Effects of LRRK2 Inhibitors in Nonhuman Primates.

Toxicology studies in nonhuman primates were conducted to evaluate selective, brain penetrant inhibitors of LRRK2. GNE 7915 was limited to 7-day administration in cynomolgus monkeys at 65 mg/kg/day or limited to 14 days in rhesus at 22.5 mg/kg b.i.d. due to physical signs. Compound 25 demonstrated acceptable tolerability at 50 and 225 mg/kg b.i.d. for 7 days in rhesus monkeys. MK-1468 was tolerated during 7-day administration at 100, 200 or 800 mg/kg/day or for 30-day administration at 30, 100, or 500 mg/kg b.i.d. in rhesus monkeys. The lungs revealed hypertrophy of type 2 pneumocytes, with accumulation of intra-alveolar macrophages. Transmission electron microscopy confirmed increased lamellar structures within hypertrophic type 2 pneumocytes. Hypertrophy and hyperplasia of type 2 pneumocytes with accumulation of intra-alveolar macrophages admixed with neutrophils were prominent at peripheral lungs of animals receiving compound 25 or MK-1468. Affected type 2 pneumocytes were immuno-positive for pro-surfactant C, but negative for CD11c, a marker for intra-alveolar macrophages. Accumulation of collagen within alveolar walls, confirmed by histochemical trichrome stain, accompanied changes described for compound 25 and MK-1468. Following a 12-week treatment-free interval, animals previously receiving MK-1468 for 30 days exhibited remodeling of alveolar structure and interstitial components that did not demonstrate reversibility.

Kidney Injury Monitoring in Tobramycin-Treated Rhesus Monkeys: Supplementing Urinary Kidney Biomarkers With Kidney Biopsy Gene Expression Profiling.

Kidney biopsies are used sparingly to diagnose kidney injury in the clinic. Here we have conducted a small exploratory study to directly compare the low-grade kidney injury monitoring performance of serum safety biomarkers, novel urine safety biomarkers, microscopic histopathology and targeted gene expression alterations in kidney biopsy specimens in rhesus monkeys treated with tobramycin. Targeted gene expression increases were observed in the kidney biopsy samples and whole kidney sections for kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN) messenger RNA transcripts. In addition, increases of the urinary kidney safety protein biomarkers including KIM-1, CLU, OPN were also observed. These increases in gene expression and urinary protein end point were in concordance with the eventual low-grade kidney lesions seen in terminal tissue sections. In contrast, conventional serum biomarkers blood urea nitrogen and serum creatinine were not as sensitive in monitoring kidney injury. Although these data do not support routinely adding kidney biopsies to regular toxicology studies, they provide evidence on the value and limitations of incorporating gene expression profiling on kidney biopsy specimens, further underscore the value of urinary kidney safety biomarkers for improved low-grade kidney injury monitoring, and open the door for future definitive studies.

Helicobacter monodelphidis sp. nov. and Helicobacter didelphidarum sp. nov., isolated from grey short-tailed opossums (Monodelphis domestica) with endemic cloacal prolapses.

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).

Identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice.

Inclusion body nephropathy (IBN) and kidney fibrosis in aged immunodeficient mice and, to lesser extent, in immunocompetent mice have been recently linked to infection of mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV). Knowledge about its prevalence and the complete genome sequence of more MKPV strains is essential for understanding phylogenetic relationships and pathogenicity among MKPV strains. In the present study using PCR and genome walking, we determined the complete 4440-nucleotide genome of a new MKPV strain, namely MIT-WI1, which was identified in IBN-affected Il2rg-/-Rag2-/- c-Kit W-sh/W-sh mice housed in the vivarium at Whitehead Institute for Biomedical Research (WI). The overall nucleotide (>94%) and deduced amino acid sequences (>98%) of p10, p15, NS1 (replicase), NS2 and VP1 (capsid protein) within the MIT-WI1 genome, are closely related to MKPV/MuCPV strains described in laboratory and wild Mus musculus mice. In addition, PCR and qPCR assays using newly designed primers conserved among the known MKPV/MuCPV genomes were developed and utilized to assess MKPV status in selected laboratory mice. MKPV was also detected in immunodeficient (NSG) and immunocompetent (Crl:CD1(ICR), UTXflox) mouse strains/stocks. The abundance of the MKPV genome copies was significantly correlated with the severity of IBN. Our data indicate that MKPV is present in selected mouse strains/stocks, and provides new insights into the genome evolution of MKPV.

