Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

A Post-Operative Follow-Up of an Endangered Saltwater Fish Lensectomy for Cataract Management in a Public Aquarium: A Case Series.

Mature cataracts can be a life-threatening condition in fish as it may result in anorexia. Lens extraction has been previously described in fish, but the long-term outcome of this procedure has not been evaluated. Eleven captive-bred adult fish housed in a public aquarium presented with unilateral (n = 3/11) or bilateral (n = 8/11) mature cataracts. All cases belonged to three endangered fish populations: striped bass (Morone saxatilis) originating from the Saint Lawrence River and Atlantic and spotted wolffish (Anarhichas lupus and Anarhichas minor). Pre-operatively, fish presented with anorexia, dark discoloration, abnormal position in the water column, skin abrasions, and/or decreased body condition scores. A lensectomy was performed in eleven fish, including a bilateral procedure in four fish, corresponding to fifteen eyes. Follow-up examinations were performed one to two weeks and one year post surgery. The main complication was self-resolving: corneal edema was present in 67% of eyes, persistent after a week in 47% of eyes, and resolved thereafter. Post-operatively, 73% of fish resumed feeding and regained a good body condition score. Persistent visual impairment post-surgery associated with anorexia required euthanasia in three out of eleven cases. The median time of death was 336 days post surgery (range: 27-1439 days) and three cases were still alive 1334 to 1425 days after the lensectomy. This was considered a favorable outcome for these older individuals with concurrent diseases.

EVALUATION OF CLINICAL DIAGNOSTICS FOR PROVENTRICULAR NEMATODIASIS DUE TO SYNHIMANTUS NASUTA IN LORIKEETS (TRICHOGLOSSUS SPP.).

In this case series, clinical investigations were pursued during a Synhimantus nasuta infection in a lorikeet (Trichoglossus spp.) flock outbreak situation to better describe and document clinical presentations. In 11 lorikeets suspected to be infected with Synhimantus based on at least one abnormal finding on their physical examination (lethargy, feather-damaging behavior on the ventrum, weight loss, pale iris), the presence of five additional parameters was documented: anemia, relative eosinophilia, increased proventricular diameter-to-keel height ratio (PKR), proventricular barium filling defect, and positive fecal occult blood detection test. A total score (X of 9) was calculated by combining all these findings. Synhimantus nasuta infection was confirmed in four of these individuals by modified Wisconsin fecal examination. Suspected cases (n = 7 of 11) presented only with low scores (1-3 of 9), whereas birds with confirmed infections (n = 4 of 11) presented with both low (1-3 of 9, n = 2 of 4) and high (6-7 of 9, n = 2 of 4) total scores. High scores were associated with clinical anemia. Fecal occult blood was present in all confirmed cases and 4 of 7 suspected cases. An enlarged proventriculus was only observed in birds with active shedding (n = 3 of 4). Follow-up evaluations after 6 mon of treatment with ivermectin and selamectin suggested complete recovery with lowered or normalized total scores. In conclusion, during an S. nasuta outbreak, a rapid physical examination helps to identify suspect cases, including individuals requiring immediate medical attention. In the absence of ova shedding, infection cannot be excluded on the basis of scarce clinical findings, but the detection of occult fecal blood and an increased PKR should raise the index of suspicion.

Vaginal foreign bodies in six nonhuman primates with underlying pathological conditions.

Four female Japanese macaques (Macaca fuscata) from the same group as well as Wolf's guenon (Cercopithecus wolfi) and a Western lowland gorilla (Gorilla gorilla gorilla) from a second institution presented with vaginal foreign bodies in parallel with diseases of the urogenital tract or with endocrine disorders. These foreign bodies were associated with a mild to marked, diffuse vaginitis in all cases. Underlying pathological conditions consisted of a cavernous uterine hemangioma in a 20-year-old macaque, diffuse endometritis in a 21-year-old macaque, an in situ endometrial carcinoma in a 24-year-old macaque, endometritis and an ovarian cyst-like structure in the 27-year-old Western lowland gorilla, chronic cystitis and chronic renal disease in a 24-year-old macaque, and a history of hypothyroidism with irregular reproductive cycles in the 12-year-old Wolf's guenon. Vaginal foreign bodies have been reported in nonhuman primates used in biomedical research, but their concurrence with underlying conditions has not been explored. In women, vaginal foreign bodies have been linked to serious underlying medical conditions. This case series emphasizes the recommendation to investigate this abnormal behavior for underlying medical or adverse psychosocial conditions in primates under human care.

