Publications by authors named "Gerald E Duhamel"

39 Publications

The asymmetric Pitx2 gene regulates gut muscular-lacteal development and protects against fatty liver disease.

Cell Rep 2021 Nov;37(8):110030

Department of Molecular Medicine, College of Veterinary Medicine, Cornell, Ithaca, NY 14853, USA. Electronic address:

Intestinal lacteals are essential lymphatic channels for absorption and transport of dietary lipids and drive the pathogenesis of debilitating metabolic diseases. However, organ-specific mechanisms linking lymphatic dysfunction to disease etiology remain largely unknown. In this study, we uncover an intestinal lymphatic program that is linked to the left-right (LR) asymmetric transcription factor Pitx2. We show that deletion of the asymmetric Pitx2 enhancer ASE alters normal lacteal development through the lacteal-associated contractile smooth muscle lineage. ASE deletion leads to abnormal muscle morphogenesis induced by oxidative stress, resulting in impaired lacteal extension and defective lymphatic system-dependent lipid transport. Surprisingly, activation of lymphatic system-independent trafficking directs dietary lipids from the gut directly to the liver, causing diet-induced fatty liver disease. Our study reveals the molecular mechanism linking gut lymphatic function to the earliest symmetry-breaking Pitx2 and highlights the important relationship between intestinal lymphangiogenesis and the gut-liver axis.
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http://dx.doi.org/10.1016/j.celrep.2021.110030DOI Listing
November 2021

Comparative transcriptional profiling of canine acanthomatous ameloblastoma and homology with human ameloblastoma.

Sci Rep 2021 09 7;11(1):17792. Epub 2021 Sep 7.

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.

Ameloblastomas are odontogenic tumors that are rare in people but have a relatively high prevalence in dogs. Because canine acanthomatous ameloblastomas (CAA) have clinicopathologic and molecular features in common with human ameloblastomas (AM), spontaneous CAA can serve as a useful translational model of disease. However, the molecular basis of CAA and how it compares to AM are incompletely understood. In this study, we compared the global genomic expression profile of CAA with AM and evaluated its dental origin by using a bulk RNA-seq approach. For these studies, healthy gingiva and canine oral squamous cell carcinoma served as controls. We found that aberrant RAS signaling, and activation of the epithelial-to-mesenchymal transition cellular program are involved in the pathogenesis of CAA, and that CAA is enriched with genes known to be upregulated in AM including those expressed during the early stages of tooth development, suggesting a high level of molecular homology. These results support the model that domestic dogs with spontaneous CAA have potential for pre-clinical assessment of targeted therapeutic modalities against AM.
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http://dx.doi.org/10.1038/s41598-021-97430-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423744PMC
September 2021

DENTAL DISEASES AND OTHER ORAL PATHOLOGIES OF CAPTIVE JAGUARS () FROM BELIZE, CENTRAL AMERICA.

J Zoo Wildl Med 2021 Jan;51(4):856-867

Department of Clinical Sciences, Cornell University, NY 14853, USA,

Dental and oral diseases are prevalent in many mammalian species including wild felids. Determining the dental and oral health status of captive animal populations can help establish preventive and therapeutic strategies, leading to improved welfare and conservation efforts. The aim of this study was to assess the prevalence of periodontal disease, endodontic disease, tooth resorption, and other clinically relevant dental and maxillofacial abnormalities in a population of captive jaguars () using clinical, radiographic, and histopathological findings. Fifteen jaguars, ranging from young adult to geriatric, kept at a private zoo in Belize, Central America, had a detailed oral examination under general anesthesia between January 2015 and March 2019. Periodontitis was present in 3.8% (16/423) of examined teeth and 53.8% (7/13) of jaguars that underwent periodontal probing. Endodontic disease secondary to dentoalveolar trauma was found in 21.0% (89/423) of teeth in 73.3% (11/15) of animals. Tooth resorption, which has not been previously documented in jaguars, affected 1.4% (6/423) of teeth in 13.3% (2/15) of jaguars. Other abnormalities included metallic foreign material (gunshot) identified radiographically in 33.3% (5/15) of jaguars and nontraumatizing malocclusion in 9.1% (1/11) of jaguars that had occlusion evaluated. Much of the oral pathology identified in captive jaguars is suspected to arise from capture and/or captivity-associated behaviors, as suggested by gunshot around the oral cavity, fractures of rostral teeth (canine and incisor teeth), and abrasions consistent with cage-biting on canine teeth. Anesthetized oral examination-including full-mouth intraoral radiographs, periodontal probing, and charting-is recommended for jaguars with clinical signs of oral pain, as well as for routine systemic evaluation.
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http://dx.doi.org/10.1638/2019-0222DOI Listing
January 2021

Persistent infection and pancytopenia associated with ferret systemic coronaviral disease in a domestic ferret.

J Vet Diagn Invest 2020 Jul 26;32(4):616-620. Epub 2020 Jun 26.

Companion Exotic Animal Medicine & Surgery Service, College of Veterinary Medicine, University of California-Davis, Davis, CA (Tarbert).

