Assessing cervical dislocation as a humane euthanasia method in mice.

Research investigators often choose to euthanize mice by cervical dislocation (CD) when other methods would interfere with the aims of a research project. Others choose CD to assure death in mice treated with injected or inhaled euthanasia agents. CD was first approved for mouse euthanasia in 1972 by the AVMA Panel on Euthanasia, although scientific assessment of its humaneness has been sparse. Here we compared 4 methods of spinal dislocation--3 targeting the cervical area (CD) and one the thoracic region--in regard to time to respiratory arrest in anesthetized mice. Of the 81 mice that underwent CD by 1 of the 3 methods tested, 17 (21%) continued to breathe, and euthanasia was scored as unsuccessful. Postmortem radiography revealed cervical spinal lesions in 5 of the 17 cases of unsuccessful CD euthanasia. In addition, 63 of the 64 successfully euthanized mice had radiographically visible lesions in the high cervical or atlantooccipital region. In addition, 50 of 64 (78%) mice euthanized successfully had radiographically visible thoracic or lumbar lesions or both. Intentionally creating a midthoracic dislocation in anesthetized mice failed to induce respiratory arrest and death in any of the 18 mice subjected to that procedure. We conclude that CD of mice holds the potential for unsuccessful euthanasia, that anesthesia could be valuable for CD skills training and assessment, and that postmortem radiography has minimal promise in quality-control assessments.

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak.

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.

Effects of multimodal analgesia on the success of mouse embryo transfer surgery.

Multimodal analgesia is promoted as the best practice pain management for invasive animal research procedures. Universal acceptance and incorporation of multimodal analgesia requires assessing potential effects on study outcome. The focus of this study was to assess effects on embryo survival after multimodal analgesia comprising an opioid and nonsteroidal antiinflammatory drug (NSAID) compared with opioid-only analgesia during embryo transfer procedures in transgenic mouse production. Mice were assigned to receive either carprofen (5 mg/kg) with buprenorphine (0.1 mg/kg; CB) or vehicle with buprenorphine (0.1 mg/kg; VB) in a prospective, double-blinded placebo controlled clinical trial. Data were analyzed in surgical sets of 1 to 3 female mice receiving embryos chimeric for a shared targeted embryonic stem-cell clone and host blastocyst cells. A total of 99 surgical sets were analyzed, comprising 199 Crl:CD1 female mice and their 996 offspring. Neither yield (pups weaned per embryo implanted in the surgical set) nor birth rate (average number of pups weaned per dam in the set) differed significantly between the CB and VB conditions. Multimodal opioid-NSAID analgesia appears to have no significant positive or negative effect on the success of producing novel lines of transgenic mice by blastocyst transfer.

A watertight acrylic-free titanium recording chamber for electrophysiology in behaving monkeys.

Neurophysiological recording in alert monkeys requires the creation of a permanent aperture in the skull for repeated insertion of microelectrodes. Most laboratories use polymethyl methacrylate to attach a recording chamber over the skull opening. Here, we describe a titanium chamber that fastens to the skull with screws, using no polymethyl methacrylate. The gap between the base of the chamber and the skull is filled with hydroxyapatite, forming a watertight gasket. As the chamber base osseointegates with the skull, the hydroxyapatite is replaced with bone. Rather than having a finite lifetime, the recording chamber becomes more firmly anchored the longer it is in place. It has a small footprint, low profile, and needs little maintenance to control infection. Toilette consists of occasional application of betadine to clean the scalp margin, followed by application of neomycin, polymyxin, and bacitracin ointment. Antibiotic is also placed inside the chamber to suppress bacterial proliferation. Thickening of the dura within the chamber can be prevented by regular application of mitocycin C and/or bevacizumab, an antibody against vascular endothelial growth factor. By conducting an e-mail survey, this protocol for chamber maintenance was compared with procedures used in 37 other vision research laboratories. Refinement of appliances and techniques used for recordings in awake monkeys promises to increase the pace of scientific discovery and to benefit animal welfare.

Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis.