COMPARATIVE STUDY ON THE IMPACT OF DIFFERENT IMMOBILIZATION METHODS ON MUSCLE INTEGRITY AND STRESS INDICATORS IN BROOK TROUT (SALVELINUS FONTINALIS).

Chemical sedation of fish is often used during scientific research to reduce stress and risk of injury. Electric fish handling gloves (EFHG) have been proposed as a no-residue alternative to chemical immobilization. However, the impact of handling fish with EFHG on their physiology remains relatively poorly studied. Stress markers (plasma cortisol and lactate) and the impact on muscular integrity (plasma creatine kinase and histopathology of skeletal muscles) were assessed in brook trout (Salvelinus fontinalis) subjected to one of three immobilization methods (manual restraint, EFHG [10-16 mV], and buffered 60 mg/L tricaine methanesulfonate [MS-222] immersion anesthesia) for 1- and 5-min restraints. Plasma lactate levels were significantly higher 2 h postrestraint for all experimental groups (+242%; P < 0.001), but the magnitude of these increases was significantly lower in the MS-222 groups (P ≤ 0.0002). Plasma creatine kinase concentrations significantly increased 2 h postrestraint for the EFHG groups (+92%, P ≤ 0.0061), and this increase was significantly higher than in the MS-222 group for the 1-min restraint (P = 0.0031). Although there was no significant difference between the restraining methods tested at all time points for plasma cortisol, the EFHG and manual restraint groups showed a linear and statistically significant increase after the initial 5-min restraint, whereas all other groups presented an expected bell-shaped profile with a plasma peak 1 h after the initial restraint. One fish in the manual restraint group died during the 5-min restraint, and two fish from the EFHG group were euthanized at the end of this protocol because of buoyancy anomalies. All other fish from the 5-min restraint group developed cutaneous saprolegniasis 3 wk after the experiments. The evaluation of the safety of EFHG in brook trout showed similar impacts on the variables tested compared with manual restraint for 1- and 5-min immobilizations.

USE OF SYMMETRIC DIMETHYLARGININE TO DETECT RENAL LESIONS IN FISH: A PRELIMINARY STUDY IN BROOK TROUT (SALVELINUS FONTINALIS).

Symmetric dimethylarginine (SDMA) is an early marker for renal lesions in mammals. The objectives of this study were 1) to establish a reference interval of SDMA in healthy brook trout, Salvelinus fontinalis (Mitchill, 1814), and 2) to assess its sensibility as a marker of renal pathology. Plasma SDMA was quantified by liquid chromatography-mass spectrometry in 25 adult brook trout, including 20 fish displaying no renal histologic lesions, and five fish displaying chronic nonactive microscopic granulomas. The fish size (P = 0.30) and weight (P = 0.12) were not statistically different among groups, nor were SDMA values (P = 0.22). However, brook trout without microscopic renal lesions tended to have lower SDMA values (no lesions: mean = 24.9 µg/dL; lesions: mean = 31.4 µg/dL). The reference interval (90% confidence interval [90% CI]) for SDMA concentration in brook trout ranged between 10.0 µg/dL (90% CI: 5.4-14.7) and 39.8 µg/dL (90% CI: 34.8-43.9). These values were higher than those previously reported in other vertebrate species. Further research is needed to evaluate the use of SDMA as a marker of renal function in fishes.

BUTORPHANOL, AZAPERONE, AND MEDETOMIDINE ANESTHESIA IN FREE-RANGING EASTERN MOOSE (ALCES AMERICANUS).

Fifty-three free-ranging moose (Alces americanus) cows were darted from a helicopter with 3-4 ml of a premix combination of butorphanol (27.3 mg/ml), azaperone (9.1 mg/ml), and medetomidine (10.9 mg/ml; BAM), equivalent to estimated dosages of: butorphanol 0.26 ± 0.08 (mean ± SD) mg/kg, azaperone 0.09 ± 0.03 mg/kg, and medetomidine 0.11 ± 0.03 mg/kg. After a mean chase time (from sighting to darting) of 6.1 ± 5.5 min, the mean induction time (from darting to recumbency) was 8.3 ± 2.6 min. This combination provided a safe and reliable sedation for minor procedures that lasted 30-60 min. Heart rate (50.4 ± 7.0 beats/min), respiratory rate (21.3 ± 11.1 breaths/minute), ETCO2 via nasal canula (43.2 ± 7.0 mmHg), and rectal temperature (38.5°C ± 0.7°C) mostly remained at expected values for wild cervid and bovid species anesthetized with this drug combination. SpO2 (90.0% ± 3.7%) was suggestive of moderate hypoxemia despite intranasal oxygen supplementation (1 L per 100 kg/min). The recovery time to standing was 6.7 ± 3.8 min after reversal with IM naltrexone (3 mg/mg butorphanol) and atipamezole (5 mg/mg medetomidine). Despite a larger volume to inject, this protocol offers an alternative to highly potent opioids, and should be considered for practical or staff safety reasons. On the basis of the results of this study, the use of 4 ml of BAM is considered a safe and effective protocol for immobilization of cow moose under comparable settings.

