Coinfection by Mycobacterium marinum and Mycolicibacterium fortuitum in a captive adult diamondback water snake causing disseminated mycobacteriosis with acute cutaneous ulceration.

An adult male captive diamondback water snake (Nerodia rhombifer) was found dead after a 1-d history of lethargy and cutaneous ulcers. The snake had eaten 2 sunfish (Mola spp.) 5 d before death. Gross examination revealed white-to-tan nodules in the lung and liver and segmental intestinal impactions with digested fish. Histopathology confirmed disseminated granulomas with numerous intrahistiocytic acid-fast bacteria in the skin, skeletal muscle, lung, liver, and intestines. Mycobacterium marinum and Mycolicibacterium fortuitum were identified by culture of the hepatic granuloma, followed by PCR and rpoB gene sequencing. To our knowledge, this is the first description of M. marinum and M. fortuitum coinfection in this species. Although M. fortuitum has been isolated from reptiles, lesions associated with its presence in tissues have not been described previously. Interestingly, the mineralization within granulomas that we observed in our case is not reported in mycobacterial infection in reptiles, whereas this finding is common in mammals.

Systemic Caryospora-like coccidiosis in a clutch of hatchling red-eared slider turtles (Trachemys scripta elegans).

Caryospora-like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia/Caryospora-like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora-like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity.

Neoplastic signatures: Comparative proteomics of canine hepatobiliary neuroendocrine tumors to normal niche tissue.

Hepatobiliary neuroendocrine neoplasms are rare cancers in humans and dogs. To date, no large-scale primary hepatobiliary neoplasm omics analyses exist in any species. This limits the development of diagnostic biomarkers and targeted therapeutics. Neuroendocrine cancers are a heterogenous group of neoplasms categorized by their tissue-of-origin. Because the anatomic niche of neuroendocrine neoplasms shapes tumor phenotype, we sought to compare the proteomes of 3 canine hepatobiliary neoplasms to normal hepatobiliary tissue and adrenal glands with the objective of identifying unique protein signatures. Protein was extracted from formalin-fixed paraffin-embedded samples and submitted for tandem mass spectroscopy. Thirty-two upregulated and 126 downregulated differentially expressed proteins were identified. Remarkably, 6 (19%) of the upregulated proteins are correlated to non-hepatobiliary neuroendocrine neoplasia and 16 (50%) are functionally annotated within the exosome cellular compartment key to neuroendocrine signaling. Twenty-six (21%) downregulated proteins are enriched in metabolic pathways consistent with alterations in cancer. These results suggests that characteristic neoplastic protein signatures can be gleaned from small data sets using a comparative proteomics approach.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

Locally Transmitted Trypanosoma cruzi in a Domestic Llama (Lama glama) in a Rural Area of Greater New Orleans, Louisiana, USA.

Trypanosoma cruzi-associated megaesophagus was diagnosed in a domestic Louisiana-born llama with no significant travel history. The llama resided in the same rural area of greater New Orleans, Louisiana, where the first human autochthonous case of Chagas disease was identified in the state. Venous blood from the llama tested positive for T. cruzi kinetoplastid DNA by conventional PCR. The cardiac evaluation was unremarkable, while thoracic radiographs revealed generalized megaesophagus. The llama received supportive care, but was ultimately humanely euthanized. The esophagus was severely distended throughout its length on necropsy, and histologic evaluation showed no microscopic changes in esophageal tissue and minimal to mild lymphoplasmacytic inflammation in cardiac tissue. T. cruzi DNA was detected by conventional PCR in the esophagus, small intestine, and blood despite no protozoan organisms being observed in multiple tissue sections examined. This report contributes to the growing body of evidence of local transmission of T. cruzi in the southern United States, and Chagas disease should be considered a differential diagnosis when evaluating llamas and other large animal species for esophageal dysfunction. There is little research describing megaesophagus or Chagas disease in llamas, and this report aims to increase awareness about this zoonotic disease that is becoming more frequently reported in the southern United States.

Physical Restraints in Critically Ill Children: A Multicenter Longitudinal Point Prevalence Study.

