears by Ursidibacter maritimus is unaffected by factors related to captivity, reflecting a deep symbiotic bond to the host. We argue that the study of ecological and evolutionary principles in captive wildlife is possible for host-adapted taxa such as those in the Pasteurellaceae family. Moreover, studying captive, often trained animals protects wild populations from the stress associated with obtaining samples.

PREVALENCE OF SALMONELLA SPECIES, CLOSTRIDIUM PERFRINGENS, AND CLOSTRIDIUM DIFFICILE IN THE FECES OF HEALTHY ELEPHANTS (LOXODONTA SPECIES AND ELEPHAS MAXIMUS) IN EUROPE.

Pathogenic Salmonella spp., Clostridium perfringens, and Clostridium difficile have been reported to infect and cause severe enteritis and enterotoxemia in African (Loxodonta spp.) and Asian elephants (Elephas maximus). However, little information exists on whether healthy elephants carry and possibly shed these gastrointestinal organisms. This study was conducted to investigate the prevalence of all three bacteria in feces from healthy elephants in European zoos. Bacterial identification was performed by selective culture on fecal samples and a polymerase chain reaction (PCR) amplification protocol, on the basis of primers targeting the hilA gene (Salmonella spp.), the cpa gene (C. perfringens), and the tpi gene (C. difficile) from deoxyribonucleic acid extracted from elephant feces. The PCR protocol was validated prior to initiation of the investigation. Fecal samples collected from 50 African and 86 Asian elephants originating from 30 European zoologic institutions were investigated. The PCR validation revealed detection limits ranging from 104 to 106 colony-forming units per gram of feces of each gene. Only C. perfringens (one type A and two type E) was detected in the initial sampling (2.2%, three Asian elephants), whereas no Salmonella spp. or C. difficile was detected. At a follow-up sampling from C. perfringens-positive animals and relatives, 2 mo after the initial sampling, three animals were culture positive for Salmonella enterica spp. enterica. All positive samples were obtained with bacterial culture, whereas no PCR reactions were positive. Despite carrying these pathogens, all culture-positive animals were clinically healthy and did not develop signs of gastrointestinal disease during the study period. The findings indicate that prevalence of Salmonella spp., C. perfringens, and C. difficile in feces from healthy Asian and African elephants in Europe is very low.

Alfaxalone Sedation in Black-cheeked Lovebirds (Agapornis nigrigenis) for Non-invasive Procedures.

Alfaxalone is an injectable neuroactive steroid anesthetic that is becoming more widely used as a sedative in a wide range of animals. The purpose of this study was to evaluate the efficacy of this drug for sedation during handling and noninvasive medical procedures in black-cheeked lovebirds (Agapornis nigrigenis). Based on a pilot study that showed that 5 mg/kg alfaxalone was inadequate, and that 20 mg/kg resulted in respiratory arrest in 1 bird, the effects of 12.6 ± 0.9 mg/kg alfaxalone administered subcutaneously was investigated in 9 birds. Despite minor movements and twitching, it was possible to handle all birds and to perform positioning for a ventrodorsal radiograph. A loss of reaction to noxious stimuli was not achieved during sedation. Times from injection to initial effect (mean ± SD) was 93 ± 48 seconds; to recumbency, 209 ± 70 seconds; to first handling for positioning the bird in lateral recumbency, 251 ± 68 seconds; to initial righting effort, 55 ± 8 minutes; and to perching for a minimum of 20 seconds, 76 ± 7 minutes. Median respiration rates between 5 to 45 minutes were 36 to 40 breaths/min; apnea was not noted in any bird. Birds received 0.5 L of oxygen/min via face mask. Oxygen saturation (SpO2) and pulse rate were measured via pulse oximetry in 8 birds continuously from 10 to 30 minutes, SpO2 values remained above 90%. During sedation, mean pulse rate decreased significantly over time (P = .007; 10 minutes = 409 ± 81 beats/min; 30 minutes = 324 ± 25 beats/min). The majority of birds had rough inductions and recoveries, which could have been minimized if birds had been placed in a more confined space. In summary, 12.6 mg/kg alfaxalone provided nearly 1 hour of stable, nonanalgesic sedation appropriate for noninvasive procedures in black-cheeked lovebirds.

