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.

Comparison of intramuscular alfaxalone with medetomidine-ketamine for inducing anaesthesia in Trachemys scripta spp. undergoing sterilization.

OBJECTIVE: To compare the effect of two anaesthetic protocols on heart rate (HR), time to muscle relaxation and tracheal intubation and time to surgical plane of anaesthesia, in Trachemys scripta spp. undergoing oophorectomy. STUDY DESIGN: Prospective randomized clinical study. ANIMALS: A total of 43 healthy female turtles. METHODS: Morphine (1.5 mg kg-1) was injected subcutaneously 2 hours before anaesthesia induction. The turtles were randomly administered either medetomidine (0.2 mg kg-1) and ketamine (10 mg kg-1) (group MK; n = 23) or alfaxalone (20 mg kg-1) (group A; n = 20) intramuscularly followed by bupivacaine (2 mg kg-1) administered subcutaneously along the incision site. Anaesthesia was maintained with isoflurane delivered in oxygen (100%). HR and the anaesthetic depth score (ADS) were recorded every 5 minutes from induction to recovery. A Friedman test followed by Wilcoxon tests with Bonferroni adjustment were used to compare these non-parametric data (HR and ADS) between groups and over time. Time to muscle relaxation of neck and limbs (TMR), tracheal tube insertion (TTTI) and stage of surgical anaesthesia (TADS≤3) were recorded and compared between groups using a Welch's t test after logarithmic transformation. RESULTS: Median values of TMR, TTTI and TADS≤3 were 4, 9.5 and 25 minutes in group A, respectively, and 14, 20 and 35 minutes in group MK (TMR, TTTIp ≤ 0.0001; TADS≤3p = 0.001). Plane of anaesthesia was significantly deeper in group A than in group MK for the first 20 minutes (p < 0.01). HR at 10 and 15 minutes post injection was significantly lower in group MK (28 beats minute-1) than in group A (36 and 34 beats minute-1) (p < 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: After intramuscular injection in Trachemys scripta spp., tracheal intubation, muscle relaxation and a surgical plane of anaesthesia developed faster with alfaxalone than medetomidine-ketamine.

New insights in Cercopithecinae spermatozoa.

Sperm morphometric and morphologic data have been shown to represent useful tools for monitoring fertility, improving assisted reproduction techniques and conservation of genetic material as well as detecting inbreeding of endangered primates. We provide here for the first time sperm morphologic and morphometric data from Cercopithecus neglectus, Cercopithecus cephus, Papio papio and critically endangered Cercopithecus roloway, as well as comparative data from other Cercopithecinae species, i.e. Allochrocebus lhoesti, Mandrillus sphinx and Papio anubis. Following collection from the epididymis, spermatozoa were measured for each species for the following parameters: head length, head width, head perimeter, head area, midpiece length and total flagellum length, and the head volume, ellipticity, elongation, roughness and regularity were then calculated. Our data are consistent with both the general morphology and the morphometric proportions of Cercopithecinae sperm. Some specificities were observed, with C. cephus displaying a narrow head (width = 2.76 ± 0.26 µM) and C. roloway displaying a short midpiece (6.65 ± 0.61 µM). This data set represents an important contribution, especially for Cercopithecus roloway, one of the most endangered monkeys in the world, and further data on additional specimens coupled to data on mating systems and reproductive ecology should allow a better understanding of the mechanisms underlying these morphological differences across primate species.

PREVALENCE OF LAWSONIA INTRACELLULARIS INFECTION IN NONHUMAN PRIMATES AND PEST RODENTS IN A ZOOLOGICAL COLLECTION.

