Greater Species Richness of Bacterial Skin Symbionts Better Suppresses the Amphibian Fungal Pathogen Batrachochytrium Dendrobatidis.

The symbiotic microbes that grow in and on many organisms can play important roles in protecting their hosts from pathogen infection. While species diversity has been shown to influence community function in many other natural systems, the question of how species diversity of host-associated symbiotic microbes contributes to pathogen resistance is just beginning to be explored. Understanding diversity effects on pathogen resistance could be particularly helpful in combating the fungal pathogen Batrachochytrium dendrobatidis (Bd) which has caused dramatic population declines in many amphibian species and is a major concern for amphibian conservation. Our study investigates the ability of host-associated bacteria to inhibit the proliferation of Bd when grown in experimentally assembled biofilm communities that differ in species number and composition. Six bacterial species isolated from the skin of Cascades frogs (Rana cascadae) were used to assemble bacterial biofilm communities containing 1, 2, 3, or all 6 bacterial species. Biofilm communities were grown with Bd for 7 days following inoculation. More speciose bacterial communities reduced Bd abundance more effectively. This relationship between bacterial species richness and Bd suppression appeared to be driven by dominance effects-the bacterial species that were most effective at inhibiting Bd dominated multi-species communities-and complementarity: multi-species communities inhibited Bd growth more than monocultures of constituent species. These results underscore the notion that pathogen resistance is an emergent property of microbial communities, a consideration that should be taken into account when designing probiotic treatments to reduce the impacts of infectious disease.

American Bullfrogs (Lithobates catesbeianus) Resist Infection by Multiple Isolates of Batrachochytrium dendrobatidis, Including One Implicated in Wild Mass Mortality.

The emerging amphibian disease chytridiomycosis varies in severity depending on host species. Within species, disease susceptibility can also be influenced by pathogen variation and environmental factors. Here, we report on experimental exposures of American bullfrogs (Lithobates catesbeianus) to three different isolates of Batrachochytrium dendrobatidis (Bd), including one implicated in causing mass mortality of wild American bullfrogs. Exposed frogs showed low infection prevalence, relatively low infection load, and lack of clinical disease. Our results suggest that environmental cofactors are likely important contributors to Bd-associated American bullfrog mortality and that this species both resists and tolerates Bd infection.

Correlates of virulence in a frog-killing fungal pathogen: evidence from a California amphibian decline.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused declines and extinctions in amphibians worldwide, and there is increasing evidence that some strains of this pathogen are more virulent than others. While a number of putative virulence factors have been identified, few studies link these factors to specific epizootic events. We documented a dramatic decline in juvenile frogs in a Bd-infected population of Cascades frogs (Rana cascadae) in the mountains of northern California and used a laboratory experiment to show that Bd isolated in the midst of this decline induced higher mortality than Bd isolated from a more stable population of the same species of frog. This highly virulent Bd isolate was more toxic to immune cells and attained higher density in liquid culture than comparable isolates. Genomic analyses revealed that this isolate is nested within the global panzootic lineage and exhibited unusual genomic patterns, including increased copy numbers of many chromosomal segments. This study integrates data from multiple sources to suggest specific phenotypic and genomic characteristics of the pathogen that may be linked to disease-related declines.

Prevalence of selected pathogens in western pond turtles and sympatric introduced red-eared sliders in California, USA.

Pathogen introduction by invasive species has been speculated to be a cause of declining western pond turtle Emys marmorata populations in California, USA. This study determined the prevalence of Ranavirus spp., Herpesvirus spp., Mycoplasma spp. (via polymerase chain reaction of blood and nasal flush contents), and Salmonella spp. infection (via fecal culture) in native E. marmorata and invasive red-eared sliders Trachemys scripta elegans and compared infection prevalence in E. marmorata populations sympatric with T. scripta elegans to E. marmorata populations that were not sympatric by sampling 145 E. marmorata and 33 T. scripta elegans at 10 study sites throughout California. Mycoplasma spp. were detected in both species: prevalence in E. marmorata was 7.8% in the northern, 9.8% in the central, and 23.3% in the southern California regions. In T. scripta elegans, Mycoplasma spp. were not detected in the northern California region but were detected at 4.5 and 14.3% in the central and southern regions, respectively. All turtles tested negative for Herpesvirus spp. and Ranavirus spp. Enteric bacteria but not Salmonella spp. were isolated from feces. E. marmorata populations that were sympatric with T. scripta elegans did not have increased risk of Mycoplasma spp. infection. For E. marmorata, there was a significant association between Mycoplasma spp. infection and lower body weight and being located in the southern California region. This study is the first of its kind to document pathogen prevalence in native E. marmorata habitats and those sympatric with T. scripta elegans in California.