Ferret systemic coronaviral disease (FSCD) is a well-established cause of mortality in domestic ferrets. We describe herein novel findings in a case of FSCD that was diagnosed and medically managed following virus detection by immunohistochemical (IHC) staining of surgical biopsy samples. Hematologic changes in this ferret suggested spread of the virus to the bone marrow, which was confirmed by IHC staining of a postmortem sample. Genotyping of the virus indicated that the virus grouped with alphacoronaviruses and was most closely related to ferret enteric coronavirus (FRECV) MSU-2. Our clinical case demonstrates that a FRECV MSU-2-like ferret coronavirus associated previously with the enteric pathotype may cause systemic disease, including bone marrow involvement causing persistent pancytopenia.
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http://dx.doi.org/10.1177/1040638720937105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438646PMC
July 2020

Pathology in Practice.

J Am Vet Med Assoc 2019 09;255(5):543-545

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http://dx.doi.org/10.2460/javma.255.5.543DOI Listing
September 2019

Pathology in Practice.

J Am Vet Med Assoc 2019 Jun;254(11):1287-1290

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http://dx.doi.org/10.2460/javma.254.11.1287DOI Listing
June 2019

Ultra-frequent HRAS p.Q61R somatic mutation in canine acanthomatous ameloblastoma reveals pathogenic similarities with human ameloblastoma.

Vet Comp Oncol 2019 Sep 13;17(3):439-445. Epub 2019 Jun 13.

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York.

Ameloblastoma is a locally aggressive odontogenic tumour that occurs in humans and dogs. Most ameloblastomas (AM) in humans harbour mutually-exclusive driving mutations in BRAF, HRAS, KRAS, NRAS or FGFR2 that activate MAPK signalling, and in SMO that activates Hedgehog signalling. The remarkable clinical and histological similarities between canine acanthomatous ameloblastoma (CAA) and AM suggest they may harbour similar driving mutations. In this study, aimed at characterizing the mutational status of SMO, BRAF, HRAS, KRAS, NRAS and FGFR2 in CAA, we used RNA sequencing, Sanger sequencing and restriction fragment length polymorphism assays to demonstrate that 94% of CAA (n = 16) harbour a somatic HRAS p.Q61R mutation. The similarities in MAPK-activating mutational profiles between CAA and AM implicate conserved molecular mechanisms of tumorigenesis, thus, qualifying the dog as a potentially useful model of disease. Given the relevance of RAS mutations in the pathogenesis of odontogenic tumours and other types of cancer, the results of this study are of comparative, translational, and veterinary value.
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http://dx.doi.org/10.1111/vco.12487DOI Listing
September 2019

Neuroborreliosis in a horse with common variable immunodeficiency.

J Vet Diagn Invest 2019 Mar 19;31(2):241-245. Epub 2019 Jan 19.

Departments of Biomedical Sciences, Section of Anatomic Pathology (Pecoraro, Miller, Duhamel), College of Veterinary Medicine, Cornell University, Ithaca, NY.

Common variable immunodeficiency (CVID) is a rare condition in adult horses characterized by hypogammaglobulinemia and increased susceptibility to parasitic and bacterial infections, including recurrent respiratory diseases, septicemia, and meningitis. Lyme disease is often included as a differential diagnosis in CVID horses with signs of meningitis; however, the Borrelia burgdorferi organism has not been demonstrated previously within central nervous system tissues of CVID horses with neurologic disease, to our knowledge. We report herein a case of neuroborreliosis in a CVID horse, confirmed by combined immunologic testing, histopathology, real-time PCR assay, fluorescent in situ hybridization, and immunohistochemical staining. Implications of these findings include heightened monitoring of CVID horses for Lyme disease in endemic areas and appropriate therapy in the case of neurologic disease.
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http://dx.doi.org/10.1177/1040638718824146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838834PMC
March 2019

The Typhoid Toxin Produced by the Nontyphoidal Serotype Javiana Is Required for Induction of a DNA Damage Response and Systemic Spread .

mBio 2018 03 27;9(2). Epub 2018 Mar 27.

Department of Food Science, Cornell University, Ithaca, New York, USA

The cytolethal distending toxin (S-CDT), first described as the "typhoid toxin" in subsp. serotype Typhi, induces DNA damage in eukaryotic cells. Recent studies have shown that more than 40 nontyphoidal (NTS) serotypes carry genes that encode S-CDT, yet very little is known about the activity, function, and role of S-CDT in NTS. Here we show that deletion of genes encoding the binding subunit () and a bacteriophage muramidase predicted to play a role in toxin export () does not abolish toxin activity in the S-CDT-positive NTS subsp. serotype Javiana. However, Javiana strains harboring deletions of both and its homolog , had a complete loss of S-CDT activity, suggesting that Javiana carries genes encoding two variants of the binding subunit. S-CDT-mediated DNA damage, as determined by phosphorylation of histone 2AX (H2AX), producing phosphorylated H2AX (γH2AX), was restricted to epithelial cells in S and G/M phases of the cell cycle and did not result in apoptosis or cell death. Compared to mice infected with a Δ strain, mice infected with wild-type Javiana had significantly higher levels of Javiana in the liver, but not in the spleen, ileum, or cecum. Overall, we show that production of active S-CDT by NTS serotype Javiana requires different genes (, , and either or ) for expression of biologically active toxin than those reported for S-CDT production by Typhi (, , , and ). However, as in Typhi, NTS S-CDT influences the outcome of infection both and Nontyphoidal (NTS) are a major cause of bacterial food-borne illness worldwide; however, our understanding of virulence mechanisms that determine the outcome and severity of nontyphoidal salmonellosis is incompletely understood. Here we show that S-CDT produced by NTS plays a significant role in the outcome of infection both and , highlighting S-CDT as an important virulence factor for nontyphoidal serotypes. Our data also contribute novel information about the function of S-CDT, as S-CDT-mediated DNA damage occurs only during certain phases of the cell cycle, and the resulting damage does not induce cell death as assessed using a propidium iodide exclusion assay. Importantly, our data support that, despite having genetically similar S-CDT operons, NTS serotype Javiana has different genetic requirements than Typhi, for the production and export of active S-CDT.
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http://dx.doi.org/10.1128/mBio.00467-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874915PMC
March 2018

