Semi-quantitative Assessment of Alzheimer's-like Pathology in Two Aged Polar Bears (Ursus maritimus).

Age-associated neurodegenerative changes, including amyloid ß (Aß) plaques, neurofibrillary tangles (NFTs), and amyloid angiopathy comparable to those seen in the brains of human patients with Alzheimer's disease (AD), have been reported in the brains of aged bears. However, the significance of these findings in bears is unclear due to the difficulty in assessing cognitive impairment and the lack of standardized approaches for the semiquantitative evaluation of Aß plaques and NFTs. In this study, we evaluate the neuropathologic changes in archival brain tissue of 2 aged polar bears (Ursus maritimus, ages 28 and 37) using the National Institute of Aging-Alzheimer Association (NIA-AA) consensus guidelines for the neuropathologic assessment of Alzheimer's Disease (AD). Both bears had an Aß (A) score of 3 of 3, Braak stage (B score) of 2 of 3, and neuritic plaque (C) score of 3 of 3. These findings are consistent with the neurodegenerative changes observed in brains of patients with AD. The application of NIA-AA consensus guidelines, as applied to the neuropathologic assessment of the aged bears in this report, demonstrates the use of standardized semiquantitative assessment systems for comparative, translational studies of aging in a vulnerable wildlife species.

Degenerative Osteoarthropathy in Laboratory Housed Xenopus (Silurana) tropicalis.

In this case study, 15 adult laboratory Xenopus (Silurana) tropicalis (7 adult males and 8 adult females) were examined for nodular enlargements of the clawed digits (digits 0, I, II, and III) on the hind feet. Radiographs showed smoothly margined, rounded, peripherally mineralized lesions arising from the distal phalanges of digits 0-III with osteoproductive and osteolytic components in all frogs. Micro computed tomography (microCT) scans further revealed interphalangeal (IP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joint osteoarthritis characterized by periarticular new bone formation, rounded mineral foci both peripherally and centrally within the joints, and more rarely, linear mineralization palmar/plantar to the joints in the flexor tendons. In the nonclawed digits, the shape of the distal phalanx was variably distorted and both subluxation and malangulation of IP joints were identified. Histologically, nodules corresponded to a peripheral rim of mature cortical bone surrounding central adipose tissue, scattered hematopoietic elements, and residual bone of the distal phalanx. Occasionally, the peripheral rim of cortical bone extended proximally to encompass the distal aspect of adjacent phalanx. MCP, MTP and IP joint spaces of most digits exhibited widespread osteoarthritis characterized by periarticular cartilaginous or osseous metaplasia, bony remodeling, and less frequently, granulomatous osteomyelitis. Nutritional analyses of the feed did not indicate imbalances nor were the lesions consistent with metabolic bone disease. The exact etiopathogenesis of these lesions is unknown; however, we hypothesize that the osteoarthritic changes are due to a combination of the frogs' mature age, the unique structure of the Xenopus spp. claw, genetics and biomechanical forces on the digits and distal phalanges of the hind feet.

Axial Skeletal Malformations in Genetically Modified Xenopus laevis and Xenopus tropicalis.

Skeletal malformations in captive-bred, adult Xenopus spp., have not previously been reported. Here we describe 10 sexually mature, genetically modified laboratory frogs (6 Xenopus laevis and 4 Xenopus tropicalis) with axial skeletal abnormalities. The young adult frogs were described by veterinary staff as presenting with "hunchbacks," but were otherwise considered to be in good health. All affected frogs were genetically engineered using various techniques: transcription activator-like effector nucleases (TALEN) editing using thyroid hormone receptor α TALEN mRNA, restriction enzyme-mediated integration methods involving insertion of the inducible transgene pCAR/TRDN, or via I-SceI meganuclease transgenesis using either pDRTREdpTR-HS4 or pDPCrtTA-TREG-HS4 plasmid sequences. Radiographic findings (6 frogs) and gross necropsy (10 frogs) revealed vertebral column malformations and sacroiliac deformities that resulted in moderate to severe kyphosis and kyphoscoliosis. These findings were confirmed and additional skeletal abnormalities were identified using computed tomography to create a 3D reconstruction of 4 frogs. Additional findings visible on the 3D reconstructions included incomplete vertebral segmentation, malformed transverse processes, and a short and/or curved urostyle. Histopathologic findings included misshapen intervertebral joints with nonconforming articular surfaces, narrowed joint cavities, flattened or irregularly-formed articular cartilage, irregular maturation lines and nonpolarized chondrocytes, excess fibrocartilage, and evidence of irregular bone resorption and growth. While the specific etiology of the vertebral skeletal abnormalities remains unclear, possibilities include: 1) egg/oocyte physical manipulation (dejellying, microinjection, fertilization, etc.), 2) induction and expression of the transgenes, 3) inactivation (knockout) of existing genes by insertional mutagenesis, or 4) a combination of the above. Furthermore, the possibility of undetected changes in the macro or microenvironment, or a feature of the genetic background of the affected frogs cannot be ruled out.