Endothelial siRNA delivery in nonhuman primates using ionizable low-molecular weight polymeric nanoparticles.

Dysfunctional endothelial cells contribute to the pathophysiology of many diseases, including vascular disease, stroke, hypertension, atherosclerosis, organ failure, diabetes, retinopathy, and cancer. Toward the goal of creating a new RNA-based therapy to correct aberrant endothelial cell gene expression in humans, efficient gene silencing in the endothelium of nonhuman primates was achieved by delivering small interfering RNA (siRNA) with 7C1, a low-molecular weight, ionizable polymer that forms nanoparticles. After a single intravenous administration of 1 mg of siRNA per kilogram of animal, 7C1 nanoparticles delivering Tie2 siRNA caused Tie2 mRNA levels to decrease by approximately 80% in the endothelium of the lung. Significant decreases in Tie2 mRNA were also found in the heart, retina, kidney, pancreas, and bone. Blood chemistry and liver function analysis before and after treatment all showed protein and enzyme concentrations within the normal reference ranges. Furthermore, after controlling for siRNA-specific effects, no significant increases in inflammatory cytokine concentrations were found in the serum. Similarly, no gross lesions or significant underlying pathologies were observed after histological examination of nonhuman primate tissues. This study is the first demonstration of endothelial gene silencing in multiple nonhuman primate organs using systemically administered siRNA nanoparticles and highlights the potential of this approach for the treatment of disease in humans.

DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling.

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

Spontaneous Urinary Bladder Leiomyoma in a Rhesus Macaque (Macaca mulatta).

Here we report the case of a urinary bladder leiomyoma in a rhesus macaque. The animal was clinically normal and had a lipoma localized to the stifle. Endovesicular leiomyomas are the most common form of urinary bladder leiomyoma in humans. In contrast, this macaque's tumor exhibited extravesicular localization in the bladder. Urinary bladder leiomyomas account for less than 0.5% of all bladder tumors in humans, with only 250 cases reported in total.

Prolonged restraint stressor exposure in outbred CD-1 mice impacts microbiota, colonic inflammation, and short chain fatty acids.

Stressor-exposure has been shown to exacerbate inflammation and change the composition of the gastrointestinal microbiota; however stressor-induced effects on microbiota-derived metabolites and their receptors are unknown. Thus, bacterial-produced short chain fatty acids (SCFAs), as well as microbial community composition, were assessed in the colons of mice exposed to stress during infection with Citrobacter rodentium. Mice were exposed to overnight restraint on 7 consecutive nights, or left undisturbed as a control. After the first exposure of restraint, mice were orally challenged with C. rodentium or with vehicle. Microbial community composition was assessed using 16S rRNA gene sequencing and SCFA levels measured using gas chromatography-mass spectrometry (GC-MS). Pathogen levels and colonic inflammation were also assessed 6 days post-infection. Results demonstrated that the microbial community structure and SCFA production were significantly affected by both stressor exposure and C. rodentium-infection. Exposure to prolonged restraint in the absence of infection significantly reduced SCFAs (acetic acid, butyric acid, and propionic acid). Multiple bacterial taxa were affected by stressor exposure, with the relative abundance of Lactobacillus being significantly reduced and directly correlated with propionic acid. Lactobacillus abundances were inversely correlated with colonic inflammation, supporting the contention that Lactobacillus helps to regulate mucosal inflammatory responses. Our data indicates that restraint stressor can have significant effects on pathogen-induced colonic inflammation and suggest that stressor-induced changes in the microbiota, microbial-produced SCFAs and their receptors may be involved.

Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis.

Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

Cutaneous Dermatophilosis in a Meadow Jumping Mouse (Zapus hudsonius).