Development and validation of a fecal PCR assay for Notoedres cati and application to notoedric mange cases in bobcats (Lynx rufus) in Northern California, USA.

Notoedric mange in felids is a devastating disease caused by a hypersensitivity reaction to the mite Notoedres cati. The burrowing of the mite causes intense pruritis resulting in self-mutilation, secondary bacterial infection, and often death of affected felids if left untreated. Our understanding of how notoedric mange is maintained in felid populations, and the true geographic extent of infestations, has been hampered because wild felids are elusive and, thus, traditional diagnostic methods are difficult to implement. To create a noninvasive diagnostic test, we developed and validated a novel PCR assay to detect N. cati DNA in fecal samples of bobcats (Lynx rufus) and used this assay to investigate a recent outbreak of mange in northern California, United States. Although the fecal PCR assay was 100% specific and could detect as few as 1.9 mites/200 µg of feces, it had a moderate sensitivity of 52.6%, potentially due to intermittent shedding of mites in feces or fecal PCR inhibitors. In a field investigation, 12% (95% confidence interval [CI]: 0.06, 0.23) of fecal samples (n=65) collected from Rancho San Antonia County Park and Open Space Preserve in Santa Clara County, California were PCR-positive for N. cati. When this estimate was adjusted for test sensitivity, the corrected proportion for fecal samples containing N. cati was 23% (95% CI: 0.14, 0.36), suggesting widespread mange in this area. This novel PCR assay will be an important tool to assess the distribution and spread of notoedric mange in bobcats and could be validated to test other wild felids such as mountain lions (Puma concolor). The assay could also be used to detect notoedric mange in domestic cats (Felis catus), particularly feral cats, which may also suffer from mange and could represent an important contributor to mange in periurban bobcat populations.

Feline infectious peritonitis in a mountain lion (Puma concolor), California, USA.

Feline infectious peritonitis (FIP) is a fatal immune-mediated vasculitis of felids caused by a mutant form of a common feline enteric virus, feline enteric coronavirus. The virus can attack many organ systems and causes a broad range of signs, commonly including weight loss and fever. Regardless of presentation, FIP is ultimately fatal and often presents a diagnostic challenge. In May 2010, a malnourished young adult male mountain lion (Puma concolor) from Kern County, California, USA was euthanized because of concern for public safety, and a postmortem examination was performed. Gross necropsy and histopathologic examination revealed necrotizing, multifocal myocarditis; necrotizing, neutrophilic, and histiocytic myositis and vasculitis of the tunica muscularis layer of the small and large intestines; and embolic, multifocal, interstitial pneumonia. Feline coronavirus antigen was detected in both the heart and intestinal tissue by immunohistochemistry. A PCR for coronavirus performed on kidney tissue was positive, confirming a diagnosis of FIP. Although coronavirus infection has been documented in mountain lions by serology, this is the first confirmed report of FIP.

Eighth nerve aplasia and hypoplasia in cochlear implant candidates: the clinical perspective.

OBJECTIVE: To identify the clinical and radiologic characteristics of aplasia and hypoplasia of the eighth nerve. STUDY DESIGN: Retrospective case-note review. SETTING: Cochlear implant program. PATIENTS: All children at the authors' institution in whom the cochlear implant assessment failed because of absence or hypoplasia of the eighth nerve. INTERVENTION: Computed tomography of petrous bones and magnetic resonance imaging of the brain. MAIN OUTCOME MEASURES: Presence or absence of eighth nerve and other radiologic factors contraindicating implantation. RESULTS: Of 143 cochlear implant candidates, 237 were judged ineligible for cochlear implantation. The preimplant assessment failed in 10 candidates of 143 because of bilateral aplasia or hypoplasia of the eighth nerve (7 cases) or unilateral aplasia or hypoplasia of the eighth nerve and a contraindication to operation on the other side (3 cases). The aplasia or hypoplasia of the eighth nerve was confirmed by magnetic resonance imaging in seven cases (5%): six were syndromic (3 CHARGE, 1 VATER-RAPADILLINO, 1 Möbius, 1 Okihiro), and one was nonsyndromic autosomal-recessive. All seven children had delayed motor milestones and absence of auditory brainstem responses. CONCLUSION: Aplasia and hypoplasia of the eighth nerve are not uncommon in pediatric cochlear implant candidates, particularly in the presence of a syndrome such as CHARGE. Magnetic resonance imaging of the brain is mandatory before implantation because it can identify the presence or absence of the eighth nerve. Parents of children with profound hearing loss, delayed motor milestones, absence of auditory brainstem responses, and a syndromic diagnosis, should be made aware of this possible abnormality.