Global amphibian decline by chytridiomycosis is a major environmental disaster that has been attributed to either recent fungal spread or environmental change that promotes disease. Here, we present a population genetic comparison of Batrachochytrium dendrobatidis isolates from an intensively studied region of frog decline, the Sierra Nevada of California. In support of a novel pathogen, we find low diversity, no amphibian-host specificity, little correlation between fungal genotype and geography, local frog extirpation by a single fungal genotype, and evidence of human-assisted fungus migration. In support of endemism, at a local scale, we find some diverse, recombining populations. Therefore neither epidemic spread nor endemism alone explains this particular amphibian decline. Recombination raises the possibility of resistant sporangia and a mechanism for rapid spread as well as persistence that could greatly complicate global control of the pathogen.

Effects of chytrid and carbaryl exposure on survival, growth and skin peptide defenses in foothill yellow-legged frogs.

Environmental contaminants and disease may synergistically contribute to amphibian population declines. Sub-lethal levels of contaminants can suppress amphibian immune defenses and, thereby, may facilitate disease outbreaks. We conducted laboratory experiments on newly metamorphosed foothill yellow-legged frogs (Rana boylii) to determine whether sublethal exposure to the pesticide carbaryl would increase susceptibility to the pathogenic chytrid fungus Batrachochytrium dendrobatidis that is widely associated with amphibian declines. We examined the effect of carbaryl alone, chytrid alone, and interactions of the two on individual survival, growth, and antimicrobial skin defenses. We found no effect of chytrid, carbaryl, or their interaction on survival. However, chytrid infection reduced growth by approximately one-half. This is the first report of suppressed growth in post-metamorphic amphibians due to infection with chytrid. Rana boylii skin peptides strongly inhibited chytrid growth in vitro, which may explain why chytrid exposure did not result in significant mortality. Skin peptide defenses were significantly reduced after exposure to carbaryl suggesting that pesticides may inhibit this innate immune defense and increase susceptibility to disease.

Emerging infectious disease as a proximate cause of amphibian mass mortality.

A newly discovered infectious disease of amphibians, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis, is implicated in population declines and possible extinctions throughout the world. The purpose of our study was to examine the effects of B. dendrobatidis on the mountain yellow-legged frog (Rana muscosa) in the Sierra Nevada of California (USA). We (1) quantified the prevalence and incidence of B. dendrobatidis through repeat surveys of several hundred R. muscosa populations in the southern Sierra Nevada; (2) described the population-level effects of B. dendrobatidis on R. muscosa population abundance; and (3) compared the mortality rates of infected and uninfected R. muscosa individuals from pre- through post-metamorphosis using both laboratory and field experiments. Mouthpart inspections conducted in 144 and 132 R. muscosa populations in 2003 and 2004, respectively, indicated that 19% of R. muscosa populations in both years showed indications of chytridiomycosis. Sixteen percent of populations that were uninfected in 2003 became infected by 2004. Rana muscosa population sizes were reduced by an average of 88% following B. dendrobatidis outbreaks at six sites, but at seven B. dendrobatidis-negative sites, R. muscosa population sizes increased by an average of 45% over the same time period. In the laboratory, all infected R. muscosa developed fatal chytridiomycosis after metamorphosis, while all uninfected individuals remained healthy. In the field experiment in which R. muscosa tadpoles were caged at infected and uninfected sites, 96% of the individuals that metamorphosed at infected sites died vs. 5% at the uninfected sites. These studies indicate that chytridiomycosis causes high mortality in post-metamorphic R. muscosa, that this emerging disease is the proximate cause of numerous observed R. muscosa population declines, and that the disease threatens this species with extirpation at numerous sites in California's Sierra Nevada.

A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis. The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii, is closely related to M. ulcerans and Mycobacterium marinum, and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans, strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis).

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mug ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T).

Columbian black-tailed deer (Odocoileus hemionus columbianus) as hosts for Borrelia spp. in northern California.

