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.

Airborne environmental DNA for terrestrial vertebrate community monitoring.

Biodiversity monitoring at the community scale is a critical element of assessing and studying species distributions, ecology, diversity, and movements, and it is key to understanding and tracking environmental and anthropogenic effects on natural ecosystems.1-4 Vertebrates in terrestrial ecosystems are experiencing extinctions and declines in both population numbers and sizes due to increasing threats from human activities and environmental change.5-8 Terrestrial vertebrate monitoring using existing methods is generally costly and laborious, and although environmental DNA (eDNA) is becoming the tool of choice to assess biodiversity, few sample types effectively capture terrestrial vertebrate diversity. We hypothesized that eDNA captured from air could allow straightforward collection and characterization of terrestrial vertebrate communities. We filtered air at three localities in the Copenhagen Zoo: a stable, outside between the outdoor enclosures, and in the Rainforest House. Through metabarcoding of airborne eDNA, we detected 49 vertebrate species spanning 26 orders and 37 families: 30 mammal, 13 bird, 4 fish, 1 amphibian, and 1 reptile species. These spanned animals kept at the zoo, species occurring in the zoo surroundings, and species used as feed in the zoo. The detected species comprise a range of taxonomic orders and families, sizes, behaviors, and abundances. We found shorter distance to the air sampling device and higher animal biomass to increase the probability of detection. We hereby show that airborne eDNA can offer a fundamentally new way of studying and monitoring terrestrial communities.

Did giraffe cardiovascular evolution solve the problem of heart failure with preserved ejection fraction?

The evolved adaptations of other species can be a source of insight for novel biomedical innovation. Limitations of traditional animal models for the study of some pathologies are fueling efforts to find new approaches to biomedical investigation. One emerging approach recognizes the evolved adaptations in other species as possible solutions to human pathology. The giraffe heart, for example, appears resistant to pathology related to heart failure with preserved ejection fraction (HFpEF)-a leading form of hypertension-associated cardiovascular disease in humans. Here, we postulate that the physiological pressure-induced left ventricular thickening in giraffes does not result in the pathological cardiovascular changes observed in humans with hypertension. The mechanisms underlying this cardiovascular adaptation to high blood pressure in the giraffe may be a bioinspired roadmap for preventive and therapeutic strategies for human HFpEF.

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.

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.

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.

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 microbiome of captive hamadryas baboons.

BACKGROUND: The hamadryas baboon (Papio hamadryas) is a highly social primate that lives in complex multilevel societies exhibiting a wide range of group behaviors akin to humans. In contrast to the widely studied human microbiome, there is a paucity of information on the host-associated microbiomes of nonhuman primates (NHPs). Here, our goal was to understand the microbial composition throughout different body sites of cohabiting baboons. RESULTS: We analyzed 170 oral, oropharyngeal, cervical, uterine, vaginal, nasal and rectal samples from 16 hamadryas baboons via 16S rRNA gene sequencing. Additionally, raw Miseq sequencing data from 1041 comparable publicly available samples from the human oral cavity, gut and vagina were reanalyzed using the same pipeline. We compared the baboon and human microbiome of the oral cavity, gut and vagina, showing that the baboon microbiome is distinct from the human. Baboon cohabitants share similar microbial profiles in their cervix, uterus, vagina, and gut. The oral cavity, gut and vagina shared more bacterial amplicon sequence variants (ASVs) in group living baboons than in humans. The shared ASVs had significantly positive correlations between most body sites, suggesting a potential bacterial exchange throughout the body. No significant differences in gut microbiome composition were detected within the maternity line and between maternity lines, suggesting that the offspring gut microbiota is shaped primarily through bacterial exchange among cohabitants. Finally, Lactobacillus was not so predominant in baboon vagina as in the human vagina but was the most abundant genus in the baboon gut. CONCLUSIONS: This study is the first to provide comprehensive analyses of the baboon microbiota across different body sites. We contrast this to human body sites and find substantially different microbiomes. This group of cohabitating baboons generally showed higher microbial diversity and remarkable similarities between body sites than were observed in humans. These data and findings from one group of baboons can form the basis of future microbiome studies in baboons and be used as a reference in research where the microbiome is expected to impact human modeling with baboons.

Morphology and evolution of the snake cornea.

To investigate whether the thickness of the cornea in snakes correlates with overall anatomy, habitat or daily activity pattern, we measured corneal thickness using optical coherence tomography scanning in 44 species from 14 families (214 specimens) in the collection at the Natural History Museum (Denmark). Specifically, we analyzed whether the thickness of the cornea varies among species in absolute terms and relative to morphometrics, such as body length, spectacle diameter, and spectacle thickness. Furthermore, we examined whether corneal thickness reflects adaptation to different habitats and/or daily activity patterns. The snakes were defined as arboreal (n = 8), terrestrial (n = 22), fossorial (n = 7), and aquatic (n = 7); 14 species were classified as diurnal and 30 as nocturnal. We reveal that the interspecific variation in corneal thickness is largely explained by differences in body size, but find a tendency towards thicker corneas in diurnal (313 ± 227 µm) compared to nocturnal species (205 ± 169 µm). Furthermore, arboreal snakes had the thickest corneas and fossorial snakes the thinnest. Our study shows that body length, habitat, and daily activity pattern could explain the interspecific variation in corneal morphology among snakes. This study provides a quantitative analysis of the evolution of the corneal morphology in snakes, and it presents baseline values of corneal thickness of multiple snake species. We speculate that the cornea likely plays a role in snake vision, despite the fact that results from previous studies suggest that the cornea in snakes is not relevant for vision (Sivak, Vision Research, 1977, 17, 293-298).