In 2016 and 2017, Lawsonia intracellularis was isolated from several pileated gibbons (Hylobates pileatus) presenting with diarrhea in Mulhouse Zoo (eastern France). To this day, infection with this bacterium has rarely been described in nonhuman primates (NHP) in captivity or in the wild and there are no data about the prevalence or transmission of the disease. This study focuses on finding the prevalence of this infection amongst Mulhouse Zoo's NHP collection and trying to identify a source of contamination responsible for this epizooty. Forty-eight real-time PCR were conducted on feces from all NHP species in the zoo and on small mammals trapped in the NHP housing structures. No NHP was experiencing symptoms at the time of the study, however test results showed that Lawsonia intracellularis can be found in 61.76% (21/34) of the group total (n = 34) and the prevalence even increases to 92.3% (12/13) in the Lemuriform infraorder (n = 13). In small mammals (n = 14), prevalence of the bacterium is 57.17% (8/14) including 77.78% in rodents (7/9). The results of this study show that several NHP species are healthy carriers and some species of small mammals can be considered as a potential source of contamination. Because of the difficulty encountered trying to isolate the bacterium, it is plausible that infections caused by Lawsonia intracellularis have been underdiagnosed to this day, and that it could be an emerging disease in Europe. Therefore, using real-time PCR to search for this bacterium seems essential in case of diarrhea occurring in nonhuman primates. Moreover, even though further studies on contamination sources need to be conducted, the issue of the presence of rodents in NHP housing structures has to be taken very seriously and tackled with the utmost care.

Haemosporidian parasites from captive Strigiformes in France.

Haemosporidia infections may cause major damage to avian populations and represent a concern for veterinarians working in zoological parks or wildlife rescue centres. Following the fatal infection of 9 Great grey owls (Strix nebulosa) at Mulhouse zoological park, between summer 2013 and 2015, a prospective epidemiological investigation was performed in captive strigiform birds in France in 2016. The purpose was to evaluate the prevalence of haemosporidian parasites in captive Strigiformes and to estimate the infection dynamics around the nesting period. Blood samples were taken from 122 strigiform birds representing 14 species from 15 French zoological parks. Parasites were detected by direct examination of blood smears and by PCR targeting the mitochondrial cytochrome b gene. Haemosporidian parasites were detected in 59 birds from 11 zoos. Three distinct Haemoproteus mitochondrial cytochrome b sequences (haplotypes A and C for H. syrnii and haplotype B for Haemoproteus sp.) as well as two species of Plasmodium were detected. The overall prevalence of Haemoproteus infection was 12.8%. The percentage of birds infected by Haemoproteus varied according to the period of sampling. Nesting season seemed to be at greater risk with an average prevalence of 53.9% compared with winter season with an average prevalence of 14.8%, related to the abundance of the vectors. The prevalence of Plasmodium infection in Strigiformes did not exceed 8% throughout the year. This study confirmed how significant Haemosporidia infection could be in Strigiformes from zoological parks in France. The nesting season was identified as a period of higher risk of infection and consequently the appropriate period to apply prophylactic measures.

The body center of mass in primates: Is it more caudal than in other quadrupedal mammals?

OBJECTIVES: Whole body center of mass (BCoM) position values are lacking for a comparative sample of primates. Therefore, it still remains unknown whether the BCoM in primates is more posteriorly located than in other mammals. The aim of the present report is to provide data for a large sample of primate species and to compare the position of the BCoM in primates to non-primate mammals. MATERIALS AND METHODS: We collected morphometrics on eight primate species belonging to various families: Hylobatidae (Nomascus grabriellae, Nomascus Siki), Cercopithecidae (Cercopithecus roloway, Cercopithecus lhoesti, Colobus guereza, Trachypithecus francoisi), Cebidae (Sapajus xanthosternos), and Atelidae (Ateles fusciceps). Using a geometric model, we assessed the position of the BCoM in a natural quadrupedal posture and in a control posture. To complete our comparative sample with a wider range of morphotypes, we added the data available in the literature for hominoids (Pan paniscus, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates lar) and another cercopithecoid species (Papio anubis). We also evaluated the phylogenetic signal of the position of the BCoM in primates. RESULTS: The variation in the position of the BCoM in primates is very large, ranging from 40% of the distance between the hip and the shoulder in Ateles fusciceps to 63% in Hylobates lar. We observed a strong phylogenetic signal for this trait: hominoid species, as well as the baboon, have a cranial BCoM relative to the midline between the hip and the shoulder, arboreal cercopithecoids and the spider monkey have a caudal BCoM, and the capuchin monkey has a BCoM positioned at mid-trunk. The variation observed in non-primate quadrupedal mammals lies inside the variation range of primates, from 51% in Felis catus to 63% in Canis familiaris. DISCUSSION: The BCoM of primates is not more posteriorly located than in other quadrupedal mammals; however, there is a substantial range of variation in primates, from caudal (in arboreal quadrupeds) to cranial (in hominoids and terrestrial quadrupeds) positions. This variation is related to a phylogenetic model that suggests stabilizing selection for this trait. It seems that the BCoM position mostly depends of the size of the appendicular system (i.e., limbs) and the tail. Therefore, it may also reflect a general trend in quadrupedal mammals with arboreal species exhibiting a caudal BCoM and terrestrial species exhibiting a cranial BCoM. These results are discussed in the context of the locomotor evolution of primates including locomotor habits and gait mechanics. We also propose a new "passive" mechanism for the explanation of the particular weight support pattern observed in primates with tails.