AMYLOIDOSIS IN CARACALS (CARACAL CARACAL).

Nine cases of amyloidosis in caracals (Caracal caracal) from three different institutions in Europe were reviewed and evaluated histopathologically. The six males and three females died between 2008 and 2018 at an age of 6 yr ± 2.5 mo (median ± interquartile range). In two out of nine (2/9) animals, amyloidosis was an incidental postmortem finding; the animals died of bronchopneumonia and gastric ulceration due to Helicobacter spp., respectively. Seven (7/9) animals suffered from acute renal failure due to amyloidosis, one of them additionally of cardiac decompensation. The predominant clinical signs were weight loss, lethargy, dys- or anorexia, dehydration, increased BUN and creatinine, and azotemia. The main gross lesion was a pale renal cortex on cut surface; in two animals, the kidneys appeared enlarged. Histologically, glomerular amyloid was present in every animal (9/9), and was the predominant renal manifestation of amyloidosis. Additional findings included splenic amyloid (8/8), amyloid in the lamina propria of the intestine (5/5), and amyloid in the lingual submucosa (4/4). Gastric mineralization was present in four animals suffering from renal failure. In the animal dying from bronchopneumonia, severe pancreatic amyloid deposits mainly affecting the exocrine pancreas (1/5) were identified. Immunohistochemistry was employed to identify amyloid AA in eight cases; only in the caracal dying from bronchopneumonia AA was amyloid confirmed. In several organs, especially in those where only small amyloid deposits were detected, a Congo red stain was often necessary to confirm the deposition. The etiology of the amyloidosis remains unknown. Three caracals were related within two generations, another three within four generations, so one might hypothesize a familial trait. In conclusion, amyloidosis should be considered as a significant disease in the caracal. Particularly in cases with renal disease, it should be included as a major differential diagnosis.

The effects of morphine on gas exchange, ventilation pattern and ventilatory responses to hypercapnia and hypoxia in dwarf caiman (Paleosuchus palpebrosus).

Morphine and other opioids cause respiratory depression in high doses and lower the ventilatory responses to hypoxia and hypercapnia in mammals. Recent studies indicate that turtles respond similarly, but although they are used routinely for post-surgical analgesia, little is known about the physiological effects of opioids in reptiles. We therefore investigated the effects of morphine (10 and 20 mg kg-1) on gas exchange and ventilation in six dwarf caiman (Paleosuchus palpebrosus) using pneumotachography in a crossover design. Intraperitoneal injections of morphine changed the ventilation pattern from a typical intermittent/periodic pattern with a few or several breaths in ventilatory bouts to single breaths and prolonged the apnoea, such that respiratory frequency was depressed, while tidal volume was elevated. Furthermore, the duration of inspiration and especially expiration was prolonged. The resulting decrease in minute ventilation was attended by a lowering of the respiratory exchange ratio (RER) (especially for 20 mg kg-1 dose) indicating CO2 retention with a long time constant for approaching the new steady state. The changes in ventilation pattern and gas exchange reached a new stable level approximately 3 h after the morphine injection and did not significantly affect steady state O2 uptake, i.e. O2 consumption. As expected, the ventilatory response to 5% O2 was lower in morphine-treated caimans, but minute ventilation upon exposure to 2% CO2 did not differ significantly different from control animals.

EVALUATION OF TWO ENZYME-LINKED IMMUNOSORBENT ASSAYS MEASURING PREGNANCY-ASSOCIATED GLYCOPROTEINS IN THE BLOOD OF MUSKOXEN ( OVIBOS MOSCHATUS).