OBJECTIVES: We elucidate to investigate the prevalence of and factors associated with the use of physical restraints among critically ill or injured children in PICUs. DESIGN: This was a multicenter, longitudinal point prevalence study. SETTING: We included 26 PICUs in Japan. PATIENTS: Included children were 1 month to 10 years old. We screened all admitted patients in the PICUs on three study dates (in March, June, and September 2019). INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: We collected prevalence and demographic characteristics of critically ill or injured children with physical restraints, as well as details of physical restraints, including indications and treatments provided. A total of 398 children were screened in the participating PICUs on the three data collection dates. The prevalence of children with physical restraints was 53% (211/398). Wrist restraint bands were the most frequently used means (55%, 117/211) for potential contingent events. The adjusted odds of using physical restraint in patients 1-2 years old was 2.3 (95% CI, 1.3-4.0) compared with children less than 1 year old. When looking at the individual hospital effect, units without a prespecified practice policy for physical restraints management or those with more than 10 beds were more likely to use physical restraints. CONCLUSIONS: The prevalence of physical restraints in critically ill or injured children was high, and significant variation was observed among PICUs. Our study findings suggested that patient age, unit size, and practice policy of physical restraint could be associated with more frequent use of physical restraints.

Unusual Pathologic Brain Changes Associated with Zinc Toxicosis in a Blue-Fronted Amazon Parrot (Amazona aestiva).

An adult blue-fronted Amazon parrot (Amazona aestiva) was presented for a 6-wk history of ataxia and weight loss. Complete blood count, plasma chemistry panel, bile acids, and radiographic imaging were considered normal or unremarkable. The patient was hospitalized and supported with subcutaneous fluids, vitamin B complex, meloxicam, enrofloxacin, gavage feeding, and fenbendazole. While hospitalized, the ataxia significantly improved, and the bird began eating on its own and gaining weight. The bird was discharged from the hospital and prescribed enrofloxacin, meloxicam, and fenbendazole to be administered by the owner with recommendations for routine follow-up care. Medications were discontinued before emergent representation; at the time of reevaluation, the patient's condition had deteriorated severely. Given the poor prognosis, the owners elected for euthanasia. No gross abnormalities were noted on postmortem examination. Liver tissue zinc levels measured 125 ppm; normal limit is less than or equal to 25 ppm. Histopathologic changes to the brain were consistent with severe zinc toxicosis demonstrated by vasculopathy of the cerebral arteries and arterioles with multifocal areas of hemorrhage and astrocyte swelling. These findings have been reported in humans and other mammals but not birds. Although the source of this bird's heavy metal exposure is unknown, the high tissue zinc concentrations imply chronic exposure. This case presentation and unusual pathologic findings will be beneficial to the further understanding of avian zinc toxicosis.

Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease.

The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD.

WNK1 regulates skeletal muscle cell hypertrophy by modulating the nuclear localization and transcriptional activity of FOXO4.

With-no-lysine (K) (WNK) kinases, which are mutated in the inherited form of hypertension pseudohypoaldosteronism type II, are essential regulators of membrane ion transporters. Here, we report that WNK1 positively regulates skeletal muscle cell hypertrophy via mediating the function of the pro-longevity transcription factor forkhead box protein O4 (FOXO4) independent of the conventional WNK signaling pathway linking SPS/STE20-related proline-alanine-rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) to downstream effector ion transporters. Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and remarkable increases in the mRNA expression of the muscle atrophy ubiquitin ligases MAFbx and MuRF1 in C2C12 mouse skeletal muscle cells. WNK1 silencing also increased FOXO4 nuclear localization, and co-transfection of Foxo4-targeted siRNA completely reversed the myotube atrophy and upregulation of atrogene transcription induced by WNK1 silencing. We further illustrated that WNK1 protein abundance in skeletal muscle was increased by chronic voluntary wheel running exercise (hypertrophic stimulus) and markedly decreased by adenine-induced chronic kidney disease (atrophic stimulus) in mice. These findings suggest that WNK1 is involved in the physiological regulation of mammalian skeletal muscle hypertrophy and atrophy via interactions with FOXO4. The WNK1-FOXO4 axis may be a potential therapeutic target in human diseases causing sarcopenia.

Decreased KLHL3 expression is involved in the pathogenesis of pseudohypoaldosteronism type II caused by cullin 3 mutation in vivo.