BIOMEDICAL EVALUATION OF A BROWN LEMUR (EULEMUR FULVUS SPP.) POPULATION FROM MBOUZI ISLET, MAYOTTE ISLAND.

The brown lemur population (Eulemur fulvus spp.) in Mbouzi islet is not native, and was introduced in 1997. Since then, the population has grown. In 2012 the National Council for Protection of Nature of Mayotte requested to remove this population of lemurs from Mbouzi, as they were suspected to be a threat to the protected endemic flora of the islet. The Association Francophone des Vétérinaires de Parcs Zoologiques (French-speaking Zoo Veterinarians Association, AFVPZ) was asked to conduct a biomedical evaluation of the population. Fifty-two animals were captured, anesthetized, and weighed. They all underwent a general physical examination. Feces were sampled for bacterial and parasitological screening. Hair was sampled for genetic studies and blood was sampled for hematology, biochemistry, viral serology, and haemoparasitology. Results showed that three individuals had a positive feces culture for Salmonella enterica and six had Lemuricola or Callistoura parasite infestations. Blood analyses for hematology and biochemistry showed 46 animals with elevated transferrin, 42 with low ferritin levels, 19 with hyperglycemia, and 10 with neutrophilia. Finally, 10 were positive for Toxoplasma serology, one was positive for α herpesvirus, five for pox virus, five for simian virus 40, and two for flavivirus. This publication reports the first complete biomedical evaluation of lemurs on Mayotte Island.

INVESTIGATION OF THE PRESENCE OF ATOXOPLASMA SPP. IN BLUE-CROWNED LAUGHINGTHRUSH (DRYONASTES COURTOISI) ADULTS AND NEONATES.

Between 1996 and 2013, 71 blue-crowned laughingthrush (Dryonastes courtoisi) chicks, a small passerine bird endemic to China, were born at Mulhouse Zoo in northeast France. None of them survived past 1 yr, and 82% died between 0 and 6 days old of an unidentified cause and despite an attempt to establish an artificial breeding protocol. Atoxoplasma spp., causing a disease known as systemic isosporosis, is a coccidian parasite that can infect several species of birds. Mulhouse's adult birds were suspected to be infected with Atoxoplasma spp. and to transmit this parasite to their offspring. A treatment with toltrazuril (Baycox® 2.5%) was implemented in the four adult birds. Coprologic examinations were performed before, during, and after the treatment to quantify the parasite load in feces. Polymerase chain reaction (PCR) assays were used to test blood samples from the adult and liver, lung, gizzard, and kidney samples from 10 chicks to detect Atoxoplasma spp. Five of the 10 chicks had some tissue samples positive for Atoxoplasma spp. in at least one of the three repeats of the atoxoplasmosis PCR. An average of 181 Isospora spp. oocysts per gram of feces were found in the group of adults before treatment. This number was reduced to zero 1 wk after the beginning of the toltrazuril treatment. The PCR results suggest a transovarian transmission of Atoxoplasma spp., but further investigation is needed for confirmation. The treatment with toltrazuril appears to allow a significant reduction of the parasite excretion.

SQUAMOUS CELL CARCINOMA IN TWO SNOW LEOPARDS (UNCIA UNCIA) WITH UNUSUAL AURICULAR PRESENTATION.