Pregnancy-associated glycoproteins (PAGs) are expressed by the ruminal placenta, making their detection in blood an accurate indicator of pregnancy. This study aimed to evaluate two commercially available PAG enzyme-linked immunosorbent assays (ELISAs) in muskoxen ( Ovibos moschatus). The two tests are based on the same principles; however, one is evaluated photometrically and the other visually. Sixteen samples covering all trimesters of pregnancy, and 16 nonpregnant samples were included to evaluate test performance. Both tests reliably detected pregnancy. The photometric ELISA showed a sensitivity and specificity of 94% and 100%, respectively. Although the visual ELISA depends on somewhat subjective interpretations, it came up with a sensitivity of 81% and a specificity of 100%, and might thus provide a useful in-house tool when limited laboratory equipment is available. Analysis of additional samples showed consistent results during pregnancy and circulating PAGs for at least 18 days postpartum.

THE ACUTE-PHASE AND HEMOSTATIC RESPONSE IN DROMEDARY CAMELS ( CAMELUS DROMEDARIUS).

Acute-phase reactants indicate inflammation and are increasingly used in veterinary medicine to indicate and to monitor progression of disease. Hemostasis and inflammation have interconnected pathophysiologic pathways and influence each other on different levels. This study established observed normal ranges for acute-phase reactants and for coagulation and thromboelastographic (TEG) parameters in 49 dromedary camels ( Camelus dromedarius) and assessed the response to chronic and acute inflammation. Chronically infected animals suffering from lymph abscessation due to Corynebacterium spp. had significantly higher concentrations of the acute-phase reactants haptoglobin ( P < 0.005) and fibrinogen ( P < 0.013) and an increased clot strength characterized by an increase of the TEG parameters MA ( P < 0.039), representing the maximum amplitude of the clot strengths, and G, the global clot strength ( P < 0.022), compared to healthy animals. When the acute-phase and hemostatic responses of 10 males receiving a gonadotropin-releasing hormone vaccine and of 9 males that were surgically castrated over 7 days were studied, haptoglobin proved to be a minor positive acute-phase protein, with moderate levels in healthy animals. It increased significantly after both vaccination and castration and remained elevated 7 days postinsult. The negative reactant iron significantly decreased over the 7-day period after castration, whereas a similar decrease following vaccination lasted less than 3 days. Fibrinogen reacted as a positive, minor reactant, with a significant increase and a peak on days 3-5, with higher values seen after castration. Prothrombin time showed a slight shortening at days 5-7, and the TEG parameters MA and G showed significantly increased values, similar to fibrinogen. The acute-phase protein serum amyloid A showed poor repeatability, suggesting that the assay was not reliable.

Morphology of the snake spectacle reflects its evolutionary adaptation and development.

BACKGROUND: Covering the eye of all snakes is a transparent integumental structure known as the spectacle. In order to determine variations in spectacle thickness among species, the spectacles of 217 alcohol-preserved museum specimens of 44 species belonging to 14 different families underwent optical coherence tomography (OCT) to measure spectacular thickness. Multivariable analyses were made to determine whether family, activity period (diurnal/nocturnal) and habitat (arboreal/terrestrial/fossorial/aquatic) influenced spectacle thickness. RESULTS: The thinnest spectacles in absolute terms were found in the Usambara bush viper (Viperidae) with a thickness of 74 ± 9 µm and the absolute thickest spectacle was found in the red-tailed pipe snake (Cylindrophiidae) which had a spectacle thickness of 244 ± 57 µm. Fossorial and aquatic snakes had significantly thicker spectacles than arboreal and terrestrial snakes. When spectacle thickness was correlated to eye size (horizontal spectacle diameter), Gray's earth snake (Uropeltidae) had the lowest ratio (1:7) and the cottonmouth (Viperidae) had the highest ratio (1:65). Multivariable and phylogenetic analyses showed that spectacular thickness could be predicted by taxonomic family and habitat, but not activity period. CONCLUSION: This phylogenetically broad systematic study of the thickness of the snake spectacle showed that spectacular thickness varies greatly across snake species and may reflect evolutionary adaptation and development.

Speciation with gene flow in equids despite extensive chromosomal plasticity.

Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0-4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1-3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

EVALUATION OF A COMMERCIAL COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF AVIAN INFLUENZA VIRUS SUBTYPE H5 ANTIBODIES IN ZOO BIRDS.