BACKGROUND: Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four genes: WNK1, WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Recently, it was revealed that CUL3-KLHL3 E3 ligase complex ubiquitinates WNK1 and WNK4, leading to their degradation, and that a common pathogenesis of PHAII is defective WNK degradation due to CUL3-KLHL3 E3 ligase complex impairment. PHAII-causing CUL3 mutations mediate exon9 skipping, producing a CUL3 protein with a 57-amino acid deletion (Δ403-459). However, the pathogenic effects of KLHL3, an adaptor protein that links WNKs with CUL3, in PHAII caused by CUL3 mutation remain unclear. METHODS: To clarify detailed pathophysiological mechanisms underlying PHAII caused by CUL3 mutation in vivo, we generated and analyzed knock-in mice carrying the same CUL3 exon9 deletion (CUL3WT/Δex9) as that reported in PHAII patients. RESULTS: CUL3WT/Δex9 mice exhibited a PHAII-like phenotype. Interestingly, we confirmed markedly decreased KLHL3 expression in CUL3WT/Δex9 mice by confirming the true KLHL3 band in vivo. However, the expression of other KLHL family proteins, such as KLHL2, was comparable between WT and mutant mice. CONCLUSION: KLHL3 expression was decreased in CUL3WT/Δex9 mice. However, expression levels of other KLHL family proteins were comparable between the wild-type and mutant mice. These findings indicate that the decreased abundance of KLHL3 is a specific phenomenon caused by mutant CUL3 (Δexon9). Our findings would improve our understanding of the pathogenesis of PHAII caused by CUL3 mutation in vivo.

Thioacetamide-induced hepatic fibrosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a nonhuman primate that is used for preclinical research on stem cell transplantation therapies due to its similarity to human beings as well as its small size, enabling researchers to perform experiments without preparing a large number of cells. In this study, we developed a marmoset hepatic fibrosis model for regenerative medicine research. Six female marmosets aged 4-6 years were administered thioacetamide (TAA) at a dose of 2.5-40 mg/kg two or three times a week. Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage. Administration of TAA increased total bile acid, aspartate aminotransferase, and total bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver biopsies following the last administration of TAA revealed that induced fibrosis remained up to 11 weeks. The results suggest that continuous TAA administration induces persistent hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model have the possibility to evaluate the therapeutic effects of test samples to ameliorate hepatic fibrosis.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Impaired degradation of medullary WNK4 in the kidneys of KLHL2 knockout mice.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), and Cullin3 (CUL3) genes were identified as being responsible for hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). Normally, the KLHL3/CUL3 ubiquitin ligase complex degrades WNKs. In PHAII, the loss of interaction between KLHL3 and WNK4 increases levels of WNKs because of impaired ubiquitination, leading to abnormal over-activation of the WNK-OSR1/SPAK-NCC cascade in the kidney's distal convoluted tubules (DCT). KLHL2, which is highly homologous to KLHL3, was reported to ubiquitinate and degrade WNKs in vitro. Mutations in KLHL2 have not been reported in patients with PHAII, suggesting that KLHL2 plays a different physiological role than that played by KLHL3 in the kidney. To investigate the physiological roles of KLHL2 in the kidney, we generated KLHL2-/- mice. KLHL2-/- mice did not exhibit increased phosphorylation of the OSR1/SPAK-NCC cascade and PHAII-like phenotype. KLHL2 was predominantly expressed in the medulla compared with the cortex. Accordingly, medullary WNK4 protein levels were significantly increased in the kidneys of KLHL2-/- mice. KLHL2 is indeed a physiological regulator of WNK4 in vivo; however, its function might be different from that of KLHL3 because KLHL2 mainly localized in medulla.

Loop diuretics affect skeletal myoblast differentiation and exercise-induced muscle hypertrophy.

Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl- cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks' voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3-/- mice that expressed ß-galactosidase (ß-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ß-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3-/- mice but not in KLHL3+/- mice. KLHL3-/- mice also showed PHAII-like phenotypes, whereas KLHL3+/- mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.

An adult case of severe steroid-resistant Henoch-Schönlein purpura nephritis treated with intravenous cyclophosphamide and tonsillectomy.

A previously healthy 37-year-old Canadian man living in Japan visited a hospital in Thailand while traveling because of edematous legs, purpura, arthralgia, bloody stool, and fever after an insect bite. Henoch-Schönlein purpura (HSP) was suspected. His creatinine level was 5.2 mg/dL. He was treated with oral prednisolone (PSL) and oral cyclophosphamide (CPA); after treatment, his creatinine level improved to 2.4 mg/dL. Upon returning to Japan, he was admitted to the National Center for Global Health and Medicine Hospital in Tokyo. A kidney biopsy was performed, and HSP nephritis (HSPN) was diagnosed. Renal dysfunction and proteinuria persisted despite 4 administrations of steroid-pulse therapy and 3 sessions of plasma exchange. Finally, he was treated with intravenous cyclophosphamide (IVCY). His creatinine level and proteinuria markedly improved. His microscopic hematuria disappeared after he underwent tonsillectomy. There have been only a few case reports describing patients with adult-onset HSPN necessitating IVCY. We present here a rare case of steroid-resistant HSPN treated with IVCY and tonsillectomy, with reference to some recent findings.