Squamous cell carcinoma (SCC) is well documented in snow leopards ( Uncia uncia ) and most common locations are oral, facial, or pedal. These two cases illustrate an unusual auricular presentation, which is more often reported in white domestic cats. The animals were aged and presented clinical signs of otitis such as head shaking and ear scratching. Clinical examinations showed auricular canal masses with chronic purulent otitis. In both cases, clinical deterioration led to euthanasia and histology of the ear canal was consistent with SCC and showed numerous vascular emboli. These cases illustrate an unreported aggressive localization for SCC in snow leopards, which should be included in the differential diagnosis of otitis in this species. Auricular SCC may be underdiagnosed as the ear canal is infrequently sampled for histopathology. This auricular localization should be considered when metastases are found upon necropsy without internal primary tumor.

A CASE OF TRYCHOPHYTON RUBRUM DERMATOPHYTOSIS IN A PATAGONIAN SEA LION (OTARIA BYRONIA).

A 23-yr old female Patagonian sea lion (Otaria byronia) presented multifocal to coalescing and ulcerative skin lesions on the lumbar region. Skin scrapings were collected and a microscopic examination was conducted followed by a fungal culture that revealed a Trychophyton rubrum infection, an anthropophilic dermatophytosis agent. Oral terbinafine and topical eniconazole were used as a treatment for a period of 75 days and complete recovery was achieved. Epidemiological analysis revealed a dermatophytosis case in one of the carnivore section keepers a few weeks before the lesions were diagnosed in the sea lion.

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species.

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.

Gastrointestinal parasites in non-human primates in zoological institutions in France.

Gastrointestinal parasites are frequently encountered in captive non-human primates and infestation may have severe consequences on the animal's health status. Most of these parasites are also transmissible to humans. Nevertheless, little is known about the prevalence and monitoring modalities of gastrointestinal parasitoses in non-human primates housed in zoological institutions and there are currently no guidelines available for their detection and identification. The objective of this study was to identify the main gastrointestinal parasites that may be observed in non-human primates in zoological institutions in France, as well as to develop a decision-making tree to ease their identification. Twenty-four zoological institutions were surveyed, most of which performed fecal examinations routinely on their non-human primates (91.7%). Most institutions used flotation enrichment protocols to detect gastrointestinal parasites (95.2%) and nematodes were the most frequently encountered parasites (73.0%). A total of 252 fecal samples corresponding to 68 different non-human primate species from these institutions were analyzed using sedimentation and flotation protocols. Protozoa (47.3%) were found to be more frequent than helminths (15.6%). Furthermore, old-world monkeys exhibited a higher parasite load (93.6%) than any other non-human primate species category. Compiled data from fecal examinations allowed the development of a decision-making tree and diagnostic atlas to facilitate parasite diagnosis in captive non-human primates.

Title: Parasites gastro-intestinaux chez les primates non-humains dans les parcs zoologiques français. Abstract: Les parasites gastro-intestinaux sont fréquemment rencontrés chez les primates non-humains en captivité et peuvent avoir des conséquences graves sur l'état de santé de l'animal. La plupart sont également transmissibles à Homme. Néanmoins, on sait peu de choses sur la prévalence et les modalités de surveillance des parasitoses gastro-intestinales chez les primates non-humains hébergés dans des institutions zoologiques et il n'existe actuellement aucune ligne directrice disponible pour leur détection et leur identification. L'objectif de cette étude était d'identifier les principaux parasites gastro-intestinaux pouvant être observés chez les primates non-humains dans les institutions zoologiques françaises, ainsi que de développer un arbre décisionnel pour faciliter leur identification. Vingt-quatre institutions zoologiques françaises ont été interrogées : la plupart (91,7 %) effectuaient des examens fécaux en routine sur leurs primates non-humains. La plupart des établissements utilisaient des protocoles d'enrichissement par flottation pour détecter les parasites gastro-intestinaux (95,2 %) et les nématodes étaient les parasites les plus fréquemment rencontrés (73,0 %). Un total de 252 échantillons fécaux correspondant à 68 espèces différentes de primates non-humains provenant de ces institutions ont été analysés à l'aide de protocoles de sédimentation et de flottation. Les protozoaires (47,3 %) étaient plus fréquents que les helminthes (15,6 %). De plus, les singes de l'ancien monde présentaient une charge parasitaire plus élevée (93,6 %) que toute autre catégorie d'espèces de primates non-humains. Les données compilées des examens fécaux ont permis le développement d'un arbre décisionnel et d'un atlas diagnostique pour faciliter le diagnostic des parasites chez les primates non-humains en captivité.