Scuticociliate (Philasterides dicentrarchi) infection cluster in a multispecies marine aquarium system.

Scuticociliatosis, caused by ciliated protozoa of the subclass Scuticociliatia, has been associated with high mortalities in marine fish. Environmental factors such as an increase in water temperature can enhance this disease. The aim of the present report is to describe the occurrence of a cluster of cases of scuticociliatosis in a multispecies marine cold-water system in a public aquarium. Philasterides dicentrarchi was identified by PCR in formalin-fixed tissues of some of the fish showing meningitis or meningoencephalitis, dermatitis and myositis with intralesional protozoa. An increase in water temperature of approximately 2°C was identified as a potential contributing factor for this cluster of infections. Higher temperature may have enhanced the propagation or pathogenicity of scuticociliates or increased host susceptibility of some species of fish, especially wolf-eel Anarrhichthys ocellatus and spotted ratfish Hydrolagus colliei. This report also highlights the complexity of dealing with mixed species systems housing fish from different natural ecozones.

A PRELIMINARY STUDY OF INTRINSIC AND EXTRINSIC FACTORS INFLUENCING INTRAOCULAR PRESSURE IN BROOK TROUT (SALVELINUS FONTINALIS).

Reference intervals of intraocular pressure (IOP) are poorly described in piscine species as the factors that may influence it. Rebound tonometry was used to measure IOP in 28 adult brook trout (Salvelinus fontinalis) anesthetized in a buffered solution of 60 mg/L tricaine methanesulfonate (n = 16) or restrained with electronarcosis (n = 12) at 16 mA. There was no significant effect of the eye side, sex, fish origin, and body weight, but IOP values were significantly higher with electronarcosis (mean ± SD: 16.4 ± 5.0 mm Hg) than with immersion anesthesia (10.8 ± 3.3 mm Hg; P = 0.0017). The same restraint method should be used for comparison with previously published IOP values or when evaluating individual variations over time.

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant (Loxodonta africana) occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats (Rattus rattus) captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.

HEMATOLOGY AND SERUM BIOCHEMISTRY VALUES OF CAPTIVE SOUTHERN LECHWES (KOBUS LECHE).

Blood samples collected from captive Southern lechwes (Kobus leche) were used to develop reference intervals for complete blood count and serum biochemistry values. The study population consisted in 108 healthy individuals, including 62 females and 46 males and 38 neonates (<2 days old), 27 juveniles (2 mo to 2 yr old), and 43 adults (>2 yr old) from the Réserve Africaine de Sigean, Sigean, France, between 2014 and 2016. No significant effects of sex and season of sampling were found. No significant differences were found for all hematology and biochemistry parameters between juveniles and adults, and their results were then combined. Significant differences were found between animals >2 mo old and neonates for most tested parameters; in particular, neonates had lower hemoglobin, eosinophil count, alanine transaminase, and total protein values, but higher alkaline phosphatase values. The reference intervals obtained were similar to values previously reported for other Bovidae species.

Fatal West Nile Virus Infection in a Virginia Opossum (Didelphis virginiana) with Pulmonary Lepidic-Predominant Adenocarcinoma.

A fatal case of West Nile virus (WNV) infection was diagnosed based on histopathologic findings and the presence of WNV RNA by reverse transcription PCR in the affected organs in a Virginia opossum (Didelphis virginiana) in Quebec, Canada in 2017. Disease caused by WNV has not been described in a marsupial species.

RETROSPECTIVE STUDY OF MORTALITY IN CAPTIVE STRUTHIONIFORMES IN A FRENCH ZOO (1974-2015).

