Adenomatous hyperplasia of the mucous glands in captive Archey's frogs (Leiopelma archeyi).

AIMS: To describe the gross and light microscopic characteristics of skin lesions observed on the ventral skin of captive Archey's frogs (Leiopelma archeyi) between 2000 and 2012, and to investigate their occurrence, possible aetiology and association with survival. METHODS: Postmortem skin samples were obtained for histological evaluation from 37 frogs, with and without skin lesions, that died while in captivity at Auckland Zoo between 2000 and 2012. Four frogs with skin lesions were biopsied under general anaesthesia and samples used for both light and transmission electron microscopy. The records of 94 frogs held at the University of Otago and Auckland Zoo between 2000-2012 were reviewed, which included some frogs recently collected from the wild. Information about the occurrence of skin lesions, and mortality associated with skin lesions was collated. RESULTS: Grossly the skin lesions varied in appearance; most were circular, pale grey papules, which measured from <0.5-1.5 mm in diameter with no umbilication. The overlying epidermis was not fragile and there was no associated inflammation. Contents often appeared clear or semi-transparent. Lesions were located predominantly on ventral surfaces including trunk, thighs, lower legs and forearms, and gular region, but not on digits. The number ranged from single to multiple, often confluent lesions covering the entire ventral surface of the frog. Histologically the lesions consisted of enlarged proliferating mucous glands that expanded the dermis and elevated the epidermis. They were semi-organised, solid or occasionally cavitated acinar structures with central lumina which sometimes contained mucus. Nuclei showed moderate anisokaryosis and mitotic figures were uncommon. Transmission electron microscopy did not show any infectious agents. Between 2000 and 2012, skin lesions were recorded in 35/94 (37%) frogs. The size and location of skin lesions varied over time, with some resolving and sometimes reappearing. Skin lesions were not associated with an increased risk of death. CONCLUSIONS: The skin lesions had the gross and microscopic characteristics of adenomatous hyperplasia of the dermal mucous glands. CLINICAL RELEVANCE: The aetiology of this adenomatous hyperplasia is unknown, but factors associated with the captive environment are the most likely cause. This is the first description of adenomatous hyperplasia of the cutaneous mucous glands in amphibians.

Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals.

BACKGROUND: Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test. OBJECTIVE: The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals. ANIMALS: One hundred and twenty-five foals born in 2011 at a ranch in Texas. METHODS: Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, 5, and 7 weeks. Affected (pneumonic) foals (n = 25) were matched by age and date-of-birth to unaffected (n = 25) and subclinical (ie, having thoracic TUS lesions but no clinical signs of pneumonia) foals (n = 75). DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. RESULTS: Subsequently affected foals had significantly greater concentrations of vapA in feces than foals that did not develop pneumonia (unaffected and subclinical foals) at 5 and 7 weeks of age. Accuracy of fecal qPCR, however, was poor as a screening test to differentiate foals that would develop clinical signs of pneumonia from those that would remain free of clinical signs (including foals with subclinical pulmonary lesions attributed to R. equi) using receiver operating characteristic (ROC) methods. CONCLUSIONS AND CLINICAL IMPORTANCE: In the population studied, serial qPCR on feces lacked adequate accuracy as a screening test for clinical R. equi foal pneumonia.

Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals.

BACKGROUND: Real-time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals. OBJECTIVE: The objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods. ANIMALS: 186 foals born in 2011 at an R. equi-endemic ranch in Texas. METHODS: Fecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 31). Foals with pneumonia were matched by age and birth date to healthy (n = 31) and subclinical (n = 124) control foals; fecal samples were collected from these controls. DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. RESULTS: Concentration of R. equi in feces differed significantly (P < .05) among groups. The area under the ROC curve for fecal qPCR for diagnosis of R. equi pneumonia was 89% (95% CI, 83-99), with a sensitivity of 94% and specificity of 72%. CONCLUSIONS AND CLINICAL IMPORTANCE: qPCR of feces can be useful as an alternative to tracheobronchial aspiration for the diagnosis of R. equi in foals with clinical signs of pneumonia. Caution should be used in extrapolating results of this study to other populations because fecal concentration of R. equi might vary by geographic location or management practices.

Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells.

Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lines. The mechanism(s) of this effect is not known, although the focus has been on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the present study, NDGA-induced apoptosis was studied in a murine hematopoietic cell line, FL5.12. Although this cell line lacks detectable LOX protein or activities, NDGA (10 microM) was able to induce apoptosis. There was a massive loss of mitochondrial membrane potential by 4 h after the addition of NDGA, suggesting that this organelle might be targeted by NDGA. A pro-oxidant NDGA effect has been suggested as playing a role in apoptosis. This was supported by the findings that glutathione disulfide levels were increased by 4 h following treatment with 10 microM NDGA, that pretreatment with N-acetylcysteine completely blocked the NDGA-induced loss of membrane potential and apoptosis, and that lipid peroxidation was enhanced in cells treated with NDGA. However, no evidence of increased levels of reactive oxygen could be seen in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihydrorhodamine and analyzed by flow cytometry. Bcl-X(L) protein levels were unaffected by NDGA treatment. Caspase-3 was rapidly activated with a peak at 8 h after FL5.12 cells were treated with NDGA. Ac-DEVD-CHO (25 microM) and boc-asp-FMK (20 microM) both inhibited caspase-3 enzyme activity by 97% 8 h after NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed NDGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspase-3, had no effect. These results suggest that NDGA-induced apoptosis happens through reactions that depolarize mitochondria, oxidize glutathione and lipids, but do not generate significant amounts of free reactive oxygen species.

Environmental occurrence and potential toxicity of planar, mono-, and di-ortho polychlorinated biphenyls in the biota.

Four non-ortho-, eight mono-ortho-, and two di-ortho-chlorinated congeners have been determined in fresh water and salt water mussels, fish, snapping turtles, mallard, seals, and in human milk and adipose tissue. The planar PCB congeners are separated from the remainder of PCBs by activated carbon chromatography or HPLC on porous graphitic carbon followed by gas chromatography with electron capture detection. PCB toxic equivalency factors (TEFs) recommended by WHO [1] for 3 non-ortho, 8 mono-ortho, and 2 di-ortho PCBs and a TEF for congener 81 suggested by Harris et al. [2] were used for calculation of the contribution to dioxin-like toxicity to each life form. In all the biota examined, PCB congener IUPAC number 126 was the major contributor to PCB toxic equivalents. Congeners IUPAC number 118, 114, 105, 156, 157, 77, 81, and 170 also contributed significantly to PCB toxic equivalents. The ability to separate out planar PCBs from the majority of PCBs has allowed the use of TCDD toxicity equivalence to compare the relative dioxin-like potency of PCB residues in various species from different locations.