The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study compared a commercial ELISA for detection of AIV subtype H5 antibodies with HI test of 572 serum samples from zoo birds. There was no significant difference between the results of the two tests when statistically compared by a McNemar χ2 test (P = 0.86) and assessment of κ (κ = 0.87). With a specificity of 94.2% (95% confidence interval [CI], 0.92-0.97), a sensitivity of 93.9% (95% CI, 0.91-0.97), and an excellent correlation between the two tests, this ELISA can be recommended as an alternative to the HI test for preliminary screening of zoo bird sera for antibodies to AIV subtype H5.

The thick left ventricular wall of the giraffe heart normalises wall tension, but limits stroke volume and cardiac output.

Giraffes--the tallest extant animals on Earth--are renowned for their high central arterial blood pressure, which is necessary to secure brain perfusion. Arterial pressure may exceed 300 mmHg and has historically been attributed to an exceptionally large heart. Recently, this has been refuted by several studies demonstrating that the mass of giraffe heart is similar to that of other mammals when expressed relative to body mass. It thus remains unexplained how the normal-sized giraffe heart generates such massive arterial pressures. We hypothesized that giraffe hearts have a small intraventricular cavity and a relatively thick ventricular wall, allowing for generation of high arterial pressures at normal left ventricular wall tension. In nine anaesthetized giraffes (495±38 kg), we determined in vivo ventricular dimensions using echocardiography along with intraventricular and aortic pressures to calculate left ventricular wall stress. Cardiac output was also determined by inert gas rebreathing to provide an additional and independent estimate of stroke volume. Echocardiography and inert gas-rebreathing yielded similar cardiac outputs of 16.1±2.5 and 16.4±1.4 l min(-1), respectively. End-diastolic and end-systolic volumes were 521±61 ml and 228±42 ml, respectively, yielding an ejection fraction of 56±4% and a stroke volume of 0.59 ml kg(-1). Left ventricular circumferential wall stress was 7.83±1.76 kPa. We conclude that, relative to body mass, a small left ventricular cavity and a low stroke volume characterizes the giraffe heart. The adaptations result in typical mammalian left ventricular wall tensions, but produce a lowered cardiac output.

Testudinibacter aquarius gen. nov., sp. nov., a member of the family Pasteurellaceae isolated from the oral cavity of freshwater turtles.

A total of 13 Pasteurellaceae isolates from healthy freshwater turtles were characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group. The closest related species based on 16S rRNA gene sequencing was Chelonobacter oris CCUG 55632T with 94.4 % similarity and the closest related species based on rpoB gene sequence comparison was [Pasteurella] testudinis CCUG 19802T with 91.5 % similarity. All the investigated isolates exhibited phenotypic characteristics of the family Pasteurellaceae. However, they could be separated from existing genera of the Pasteurellaceae by the following test results: indole, ornithine decarboxylase and Voges-Proskauer positive; and methyl red, urease and PNPG (α-glucosidase) negative. No X- or V-factor requirement was observed. A zone of ß-haemolysis surrounded the colonies after 24 h of incubation on bovine blood agar at 37 °C. Acid was produced from l-arabinose, dulcitol, d-mannitol, sucrose and trehalose. Representative strain ELNT2xT had a fatty acid profile that was characteristic for members of the Pasteurellaceae. ELNT2xT expressed only one respiratory quinone, ubiquinone-8 (100 %). The DNA G+C content of strain ELNT2xT was 42.8 mol%. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the strains should be classified as representatives of a novel species of a new genus, Testudinibacter aquarius gen. nov., sp. nov. The type strain of Testudinibacter aquarius is ELNT2xT ( = CCUG 65146T = DSM 28140T), which was isolated from the oral cavity of a captive eastern long-necked turtle (Chelodina longicollis) in Denmark in 2012.

Bisgaardia miroungae sp. nov., a new member of the family Pasteurellaceae isolated from the oral cavity of northern elephant seals (Mirounga angustirostris), and emended description of the genus Bisgaardia.