The prevalence of infection of Columbian black-tailed deer (Odocoileus hemionus columbianus) with Borrelia spp. was evaluated in an area of northwestern California (USA) where Lyme disease is endemic and the relapsing-fever group spirochete Borrelia coriaceae is enzootic, and in a far-removed comparison area having a disparate climate and lower density of vector ticks. Blood samples collected from both deer herds in 1987, 1988, and from 2000-02 were assayed for borrelial infection with microscopic and molecular methods. Serum specimens from two (5%) of 39 deer from the Dye Creek Preserve in Tehama County versus 13 (20%) of 64 animals from the Hopland Research and Extension Center (HREC) in Mendocino County, California were polymerase chain reaction (PCR) test positive for B. burgdorferi sensu lato. DNA sequencing analyses revealed that eight animals were infected with B. bissettii, six with three unclassified genotypes, and one with B. burgdorferi sensu stricto. One serum sample (2%) from HREC was positive for a relapsing-fever group spirochete that had a 16S rRNA sequence homology of 99% with the C053 type strain of B. coriaceae. Spirochetes undetermined to geno-species were detected in thick-blood drops prepared from three (8%) of 36 deer from the HREC by direct immunofluorescence. Adults of the hippoboscid flies Lipoptena depressa (n=73) and Neolipoptena ferrisi (n=24), the Pacific Coast tick (Dermacentor occidentalis) (n=22), and the western black-legged tick (Ixodes pacificus) (n=1) that had been removed from deer from both study areas in 2002 were PCR test negative for borreliae. The occurrence of diverse borreliae in deer from northern California confounds and, consequently, reduces the utility of borrelial serosurveys for detecting specific genospecies, unless they are complemented by more specific assays (e.g., immunoblotting, PCR/sequencing analysis).

Amphibian oncology.

Spontaneous neoplasia is rare in all three orders of Amphibia. Tumors are documented in most major organ systems, and some have various underlying etiologies, including viral infection, environmental contaminants, and genetic predisposition. Currently,treatment options are limited to removal of the predisposing condition(s), palliative care, surgical excision, and, when necessary,humane euthanasia may be elected. Neoplasia must be distinguished from common infectious, nonneoplastic conditions that can negatively impact population health. This article is a review of the more common types of neoplasia in amphibians, and includes clinically relevant information, such as biologic behavior,anatomy, associated etiologies, major differential diagnoses, and clinical management.

Characterization of a Mycobacterium ulcerans-like infection in a colony of African tropical clawed frogs (Xenopus tropicalis).

A nontuberculous Mycobacterium ulcerans-like organism was identified as the causative agent of an epizootic of mycobacteriosis in a colony of African tropical clawed frogs, Xenopus (Silurana) tropicalis, at the University of California, Berkeley. Diverse clinical signs of disease were observed, including lethargy, excess buoyancy, coelomic effusion, cutaneous ulcers, and granulomas. Visceral granulomas, ulcerative and granulomatous dermatitis, coelomitis, and septicemia were common findings at necropsy. Identification of M. ulcerans-like organisms was based on molecular and phenotypical characteristics. The findings of this investigation indicate that this M. ulcerans-like organism is a primary cause of morbidity and mortality in aquatic anurans and should be considered in the differential diagnosis of coelomic effusion in amphibians. Furthermore, if this Mycobacterium species ultimately is identified as M. ulcerans, X. tropicalis should be considered a potential source of this important public health pathogen.

Clinical diagnosis and treatment of epidermal chytridiomycosis in African clawed frogs (Xenopus tropicalis).

An investigation was conducted to determine the cause of morbidity and mortality in a collection of 55 adult male Xenopus (Silurana) tropicalis at the University of California, Berkeley. More than 80% of affected frogs died during the epizootic. All frogs were anorectic and lethargic, had dark pigmentation and excess skin sloughing, and lacked a slime layer. Histologic examination revealed severe hyperplastic and spongiotic dermatitis associated with colonization of the stratum corneum by large numbers of zoosporangia diagnostic of Batrachochytrium dendrobatidis. Treatment with a commercial formalin/malachite green solution at a dilution of 0.007 ml/L of tank water for 24 h, repeated every other day for four treatments, eliminated the organism and was curative. These findings are indicative of epidermal chytridiomycosis as a primary cause of death in this collection of X. tropicalis.