Acute and Chronic Outcomes of Gas-Bubble Disease in a Colony of African Clawed Frogs (Xenopus laevis).

Gas-bubble disease occurs in aquatic species that are exposed to water that is supersaturated with gases. In February 2007, municipal water supersaturated with gas was inadvertently pumped into the vivarium's aquatic housing systems and affected approximately 450 adult female Xenopus laevis. The inflow of supersaturated water was stopped immediately, the holding tanks aggressively aerated, and all experimental manipulations and feeding ceased. Within the first 6 h after the event, morbidity approached 90%, and mortality reached 3.5%. Acutely affected frogs showed clinical signs of gas-bubble disease: buoyancy problems, micro- and macroscopic bubbles in the foot webbing, hyperemia in foot webbing and leg skin, and loss of the mucous slime coat. All of the frogs that died or were euthanized had areas of mesenteric infarction, which resulted in intestinal epithelial necrosis and degeneration of the muscular tunic. Over the subsequent 2 wk, as gas saturation levels returned to normal, the clinical symptoms resolved completely in the remaining frogs. However, 3 mo later, 85% of them failed to lay eggs or produce oocytes, and the remaining 15% produced oocytes of low number and poor quality, yielding cytosolic extracts with poor to no enzymatic activity. Histology of the egg mass from a single 2- to 3-y-old frog at 3 mo after disease resolution revealed irregularly shaped oocytes, few large mature oocytes, and numerous small, degenerating oocytes. At 6 mo after the incident, the remaining frogs continued to fail to produce eggs of sufficient quantity or quality after hormonal priming. The researchers consequently opted to cull the remainder of the colony and repopulate with new frogs.

Prevalence of Batrachochytrium dendrobatidis in Xenopus collected in Africa (1871-2000) and in California (2001-2010).

International trade of the invasive South African clawed frog (Xenopus laevis), a subclinical carrier of the fungal pathogen Batrachochytrium dendrobatis (Bd) has been proposed as a major means of introduction of Bd into naïve, susceptible amphibian populations. The historical presence of Bd in the indigenous African population of Xenopus is well documented. However, there are no reports documenting the presence of Bd in wild Xenopus populations in the US, particularly in California where introduced populations are well-established after intentional or accidental release. In this report, a survey was conducted on 178 archived specimens of 6 species of Xenopus collected in Africa from 1871-2000 and on 23 archived specimens (all wild-caught Xenopus laevis) collected in California, USA between 2001 and 2010. The overall prevalence rate of Bd in the tested Xenopus was 2.8%. The earliest positive specimen was X. borealis collected in Kenya in 1934. The overall prevalence of Bd in the X. laevis collected in California was 13% with 2 positive specimens from 2001 and one positive specimen from 2003. The positive Xenopus (3/23) collected in California were collected in 2001 (2/3) and 2003 (1/3). These data document the presence of Bd-infected wild Xenopus laevis in California. The findings reported here support the prevailing hypothesis that Bd was present as a stable, endemic infection in Xenopus populations in Africa prior to their worldwide distribution likely via international live-amphibian trade.

Evolutionary perspectives into placental biology and disease.

In all mammals including humans, development takes place within the protective environment of the maternal womb. Throughout gestation, nutrients and waste products are continuously exchanged between mother and fetus through the placenta. Despite the clear importance of the placenta to successful pregnancy and the health of both mother and offspring, relatively little is understood about the biology of the placenta and its role in pregnancy-related diseases. Given that pre- and peri-natal diseases involving the placenta affect millions of women and their newborns worldwide, there is an urgent need to understand placenta biology and development. Here, we suggest that the placenta is an organ under unique selective pressures that have driven its rapid diversification throughout mammalian evolution. The high divergence of the placenta complicates the use of non-human animal models and necessitates an evolutionary perspective when studying its biology and role in disease. We suggest that diversifying evolution of the placenta is primarily driven by intraspecies evolutionary conflict between mother and fetus, and that many pregnancy diseases are a consequence of this evolutionary force. Understanding how maternal-fetal conflict shapes both basic placental and reproductive biology - in all species - will provide key insights into diseases of pregnancy.