The husbandry and medical records, and necropsy reports, of 1,002 captive Struthioniformes that died at the Réserve Africaine de Sigean (France) from 1974 to 2015 were examined. The goal of this study was to determine the most common causes of mortality in ostriches ( Struthio camelus), emus ( Dromaius novaehollandiae), and American rheas ( Rhea americana), in order to highlight common causes of mortality, as well as the influence of age, gender, and rearing methods on mortality. The most prevalent cause of mortality was pathology of digestive origin in the neonates of all three species, especially yolk-sac infections, which accounted for 41% of all deaths in this captive neonate population and was especially prevalent in hand-reared neonates. Other causes included musculoskeletal disorders in emus (particularly hand-reared) and rheas; trauma in neonate ostriches, mainly due to crushing by parents; leg deformities in emu chicks and juveniles; general sepsis in hand-reared ratite chicks due to a chlamydiosis outbreaks (1989-1990); trauma by conspecifics in subadult ostriches and emus; stress myopathy in subadult rheas, particularly after introduction to a new enclosure; evisceration inflicted by herbivorous enclosure mates on adult male ostriches; fatal peritonitis following salpingitis in adult female ostriches; and death associated with ocular disorder in adult male emus. Although this study was conducted at one institution, and its results reflect this zoo's management and zootechnical practices, its findings could well have implications for management practices involving other captive Struthioniforme populations.

Seroprevalence of malignant catarrhal fever virus in captive wildebeest (Connochaetes sp.) in France.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeests (Connochaetes sp.) in sub-Saharan Africa. Although asymptomatic in wildebeest, AlHV-1 infection in a number of other ruminant species causes a severe and fatal lymphoproliferative disease named wildebeest-derived malignant catarrhal fever (WD-MCF). Several endangered species of captive ruminants are highly susceptible to developing WD-MCF if infected by AlHV-1, which is a critical concern in zoos, game reserves and wildlife parks where wildebeests are also kept in captivity. Here, we investigated the seroprevalence of AlHV-1 in 52 captive wildebeests randomly sampled from five different zoos in France. We found 46% (24/52) seropositive animals and detected AlHV-1 DNA in one of them, demonstrating that AlHV-1 infection is present in captive wildebeests in France. In an interesting manner, the repartition of seropositive wildebeests was not homogenous between zoos with 100% (20/20) of seronegative animals in three parks. These results further highlight the importance of considering WD-MCF as a threat for clinically susceptible species and encourage for testing AlHV-1 infection in captive wildebeests as a management control strategy.

Physiological costs of infection: herpesvirus replication is linked to blood oxidative stress in equids.

Viruses may have a dramatic impact on the health of their animal hosts. The patho-physiological mechanisms underlying viral infections in animals are, however, not well understood. It is increasingly recognized that oxidative stress may be a major physiological cost of viral infections. Here we compare three blood-based markers of oxidative status in herpes positive and negative individuals of the domestic horse (Equus ferus caballus) and of both captive and free-ranging Mongolian khulan (Equus hemionus hemionus) and plains zebra (Equus quagga). Herpes positive free-ranging animals had significantly more protein oxidative damage and lower glutathione peroxidase (antioxidant enzyme) than negative ones, providing correlative support for a link between oxidative stress and herpesvirus infection in free-living equids. Conversely, we found weak evidence for oxidative stress in herpes positive captive animals. Hence our work indicates that environment (captive versus free living) might affect the physiological response of equids to herpesvirus infection. The Mongolian khulan and the plains zebra are currently classified as near threatened by the International Union for Conservation of Nature. Thus, understanding health impacts of pathogens on these species is critical to maintaining viable captive and wild populations.

EVALUATION OF SYMMETRIC DIMETHYLARGININE AS AN EARLY BIOMARKER OF CHRONIC KIDNEY DISEASE IN CAPTIVE CHEETAHS (ACINONYX JUBATUS).

Symmetric dimethylarginine (SDMA) has been shown to be a valuable biomarker for early detection of chronic kidney disease (CKD) in canine and feline patients. Recognition of early (subclinical) kidney disease would be of value in cheetahs (Acinonyx jubatus) as prevalence of CKD is relatively high in this species in captivity. Fifty-eight banked serum and plasma samples from seven adult cheetahs that died of CKD were analyzed for creatinine, urea, and SDMA. A marked increase in SDMA was noted on five of the tested cheetahs earlier than the rise of serum creatinine and urea (estimated 8-35 mo; mean 21.4 mo; median 22 mo). SDMA appears as an early biomarker to evaluate renal function for the diagnosis of CKD in cheetahs regardless of the cause of this disease.

DESCRIPTION OF GASTRIC ULCERS AND OF THEIR SUSPECTED, ASSOCIATED RISK FACTORS IN DECEASED WILD EQUIDS AT THE RÉSERVE AFRICAINE DE SIGEAN, FRANCE (2010-2016).

