John M. Parker

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 Californias Sierra Nevada.

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.

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

ABSTRACT 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.

Effect of Temperature on Host Response to Batrachochytrium dendrobatidis Infection in the Mountain Yellow-legged Frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17–25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the hosts resistance to chytridiomycosis, rather than an effect on the fungus alone.

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.

High Speed Striation Pattern Recognition In Contracting Cardiac Myocytes

The understanding of muscle contraction and relaxation requires the quantitation of movement at the sub-micron level in living cells. Two complementary non-RS-170 imaging systems used for authentic real time measurement of contractile dynamics are described and compared. Images from isolated skeletal or cardiac muscle cells are projected by an optical microscope onto single line or area charge-coupled device (CCD) photodiode arrays. These data are digitized and stored for subsequent image processing and analysis. The inherently low contrast muscle striation patterns are enhanced and their rapid movement measured with an accuracy at least an order of magnitude greater than traditional limits of optical resolution. The features of each image format are complementary and when combined provide the maximum overall information in time and space.

Chapter 23 – Aquatic Facilities

Publisher Summary This chapter addresses various issues that should be considered for setting up an aquatic animal facility. Aquatic animal facilities require special design considerations beyond those of conventional laboratory animal facilities. Water, alone, can cause serious damage to facility structural components through leakage and condensation. The composition, pH, and softness or hardness of water should be considered when plumbing materials and treatment processes are selected. Knowledge of water additives, such as disinfectants, is required for appropriate selection and design of water filtration systems. Water is extremely heavy, and this weight must be factored into construction of tanks, racks, support structures, and transport equipment. Serious personnel injury risks in an aquatic facility include slipping on pooled water and electrocution. Facility design precautions are critical to minimize these hazards. Facility structures, machinery, and equipment must be capable of withstanding high levels of moisture. All wall, floor, and ceiling treatments should be impervious to water. The floor surface should be evenly sloped and relatively smooth. Similar wall construction criteria apply for aquatic facilities as are recommended for conventional facilities.