A study of the application of a frequency transposition hearing system in children.

The aim of this retrospective study was to investigate the use of the frequency transposition Transonic FT 40 system in a group of 36 children with profound sensorineural hearing loss. The group comprised 36 children (11 boys, 25 girls) aged between 2.8 and 15.6 years (mean 7.6 years) at fitting of the FT 40 device. At 48 months post-FT 40 fitting, only 11 children (30%) were still wearing the device. The children discontinued wearing the FT 40 for the following reasons: ergonomic (11%); no perceived benefit from the system (11%); cosmetic (17%); and subsequent cochlear implantation (30%). The performance of the long-term FT 40 users was investigated using the following outcome measures: aided soundfield hearing thresholds: closed set speech tests (the E2L toy test and the Manchester Picture Test discrimination test) and a speech intelligibility rating score. The 11 long-term FT 40 users (three boys, eight girls) were aged from 5.3 to 12.9 years (mean 7.2 years) at the time of initial fitting of the FT 40 device. At time of fitting, the aided soundfield thresholds with the FT 40 were significantly better at 500 Hz (p<0.04), 1 kHz (p<0.019), 2 kHz (p<0.001) and 4 kHz (p <0.001) compared to thresholds with conventional hearing aids. Six of 11 children did not show any change in performance on the closed set speech tests and two children had intelligible speech at 48 months' follow-up. A small subgroup of good performers was identified. These children were younger at age of fitting (mean 6.2 years compared to a mean of 7.7 years for the remainder of the group), were predominantly oral communicators and had identified aetiologies for their deafness. The present study suggests that there is a small subgroup of hearing-impaired children who benefit from frequency transposition hearing systems, and future suggested fitting criteria and outcome measures are listed.

Barrett's esophagus: a surgical disease.

Barrett's metaplasia can develop in patients with gastroesophageal reflux disease (GERD), and metaplasia can evolve into dysplasia and adenocarcinoma. The optimal treatment for Barrett's metaplasia and dysplasia is still being debated. The study reported herein was designed to assess the following: (1) the incidence of Barrett's metaplasia among patients with GERD; (2) the ability of laparoscopic fundoplication to control symptoms in patients with Barrett's metaplasia; (3) the results of esophagectomy in patients with high-grade dysplasia; and (4) the character of endoscopic follow-up programs of patients with Barrett's disease being managed by physicians throughout a large geographic region (northern California). Five-hundred thirty-five patients evaluated between October 1989 and February 1997 at the University of California San Francisco Swallowing Center had a diagnosis of GERD established by upper gastrointestinal series, endoscopy, manometry, and pH monitoring. Thirty-eight symptomatic patients with GERD and Barrett's metaplasia underwent laparoscopic fundoplication. Eleven other consecutive patients with high-grade dysplasia underwent transhiatal esophagectomies. Barrett's metaplasia was present in 72 (13%) of the 535 patients with GERD. The following results were achieved in patients who underwent laparoscopic fundoplication (n = 38): Heartburn resolved in 95% of patients, regurgitation in 93% of patients, and cough in 100% of patients. With regard to transhiatal esophagectomy (n = 11), the average duration of the operation was 339 +/- 89 minutes. The only significant complications were two esophageal anastomotic leaks, both of which resolved without sequelae. Mean hospital stay was 14 +/- 5 days. There were no deaths. The specimens showed high-grade dysplasia in seven patients and invasive adenocarcinoma (undiagnosed preoperatively) in four (36%). These results can be summarized as follows: (1) Barrett's metaplasia was present in 13% of patients with GERD being evaluated at a busy diagnostic center; (2) laparoscopic fundoplication was highly successful in controlling symptoms of GERD in patients with Barrett's metaplasia; (3) in patients with high-grade dysplasia esophagectomy was performed safely (invasive cancer had eluded preoperative endoscopic biopsies in one third of these patients); and (4) even though periodic endoscopic examination of Barrett's disease is universally recommended, this was actually done in fewer than two thirds of patients being managed by a large number of independent physicians in this geographic area.