Gastric ulcers are common in domestic horses and foals, affecting at least 90% of unmedicated racehorses in active training. Despite these high prevalences in domestic horses, literature about this condition in wild equids is almost nonexistent. The presence of gastric ulcers was evaluated at necropsy in six species of wild equids that died at the Réserve Africane de Sigean, a safari park in the south of France from 2010 to 2016. Among the 55 individuals that died during that period, a description of the gastric mucosa was available in 82% (45/55) of cases. Considering the cases for which a description of the gastric mucosa was available, the prevalence of gastric ulcers was 64% (29/45). The highest prevalences were noted in Grant's zebra (Equus quagga boehmi) and Hartmann's mountain zebra (Equus zebra hartmannae) at 83% and 100%, respectively. In contrast to what is reported in domestic foals, gastric ulcerations were only diagnosed in one foal (out of 11 foals necropsied). The higher prevalence was noted in young individuals (3-36 mo old) at 93% (14/15); the lesions observed consisted mainly of single to multiple, superficial lesions, of which, only the mucosa was missing; these superficial lesions are often considered not clinically significant. The prevalence was lower for adults (74%; 14/19), but lesions were deeper or with a hyperemic or inflammatory appearance. All the lesions observed were located in the gastric, nonglandular, stratified squamous mucosa, along the margo plicatus. No statistical correlation could be found between the development of gastric ulcers and an ongoing, chronic pathologic process or a digestive tract pathology. The detection of gastric ulcers was, therefore, significantly greater in wild equids isolated in smaller enclosures. Nevertheless, additional larger-scale research is needed to point out predisposing factors in equids under human care.

COLONIC ADENOCARCINOMAS IN A FAMILIAL GROUP OF CAPTIVE AMUR RAT SNAKES (ELAPHE SCHRENCKI).

This article reports five cases of colonic adenocarcinomas in a family group of captive Amur rat snake (Elaphe schrenckii) from the Réserve Africaine de Sigean, France. This tumor was detected in three females and two males, all adults, and accounted for 16% of causes of death of adults of this species at this institution from 1986 to 2013. Grossly, mild to marked thickening of the intestinal wall cranially to the cloaca was found in four cases; tan to yellow firm masses were noted in the distal intestinal wall in the other case. Microscopically, neoplasms were characterized by infiltrating, poorly circumscribed, and unencapsulated nests of epithelial cells. Marked anisokaryosis and anisocytosis were seen in all neoplasms. The etiology of the neoplasms was not determined, but the familial clustering suggests a common etiologic factor.

DIAGNOSIS OF PREGNANCY IN BARBARY SHEEP (AMMOTRAGUS LERVIA) USING A BOVINE ASSAY FOR PREGNANCY-ASSOCIATED GLYCOPROTEINS.

Pregnancy diagnosis is an important part in reproduction management of wild ruminants involved in free-ranging and captive programs. Pregnancy-associated glycoproteins (PAGs) are placenta-expressed proteins released into maternal blood circulation. Tests with high specificity have been developed and validated in domestic species and have been used in some wild ungulate species. One hundred and seventeen serum samples collected from 72 mature female Barbary sheep ( Ammotragus lervia ) were tested using a commercial enzyme-linked immunosorbent assay (ELISA) test. Pregnancy was determined either postmortem (n = 5) or by visualization of parturition (n = 33). The other sera were controls from known nonpregnant females (n = 71). The following values were obtained: sensitivity = 100.0%, specificity = 95.8%. Using a commercial ELISA for the detection of PAGs appears to be a rapid, inexpensive, and accurate test for pregnancy diagnosis in the endangered Barbary sheep. The number of offspring cannot be determined with this method.

GREATER KUDU (TRAGELAPHUS STREPSICEROS) MORTALITY IN EUROPEAN ZOOLOGICAL INSTITUTIONS: A RETROSPECTIVE STUDY.

A questionnaire was sent to 39 European institutions holding greater kudus (Tragelaphus strepsiceros), in order to determine the causes of captive greater kudu mortality. All reported macroscopic lesions and histopathologic observations, as well as other information regarding individuals that died, were analyzed to determine the most affected body systems and causes of death. Overall response rate was 31%, and 131 individuals were included in the study. The most frequently affected body systems were the digestive system (47%), respiratory system (38%), musculoskeletal system (37%), and cardiovascular system (32%). Most frequent causes of death were infectious diseases (27%) and trauma/accidents (18%); the cause was undetermined in 28% of cases. Nutrition-related disorders were difficult to assess, but results highlight possible nutritional imbalances. This retrospective study represents the first overview of greater kudu mortality in a captive population.