Mortality and morbidity in African clawed frogs (Xenopus laevis) associated with construction noise and vibrations.

In Spring 2008, 175 adult female Xenopus laevis were exposed to construction-related vibrations that caused overt water rippling in the frog tanks. The 3 affected tanks were custom-built static, 300-gal 'pond-style' tanks that sat on the floor of the housing room. The water in the tank developed visible ripples as a result of the vibrations transmitted through the floor during jack-hammering in an adjacent room that was approximately 10 ftaway. All frogs in the tanks displayed buoyancy problems, excessive air gulping, and skin sloughing; ultimately 7 frogs died. In addition, these 7 animals were bloated, and 5 of these 7 had regurgitated and everted their stomach and distal esophagus into the oral cavity, resulting in airway obstruction and death. Gross pathologic findings included regurgitation and eversion of the stomach of the distal portion of the esophagus into the oral cavity, obstruction of the airway, and lung overinflation. No significant histologic lesions were observed. Construction vibrations transmitted through the water appeared to have disrupted the mechanoreceptive function of the lateral line system, resulting in overstimulation of the noxious feeding response, regurgitation, and eversion of the stomach and distal esophagus into the oral cavity and subsequent suffocation due to airway obstruction. After immediate cessation of the jack-hammering and relocation of the remaining frogs, no additional morbidities or mortalities occurred.

The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

Evaluation and refinement of euthanasia methods for Xenopus laevis.

The most common method of euthanasia for Xenopus species is by immersion in tricaine methane sulfonate solution (MS222). A wide range of doses of MS222 (0.5 to 5 g/L) have been recommended, but few reports describe dose-response testing, the time to loss of consciousness, or the reliability of euthanasia. The objective of this study is to evaluate the efficacy of immersing individual and groups of frogs in MS222 at concentrations ranging from 1 to 5 g/L for euthanasia and of 3 less-common methods: intracoelomic injection of MS222, intracoelomic injection of sodium pentobarbital with phenytoin, and ventral cutaneous application of benzocaine gel. Our results indicate that immersion for at least 1 h in a 5-g/L buffered solution of MS222, intracoelomic injection of 1100 mg/kg sodium pentobarbital with sodium phenytoin (equivalent to 0.3 mL solution per frog), or ventral cutaneous application of 182 mg/kg benzocaine (equivalent to a 2 cm x 1 mm of 20% benzocaine gel) is necessary to euthanize adult X. laevis and ensure complete cessation of the heartbeat without recovery. These doses are considerably higher than those previously recommended for this species.

Assessment of the utility of using intra- and intervertebral minimum sagittal diameter ratios in the diagnosis of cervical vertebral malformation in horses.

Cervical vertebral malformation is one of the most common causes of ataxia in horses. The most important factor in the diagnosis of cervical vertebral malformation is the identification of cervical vertebral canal stenosis, but published data for minimum sagittal diameter ratios in adult horses are only available for C4-C7 intravertebral sites. Intra- and intervertebral sagittal diameter ratios at C2-C7 were evaluated in 26 ataxic horses, for which a complete clinical and neuropathological evaluation was undertaken. Eight of these horses were diagnosed with cervical vertebral malformation. In these horses the majority of compressive lesions were intervertebral. The mean sagittal diameter ratios of horses with cervical vertebral malformation were significantly smaller than those of horses without cervical vertebral malformation, and for an individual horse in our study, the site with the smallest intervertebral sagittal diameter ratio was always the site at which the spinal cord was compressed. Mean sagittal diameter ratio intravertebral site measurements of horses with cervical vertebral malformation were smaller than those of horses without cervical vertebral malformation; however, the site of compression could not be predicted from the data. For our dataset, horses with a sagittal diameter ratio of < or = 0.485 at any inter- or intravertebral site could be correctly classified as having cervical vertebral malformation, and sagittal diameter ratio measurements were an effective tool to identify at least one site of compression in an individual case.

Refuge cover decreases the incidence of bite wounds in laboratory South African clawed frogs (Xenopus laevis).