A laboratory-housed, wild-caught, subadult, male meadow jumping mouse (Zapus hudsonius) presented with extensive scaling of the face, limbs, and tail and severe edema of the paws. Postmortem examination revealed marked distal limb edema with focal digital hematomas and white scales, scabs, and crusts affecting the majority of nonhaired skin. Histopathologic analysis revealed severe, multifocal, chronic-active exudative and proliferative dermatitis characterized by multilaminated crusts covering the epidermis. The epidermis was expanded by hyperkeratosis, acanthosis, and hyperplasia. The superficial dermis contained moderate edema, hemorrhage, and pigmentary incontinence, and was infiltrated by granulocytes and mononuclear cells. The laminated crusts contained numerous branching filaments of gram-positive coccoid bodies arranged in parallel rows, consistent with cutaneous Dermatophilus congolensis infection. This diagnosis was confirmed through bacterial culture and 16S rRNA PCR analysis. In the presented case, factors that might have contributed to disease progression include climatic conditions at the capture site and stress associated with trapping and laboratory housing.

Isolation and molecular characterization of group B Streptococcus from laboratory Long-Evans rats (Rattus norvegicus) with and without invasive group B streptococcal disease.

Purpose. Group B Streptococcus (S. agalactiae, GBS) is a Gram-positive opportunistic pathogen that inhabits the respiratory, urogenital and gastrointestinal tracts of humans and animals. Maternal colonization of GBS is a risk factor for a spectrum of clinical diseases in humans and a principle cause of neonatal meningitis and septicaemia.Methodology. We describe polymicrobial sepsis including GBS in two gravid adult female Long-Evans rats experiencing acute mortality from a colony of long-term breeding pairs. Fluorescent in situ hybridization confirmed GBS association with pathological changes in affected tissues, including the heart and uterus.Results. Characterization of seven GBS strains obtained from clinically affected and non-affected animals indicated similar antibiotic resistance and susceptibility patterns to that of human strains of GBS. The rat strains have virulence factors known to contribute to pathogenicity, and shared serotypes with human invasive isolates. Phylogenetic analyses revealed that one rat-derived GBS strain was more closely related to human-derived strains than other rat-derived strains, strengthening the notion that interspecies transmission is possible.Conclusions. To our knowledge, this is the first investigation of genotypic and phenotypic features of rat-derived GBS strains and their comparison to human- and other animal-derived GBS strains. Since GBS commonly colonizes commercially available rats, its exclusion as a potential pathogen for immunocompromised or stressed animals is recommended.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.

Adult-onset, chronic, cyclic thrombocytopenia in a Rhesus macaque (Macaca mulatta) after dengue virus vaccination and viral challenge.

An 8-year-old, male Rhesus macaque (Macaca mulatta), previously used for dengue virus (DENV) vaccine research with viral challenge, was presented with adult-onset, chronic, cyclic thrombocytopenia. Platelet number, morphology, and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both serum anti-dengue nonstructural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive DENV titer 8 years postvaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.

Local and Systemic Changes Associated with Long-term, Percutaneous, Static Implantation of Titanium Alloys in Rhesus Macaques (Macaca mulatta).

Metal alloys are frequently used as implant materials in veterinary medicine. Recent studies suggest that many alloys induce both local and systemic inflammatory responses. In this study, 37 rhesus macaques with long-term skull-anchored percutaneous titanium alloy implants (duration, 0 to 14 y) were evaluated for changes in their hematology, coagulation, and serum chemistry profiles. Negative controls (n = 28) did not have implants. Macaques with implants had higher plasma D-dimer and lower antithrombin III concentrations than nonimplanted animals. In addition, animals with implants had higher globulin and lower albumin and calcium concentrations compared with nonimplanted macaques. Many of these changes were positively correlated with duration of implantation and the number of implants. Chronic bacterial infection of the skin was present around many of the implant sites and within deeper tissues. Representative histopathology around the implant site of 2 macaques revealed chronic suppurative to pyogranulomatous inflammation extending from the skin to the dura mater. X-ray fluorescence microscopy of tissue biopsies from the implant site of the same 2 animals revealed significantly higher levels of free metal ions in the tissue, including titanium and iron. The higher levels of free metal ions persisted in the tissues for as long as 6 mo after explantation. These results suggest that long-term skull-anchored percutaneous titanium alloy implants can be associated with localized inflammation, chronic infection, and leaching of metal ions into local tissues.

Helicobacter pylori infection and low dietary iron alter behavior, induce iron deficiency anemia, and modulate hippocampal gene expression in female C57BL/6 mice.