Elimination of Intracardiac Shunting Provides Stable Gas Anesthesia in Tortoises.

Inhalant anesthesia is challenging in chelonians due to a great capacity for breath-holding and an incomplete separation of the cardiac ventricle. Deoxygenated blood can recirculate back into systemic circulation by bypassing the lung in a process referred to as intracardiac right to left (R-L) shunting. Via electrocardiogram gated magnetic resonance imaging, a novel modality to investigate arterial flows in reptiles, intracardiac shunting and its elimination via atropine during gas anesthesia in tortoises (Chelonoidis carbonaria) was demonstrated. The great vessels of the heart were visualized confirming that after shunt-elimination, the flow (mean ± sd) in the pulmonary arteries increased significantly (54.6 ± 9.5 mL min-1 kg-1 vs 10.8 ± 3.4 mL min-1 kg-1; P < 0.008). Consequently, animals required significantly lower concentrations of inhaled anesthetics to maintain a stable anesthesia. To that end, the minimum anesthetic concentration (MAC) of isoflurane needed to maintain surgical anesthesia was measured. A significantly lower MAC was found after administration of atropine (mean MAC ± sd 2.2 ± 0.3% vs 3.2 ± 0.4%; P < 0.002). Previously, MAC has been indeterminable in chelonians likely due to intracardiac shunting, so this report constitutes the first MAC study performed in a tortoise.

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.

Debugging diversity - a pan-continental exploration of the potential of terrestrial blood-feeding leeches as a vertebrate monitoring tool.

The use of environmental DNA (eDNA) has become an applicable noninvasive tool with which to obtain information about biodiversity. A subdiscipline of eDNA is iDNA (invertebrate-derived DNA), where genetic material ingested by invertebrates is used to characterize the biodiversity of the species that served as hosts. While promising, these techniques are still in their infancy, as they have only been explored on limited numbers of samples from only a single or a few different locations. In this study, we investigate the suitability of iDNA extracted from more than 3,000 haematophagous terrestrial leeches as a tool for detecting a wide range of terrestrial vertebrates across five different geographical regions on three different continents. These regions cover almost the full geographical range of haematophagous terrestrial leeches, thus representing all parts of the world where this method might apply. We identify host taxa through metabarcoding coupled with high-throughput sequencing on Illumina and IonTorrent sequencing platforms to decrease economic costs and workload and thereby make the approach attractive for practitioners in conservation management. We identified hosts in four different taxonomic vertebrate classes: mammals, birds, reptiles and amphibians, belonging to at least 42 different taxonomic families. We find that vertebrate blood ingested by haematophagous terrestrial leeches throughout their distribution is a viable source of DNA with which to examine a wide range of vertebrates. Thus, this study provides encouraging support for the potential of haematophagous terrestrial leeches as a tool for detecting and monitoring terrestrial vertebrate biodiversity.

Improved de novo genomic assembly for the domestic donkey.

Donkeys and horses share a common ancestor dating back to about 4 million years ago. Although a high-quality genome assembly at the chromosomal level is available for the horse, current assemblies available for the donkey are limited to moderately sized scaffolds. The absence of a better-quality assembly for the donkey has hampered studies involving the characterization of patterns of genetic variation at the genome-wide scale. These range from the application of genomic tools to selective breeding and conservation to the more fundamental characterization of the genomic loci underlying speciation and domestication. We present a new high-quality donkey genome assembly obtained using the Chicago HiRise assembly technology, providing scaffolds of subchromosomal size. We make use of this new assembly to obtain more accurate measures of heterozygosity for equine species other than the horse, both genome-wide and locally, and to detect runs of homozygosity potentially pertaining to positive selection in domestic donkeys. Finally, this new assembly allowed us to identify fine-scale chromosomal rearrangements between the horse and the donkey that likely played an active role in their divergence and, ultimately, speciation.

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 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.

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.

Identification of a novel species of papillomavirus in giraffe lesions using nanopore sequencing.

Papillomaviridae form a large family of viruses that are known to infect a variety of vertebrates, including mammals, reptiles, birds and fish. Infections usually give rise to minor skin lesions but can in some cases lead to the development of malignant neoplasia. In this study, we identified a novel species of papillomavirus (PV), isolated from warts of four giraffes (Giraffa camelopardalis). The sequence of the L1 gene was determined and found to be identical for all isolates. Using nanopore sequencing, the full sequence of the PV genome could be determined. The coding region of the genome was found to contain seven open reading frames (ORF), encoding the early proteins E1, E2 and E5-E7 as well as the late proteins L1 and L2. In addition to these ORFs, a region located within the E2 gene is thought, based on sequence similarities to other papillomaviruses, to encode an E4 protein, although no start codon could be identified. Based on the sequence of the L1 gene, this novel PV was found to be most similar to Capreolus capreolus papillomavirus 1 (CcaPV1), with 67.96% nucleotide identity. We therefore suggest that the virus identified here is given the name Giraffa camelopardalis papillomavirus 1 (GcPV1) and is classified as a novel species within the genus Deltapapillomavirus, in line with the current guidelines for the nomenclature and classification of PVs.

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.

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.