A total of 17 bacterial isolates from northern elephant seals, tentatively classified within the family Pasteurellaceae, were further characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group, closely related to the genus Bisgaardia within the family Pasteurellaceae. The rpoB gene sequence similarity was 97.2-100 % within the group and 16S rRNA gene sequence comparisons showed 99.2-99.8 % similarity within the group. According to 16S rRNA gene sequence analysis, the most closely related species with a validly published name was Bisgaardia hudsonensis with 96.9 % similarity and the most closely related species based on rpoB sequence comparison was Bisgaardia genomospecies 1 with an rpoB sequence similarity of 90.9 %. All the isolates investigated exhibited the phenotypic characteristics of the family Pasteurellaceae. However, these isolates could be separated from existing species of the genus Bisgaardia by the following characteristics: ability to grow at 42 °C, and acid production from lactose, melibiose, raffinose and l-rhamnose, but not from d-mannitol or trehalose. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the strains should be classified as representatives of a novel species within the genus Bisgaardia: Bisgaardia miroungae sp. nov. The type strain, Wildatric(T) ( = CCUG 65148(T) = DSM 28141(T)), was isolated from the oral cavity of a wild northern elephant seal at The Marine Mammal Center, California, USA in 2011. To include the novel species, the description of the genus Bisgaardia has been emended.

OCCURRENCE OF PASTEURELLACEAE BACTERIA IN THE ORAL CAVITY OF THE TASMANIAN DEVIL (SARCOPHILUS HARRISII).

The occurrence of bacteria belonging to the family Pasteurellaceae in the oral cavity of captive Tasmanian devils (Sarcophilus harrisii) was investigated using phenotypic and subsequent genotypic characterization and phylogenetic analyses. A total of 62 bacterial isolates obtained from Tasmanian devils, tentatively classified with the family Pasteurellaceae, were further characterized by phylogenetic analysis of rpoB gene sequence similarity, which showed that the isolates investigated formed five distinct groups. A total of 15 strains formed a novel genus-like group within Pasteurellaceae. Thirty-six strains grouped with the type strain of Frederiksenia canicola. Five strains clustered with the type strain of Pasteurella multocida . Interestingly, four of the P. multocida-like strains were ß-hemolytic when incubated on blood agar, which is atypical for this genus. Five strains grouped with a 100% rpoB similarity with Pasteurella dagmatis. Finally, a single strain showed 97.1% resemblance to Haemophilus haemoglobinophilus. The results demonstrate that Tasmanian devils are hosting a variety of bacterial taxa affiliated with the family of Pasteurellaceae as part of their oral microflora.

Inflammation-induced haemostatic response in layer chickens infected with Streptococcus equi subsp. zooepidemicus as evaluated by fibrinogen, prothrombin time and thromboelastography.

Streptococcus zooepidemicus has recently been shown to be a severe pathogen in layer chickens, where it is able to cause serious lesions in the vascular system. To evaluate the haemostatic response, 10 layer chickens were inoculated intravenously with S. zooepidemicus. Four hypotheses were tested: that the infection-induced inflammation would increase the plasma fibrinogen (Fbg) concentration, would prolong the prothrombin time (PT) and would prompt hypercoagulability or hypocoagulability as assessed by whole-blood thromboelastography (TEG), and that a possible correlation would exist between one of the TEG values and Fbg/PT. Each parameter was measured at days 1, 3 and 6 post inoculation (p.i.), and compared with the values at day 0 from each individual bird and with values obtained from non-infected control chickens (n = 10). In the infected chickens, the mean (± standard error) of Fbg was higher at day 3 p.i. (9.4 ± 1.4 g/l) and day 6 p.i. (8.0 ± 0.7 g/l) and the PT was prolonged at day 6 p.i. (168.1 ± 21.0 sec) compared with the day 0 standards (2.6 ± 0.2 g/l and 104.6 ± 2.0 sec, respectively) (P < 0.05). The majority of infected chickens demonstrated a hypercoagulable TEG result with increased mean values of the clot formation rate (α-angle) and maximal amplitude (MA) of TEG tracing at day 3 p.i. (83.1 ± 0.7°, 83.8 ± 1.4 mm) and day 6 p.i. (84.0 ± 0.4°, 89.8 ± 1.0 mm) compared with the day 0 values (75.8 ± 2.2° and 66.9 ± 1.4 mm, respectively) (P < 0.05). In control birds, the means of Fbg (1.5 ± 0.1 g/l), PT (79.4 ± 6.4 sec), TEG-α (76.7 ± 1.5°) and TEG-MA (64.0 ± 2.3 mm) were lower at day 6 compared with values observed for the infected chickens (P < 0.05). A negative correlation coefficient (-0.71) was found between the clot formation time (TEG-K) and Fbg at day 1 in the control group (P = 0.02). In conclusion, infection with S. zooepidemicus following intravenous injection in layer chickens induced haemostatic alterations including hyperfibrinogenaemia, prolonged PT, and hypercoagulability as measured by increased TEG-α and TEG-MA.