Helicobacter pylori (H.pylori), a bacterial pathogen, is a causative agent of gastritis and peptic ulcer disease and is a strong risk factor for development of gastric cancer. Environmental conditions, such as poor dietary iron resulting in iron deficiency anemia (IDA), enhance H.pylori virulence and increases risk for gastric cancer. IDA affects billions of people worldwide, and there is considerable overlap between regions of high IDA and high H.pylori prevalence. The primary aims of our study were to evaluate the effect of H.pylori infection on behavior, iron metabolism, red blood cell indices, and behavioral outcomes following comorbid H. pylori infection and dietary iron deficiency in a mouse model. C57BL/6 female mice (n = 40) were used; half were placed on a moderately iron deficient (ID) diet immediately post-weaning, and the other half were maintained on an iron replete (IR) diet. Half were dosed with H.pylori SS1 at 5 weeks of age, and the remaining mice were sham-dosed. There were 4 study groups: a control group (-Hp, IR diet) as well as 3 experimental groups (-Hp, ID diet; +Hp, IR diet; +Hp,ID diet). All mice were tested in an open field apparatus at 8 weeks postinfection. Independent of dietary iron status, H.pylori -infected mice performed fewer exploratory behaviors in the open field chamber than uninfected mice (p<0.001). Hippocampal gene expression of myelination markers and dopamine receptor 1 was significantly downregulated in mice on an ID diet (both p<0.05), independent of infection status. At 12 months postinfection, hematocrit (Hct) and hemoglobin (Hgb) concentration were significantly lower in +Hp, ID diet mice compared to all other study groups. H.pylori infection caused IDA in mice maintained on a marginal iron diet. The mouse model developed in this study is a useful model to study the neurologic, behavioral, and hematologic impact of the common human co-morbidity of H. pylori infection and IDA.

Novel urease-negative Helicobacter sp. 'H. enhydrae sp. nov.' isolated from inflamed gastric tissue of southern sea otters.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all 'H. enhydrae' isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named 'H. enhydrae', isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret.

GPR4 deficiency alleviates intestinal inflammation in a mouse model of acute experimental colitis.

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model.

Cytotoxic Escherichia coli strains encoding colibactin colonize laboratory mice.

Escherichia coli strains have not been fully characterized in laboratory mice and are not currently excluded from mouse colonies. Colibactin (Clb), a cytotoxin, has been associated with inflammation and cancer in humans and animals. We performed bacterial cultures utilizing rectal swab, fecal, and extra intestinal samples from clinically unaffected or affected laboratory mice. Fifty-one E. coli were isolated from 45 laboratory mice, identified biochemically, and selected isolates were serotyped. The 16S rRNA gene was amplified and sequenced for specific isolates, PCR used for clbA and clbQ gene amplification, and phylogenetic group identification was performed on all 51 E. coli strains. Clb genes were sequenced and selected E. coli isolates were characterized using a HeLa cell cytotoxicity assay. Forty-five of the 51 E. coli isolates (88%) encoded clbA and clbQ and belonged to phylogenetic group B2. Mouse E. coli serotypes included: O2:H6, O-:H-, OM:H+, and O22:H-. Clb-encoding O2: H6 mouse E. coli isolates were cytotoxic in vitro. A Clb-encoding E. coli was isolated from a clinically affected genetically modified mouse with cystic endometrial hyperplasia. Our findings suggest that Clb-encoding E. coli colonize laboratory mice and may induce clinical and subclinical diseases that may impact experimental mouse models.

Systemic Coronaviral Disease in 5 Ferrets.

The prevalence of reported systemic coronaviral disease in ferrets (Mustela putorius furo), which resembles the dry form of feline infectious peritonitis, has been increasing in the literature since its initial diagnosis and characterization approximately 10 y ago. Here we describe the clinical signs, pathologic findings, and diagnosis by immunohistochemistry using an FIPV3-70 monoclonal antibody of systemic coronaviral disease in 5 ferrets, 2 of which were strictly laboratory-housed; the remaining 3 were referred from veterinary private practices. This case report illustrates the importance of considering FRSCV infection as a differential diagnosis in young, debilitated ferrets with abdominal masses and other supporting clinical signs.

Helicobacter pylori Infection Induces Anemia, Depletes Serum Iron Storage, and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice.

Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world's population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6-9 weeks of age, and were necropsied at 27-29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.