Quality control guidelines for amoxicillin, amoxicillin-clavulanate, azithromycin, piperacillin-tazobactam, roxithromycin, ticarcillin, ticarcillin-clavulanate, trovafloxacin (CP 99,219), U-100592, and U-100766 for various National Committee for Clinical Laboratory Standards susceptibility testing methods. Results from multicenter trials.

Quality control guidelines for standardized antimicrobial susceptibility test methods are critical to the continuing accuracy of the tests. In this report, quality control limits were proposed for 22 organism-antimicrobial combinations with minimum inhibitory concentration (MIC) ranges of three or four log2 dilution steps. Disk diffusion zone diameter ranges were proposed for azithromycin compared with Neisseria gonorrhoeae ATCC 49226 and ticarcillin with and without clavulanic acid tested against Staphylococcus aureus ATCC 25923. The data from five or six participating laboratories produced > or = 94.7% of results within proposed MIC limits, and 94.3%-99.0% of zones were found within suggested zone guidelines. These proposed quality control ranges should be validated by in-use results from clinical laboratories.

Cellular localization of thrombomodulin in human epithelium and squamous malignancies.

Thrombomodulin is a cell surface glycoprotein that functions as an anticoagulant. Although initially identified on endothelial cells, thrombomodulin is also expressed by other vascular cells, by mesothelial cells, and by epidermal keratinocytes. To determine whether thrombomodulin is expressed by epithelial cells in locations other than skin, we conducted a survey of thrombomodulin protein and mRNA in human epithelium. Thrombomodulin protein was detected by immunohistochemistry in all samples containing stratified squamous epithelium, including oral mucosa, larynx, esophagus, uterine ectocervix, and vagina. In these tissues, thrombomodulin staining localized to the suprabasal layer, with minimal staining observed in the basal or superficial layers of epithelium. Thrombomodulin was not detected in cuboidal, simple columnar, or pseudostratified columnar epithelium and was detected variably in transitional epithelium. Thrombomodulin staining was also observed in 21 of 26 cases of invasive squamous cell carcinoma and in several examples of squamous carcinoma-in-situ and squamous metaplasia. Expression of thrombomodulin mRNA was confirmed by in situ hybridization in both normal and malignant squamous epithelium. Full-length, functionally active thrombomodulin was demonstrated in cultured squamous epithelial cells. These data demonstrate that thrombomodulin expression correlates with the squamous phenotype and suggest that hemostasis is regulated by compartmentalization of procoagulant and anti-coagulant epithelial proteins.

Hypomyelination in the neonatal rat central and peripheral nervous systems following tellurium intoxication.

Neonatal rats were exposed to Tellurium (Te), via the mother's milk, from the day of birth until sacrifice at 7, 14, 21, and 28 days of age. Light and electron microscopy revealed Schwann cell and myelin degeneration in the sciatic nerves at each age studied. These changes were similar to those described in weanling rats as a result of Te intoxication. In the CNS, hypomyelination of the optic nerves was convincingly demonstrated at 14, 21, and 28 days of age, accompanied by some evidence of myelin degeneration. These changes were also seen in the ventral columns of the cervical spinal cords, although less markedly, and were confirmed by quantitative methods. There was little evidence of oligodendrocyte pathology in the CNS, and it appears that degeneration of these cells is not the primary cause of the CNS hypomyelination, in contrast to the PNS where Schwann cell degeneration has been shown to precede the myelin pathology.

Proliferation of rat intraspinal Schwann cells following tellurium intoxication.

A stable population of intraspinal Schwann cells, which developed following early postnatal irradiation of the spinal cord, was challenged by the addition of tellurium (Te) to the diet beginning at 30 days of age. Schwann cells incorporating [3H]thymidine were identified by 1 micron autoradiographs and by conventional electron microscopy of adjacent thin sections. Autoradiographs of areas with Schwann cell myelination showed extensive labelling of cells in the Te-fed animals. In contrast, control animals which were not fed Te showed little evidence of labelled Schwann cells. These data indicate that Schwann cells in the intraspinal environment show a proliferative response to the presence of Te in the rat's diet, as do Schwann cells in their normal extraspinal milieu.