Haematological and biochemical reference intervals for free-ranging brown bears (Ursus arctos) in Sweden.

BACKGROUND: Establishment of haematological and biochemical reference intervals is important to assess health of animals on individual and population level. Reference intervals for 13 haematological and 34 biochemical variables were established based on 88 apparently healthy free-ranging brown bears (39 males and 49 females) in Sweden. The animals were chemically immobilised by darting from a helicopter with a combination of medetomidine, tiletamine and zolazepam in April and May 2006-2012 in the county of Dalarna, Sweden. Venous blood samples were collected during anaesthesia for radio collaring and marking for ecological studies. For each of the variables, the reference interval was described based on the 95% confidence interval, and differences due to host characteristics sex and age were included if detected. To our knowledge, this is the first report of reference intervals for free-ranging brown bears in Sweden. RESULTS: The following variables were not affected by host characteristics: red blood cell, white blood cell, monocyte and platelet count, alanine transaminase, amylase, bilirubin, free fatty acids, glucose, calcium, chloride, potassium, and cortisol. Age differences were seen for the majority of the haematological variables, whereas sex influenced only mean corpuscular haemoglobin concentration, aspartate aminotransferase, lipase, lactate dehydrogenase, ß-globulin, bile acids, triglycerides and sodium. CONCLUSIONS: The biochemical and haematological reference intervals provided and the differences due to host factors age and gender can be useful for evaluation of health status in free-ranging European brown bears.

Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil).

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in today's population preceded the Devil Facial Tumor Disease disease outbreak by at least 100 y. Using a genetically characterized breeding stock based on the genome sequence will enable preservation of the extant genetic diversity in future Tasmanian devil populations.

Acute-phase responses in healthy and diseased rhesus macaques (Macaca mulatta).

Five acute-phase reactants-serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron-were measured using commercially available assays in 110 healthy rhesus macaques (Macaca mulatta), and reference intervals were established for future use in health monitoring of this species. Reference intervals established were as follows: SAA, 29.5-87.7 mg/L; CRP, 0-17.5 mg/L; haptoglobin, 354.3-2,414.7 mg/ L; albumin, 36.1-53.0 g/L; and iron, 13.3-40.2 micromol/L. Furthermore, changes in the acute-phase reactants were studied in two additional groups of animals: eight rhesus macaques suffering from acute traumatic injuries and nine rhesus macaques experimentally infected with Mycobacterium tuberculosis reflecting a chronic active inflammation. In animals with inflammation, SAA and haptoglobin concentrations were moderately increased, while CRP increased more than 200-fold. In addition, marked decreases in albumin and iron concentrations were observed. These results show that SAA, CRP, and haptoglobin are positive acute-phase proteins, whereas albumin and iron are negative acute-phase reactants in rhesus macaques.

Probable transmission of coxsackie B3 virus from human to chimpanzee, Denmark.

In 2010, a chimpanzee died at Copenhagen Zoo following an outbreak of respiratory disease among chimpanzees in the zoo. Identification of coxsackie B3 virus, a common human pathogen, as the causative agent, and its severe manifestation, raise questions about pathogenicity and transmissibility among humans and other primates.