The Guide for the Care and Use of Laboratory Animals recommends environmental enrichment for all laboratory animals, including amphibians. In this study, we evaluated the effect of adding environmental enrichment in the form of acrylonitrile- butadiene-styrene (ABS) pipes as covered refuge for laboratory Xenopus laevis housed in 2 pond-style tanks (capacity, 300 l; stocking density, approximately 150 frogs/tank; dimensions, 1.3 x 1.8 x 1.3 m). Medical records from animals housed in these 2 ponds between 1 January 2001 to 31 December 2003 revealed the incidence of bite wounds to be 5.0%, 4.0%, and 5.0% annually, respectively, and indicated 2 episodes of cannibalism (in 2003). In January 2004, we added ABS pipes as refuge housing to these tanks and continued to monitor the number of bite wounds and cannibalism. Over the following 24 mo (1 January 2004 to 1 January 2006), the incidence of bite wounds declined to 0.3% and 0.7% annually, respectively; no episodes of cannibalism were reported. The results of this investigation indicate that environmental enrichment in the form of ABS pipes for refuge cover has a quantifiable beneficial effect on the physical and social wellbeing of laboratory Xenopus laevis.

Ovarian hyperstimulation syndrome in gonadotropin-treated laboratory South African clawed frogs (Xenopus laevis).

Ovarian hyperstimulation syndrome (OHS) is a rare but sometimes fatal iatrogenic complication of ovarian stimulation associated with the administration of exogenous gonadotropins to women undergoing treatment for infertility. Laboratory Xenopus spp are commonly treated with human chorionic gonadotropin (hCG) to stimulate ovulation and optimize the number of oocytes harvested for use in biomedical research. Here we report cases of OHS in 2 gonadotropin-treated laboratory Xenopus laevis. After receiving hCG, the frogs developed severe subcutaneous accumulation of fluid, coelomic distention, and whole-body edema and were unable to dive, although they continued to eat and swim. At postmortem examination, extensive subcutaneous edema was present; ascites and massive numbers of free-floating eggs were found in the coelomic cavity and in aberrant locations: around the heart-sac and adhered to the liver capsule. Whole-body edema, gross enlargement of the ovaries, ascites, and abdominal distention are findings comparable to those observed in women with OHS. The pathophysiology of OHS is thought to be related to hormonally induced disturbances of vasoactive mediators, one of which may be vascular endothelial growth factor secreted by theca and granulosa cells. We know of no other report describing OHSlike symptoms in gonadotropin-treated frogs, and the cases described here are 2 of the 3 we have observed at our respective institutions over the last 6 y. According to these results, OHS appears to be rare in gonadotropin-treated laboratory Xenopus. However, the condition should be included in the differential diagnosis for the bloated frog.

Postoperative analgesics in South African Clawed frogs (Xenopus laevis) after surgical harvest of oocytes.

Heightened awareness for the welfare of earlier-evolved laboratory species has prompted increasing inquiries by institutional animal care committees, investigators, and laboratory animal veterinarians regarding the need for post-surgical analgesics in laboratory Xenopus. Basic research into the mechanisms and regulation of pain in Rana pipiens has demonstrated the clinical potential of opioid, alpha2-adrenergic, and non-opioid analgesic agents in amphibians. However, clinical studies using objectively established indices of amphibian pain, or pharmacological studies in either Rana pipiens or laboratory Xenopus have not been conducted. As discussed above, comparison of limited lethality data suggests that the safety index for these agents is quite narrow in Rana pipiens. Analgesic use in laboratory Xenopus has the added risk of drowning due to over sedation. Drug doses extrapolated from such studies and intended to provide pain relief in Xenopus should therefore be considered very carefully. An additional concern for laboratory Xenopus is that the effects of these agents on amphibian oogenesis, oocyte quality, and embryogenesis are unknown. As the numbers of laboratory Xenopus used in basic and biomedical research continues to increase, clinical studies that address all of these issues cannot come too soon.

Cryptosporidiosis associated with emaciation and proliferative gastritis in a laboratory-reared South African clawed frog (Xenopus laevis).

A 2-year-old emaciated female South African clawed frog (Xenopus laevis) was euthanized because of chronic weight loss. At necropsy, there was no evidence of bacterial, fungal or viral disease; however, the histopathologic findings indicated a proliferative gastritis and the presence of numerous cryptosporidial stages throughout the intestinal tract. Crytosporidial oocysts were present in the water taken from the aquarium housing the infected frog and were likely shed by the sick frog; however, the exact source of the oocysts could not be identified. Water samples from other frog aquaria in the facility did not contain cryptosporidial oocysts. Some Cryptosporidium species are important zoonotic pathogens and, to our knowledge, this is the first report of disease associated with Cryptosporidium infection in a laboratory Xenopus laevis.

Factors affecting oogenesis in the South African clawed frog (Xenopus laevis).

Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.