Evidence for the possible occurrence of Grave's disease in a blue-eyed black lemur (Eulemur flavifrons).

The blue-eyed black lemur (Eulemur flavifrons) is classified by the International Union for Conservation of Nature (IUCN) as critically endangered. A 23-year-old male housed at Mulhouse Zoo presented with lethargy, polyphagia, alopecia, and chronic weight loss. Clinical examination suggested an endocrine pathology such as hyperthyroidism. Secondary examinations included cervical ultrasound, thyroid biopsy, and scintigraphy. The latter revealed elevated thyroid activity. Blood analysis was performed to measure the level of anti-receptor thyroid-stimulating hormone antibodies, which allowed us to test the autoimmune hypothesis. The high level of antibodies together with levels of thyroid-stimulating hormone and the scintigraphy images led to the diagnosis of Grave's disease. Carbimazole treatment followed by thyroidectomy resulted in a quick weight gain and general improvement in health status. The following breeding season, the treated individual sired an offspring. To the authors' knowledge, this is the first report of likely Grave's disease in a non-human primate.

Environmental stressors may cause equine herpesvirus reactivation in captive Grévy's zebras (Equus grevyi).

Equine Herpesviruses (EHV) are common and often latent pathogens of equids which can cause fatalities when transmitted to non-equids. Stress and elevated glucocorticoids have been associated with EHV reactivation in domestic horses, but little is known about the correlation between stress and viral reactivation in wild equids. We investigated the effect of an environmental stressor (social group restructuring following a translocation event) on EHV reactivation in captive Grévy's zebras (Equus grevyi). A mare was translocated by road transport from Zoo Mulhouse, France, to join a resident group of three mares in Tierpark Berlin, Germany. We used an indirect sampling method to assess the frequency of EHV shedding for 14 days immediately after the translocation event (termed the 'experimental period'). The results were compared with those from two control periods, one preceding and one subsequent to the experimental period. In addition, we measured fecal glucocorticoid metabolite (fGCM) concentrations daily in all individuals from 6 days before, to 14 days after translocation. We found significantly higher EHV shedding frequencies during the experimental period, compared to each of the two control periods. All animals showed significantly elevated fGCM concentrations, compared to fGCM levels before translocation. Finally, we found that an increase in fGCM concentration was significantly associated with an increased likelihood of EHV shedding. Although the small number of animals in the study limits the conclusions that can be drawn from the study, taken together, our results support the hypothesis that environmental stressors induce viral reactivation in wild equids. Our results suggest that potentials stressors such as group restructuring and translocation should be considered in the management of zoological collections to reduce the risk of fatal EHV infections in novel hosts. Moreover, environmental stressors may play an important role in EHV reactivation and spread in wild equid populations.

Efficacy of an oral live vaccine for veterinary use against pseudotuberculosis.

Pseudotuberculosis, an infection caused by the ubiquitous enteropathogenic bacterium Yersinia pseudotuberculosis, is a recurrent veterinary problem in livestock and zoo animals. The only vaccine currently available in zoos is Pseudovac (a mixture of killed strains of various serotypes), but its efficacy is not well established. We show here that Pseudovac does not protect guinea pigs against a severe Y. pseudotuberculosis infection. We thus evaluated the possibility of using a live attenuated Y. pseudotuberculosis strain (IP32680) as an oral vaccine against animal pseudotuberculosis. We report that IP32680 is avirulent for guinea pigs and induces a strong IgG response against various serotypes of Y. pseudotuberculosis. One and two oral inoculations of IP32680 provided 50% and 83% protection, respectively against a severe infection with a highly pathogenic strain. The avirulent Y. pseudotuberculosis IP32680 is therefore much more protective than Pseudovac and may represent a valuable oral vaccine against pseudotuberculosis in zoo animals.