Characterization of a Vesivirus Associated with an Outbreak of Acute Hemorrhagic Gastroenteritis in Domestic Dogs.

J Clin Microbiol 2018 05 25;56(5). Epub 2018 Apr 25.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

Four of eleven affected dogs died despite aggressive treatment during a 2015 focal outbreak of hemorrhagic gastroenteritis following a stay in a pet housing facility. Routine diagnostic investigations failed to identify a specific cause. Virus isolation from fresh necropsy tissues yielded a calicivirus with sequence homology to a vesivirus within the group colloquially known as the vesivirus 2117 strains that were originally identified as contaminants in CHO cell bioreactors. hybridization and reverse transcription-PCR assays of tissues from the four deceased dogs confirmed the presence of canine vesivirus (CaVV) nucleic acids that localized to endothelial cells of arterial and capillary blood vessels. CaVV nucleic acid corresponded to areas of necrosis and hemorrhage primarily in the intestinal tract, but also in the brain of one dog with nonsuppurative meningoencephalitis. This is the first report of an atypical disease association with a putative hypervirulent vesivirus strain in dogs, as all other known strains of CaVV appear to cause nonclinical infections or relatively mild disease. After identification of the CU-296 vesivirus strain from this outbreak, four additional CaVV strains were amplified from unrelated fecal specimens and archived stocks provided by other laboratories. Broader questions include the origins, reservoir(s), and potential for reemergence and spread of these related CaVVs.
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http://dx.doi.org/10.1128/JCM.01951-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925730PMC
May 2018

Gastric Dilatation Associated with Gastric Colonization with Sarcina-Like Bacteria in a Cat with Chronic Enteritis.

J Am Anim Hosp Assoc 2017 Nov/Dec;53(6):321-325. Epub 2017 Sep 11.

From the Blue Pearl Veterinary Partners, Long Island City, New York (J.Y.I.); and Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, New York (S.S., G.E.D.).

An 11 yr old spayed female domestic longhair cat was presented for an acute onset of vomiting. Abdominal radiographs and ultrasound revealed severe gastric dilatation (GD) without evidence of gastric outflow obstruction. On esophagogastroduodenoscopy, the duodenal mucosa was mildly erythematous, and a moderate, diffuse, chronic enteritis was found by histological examination of duodenal biopsies. Large numbers of Sarcina-like bacteria without associated inflammation were present in gastric mucosal biopsies. To the authors' knowledge, this is the first report of GD associated with colonization by Sarcina-like bacteria in a cat. Gastric colonization by Sarcina-like bacteria should be suspected when cats are presented with acute onset of GD and vomiting.
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http://dx.doi.org/10.5326/JAAHA-MS-6503DOI Listing
January 2019

First demonstration of equid gammaherpesviruses within the gastric mucosal epithelium of horses.

Virus Res 2017 10 18;242:30-36. Epub 2017 Sep 18.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. Electronic address:

Horses commonly develop gastric mucosal ulcers, similar to humans, a condition known as equine gastric ulcer syndrome (EGUS) that can lead to poor performance and lost training time and care expenses. Unlike humans, however, an infectious bacterial cause of ulcers has not been conclusively identified. Herpesviruses, while well-established causative agents of diseases such as cold sores, genital lesions, and certain types of cancer, have also been implicated in the development of a subset of gastric ulcers in humans. The presence of equid herpesviruses in the gastrointestinal tract and their potential contribution to EGUS has not been evaluated. Here, we provide the first evidence of equid gammaherpesviruses 2 and 5 (EHV-2 and -5) within the epithelium of the gastric mucosa of horses. These viruses were initially detected by a nested PCR screen of gastric tissue samples obtained from client- and university-owned horses with and without ulcers; however, no association with EGUS was found in this limited sample set. We then validated a highly sensitive in situ hybridization (ISH) assay and used this assay to characterize the distribution of these viruses in necropsy gastric tissue samples from five racehorses. Analyses revealed frequent EHV-2 and EHV-5 co-infections within the gastric mucosal epithelium, regardless of the ulcer status. These results are the first to demonstrate the presence of equid gammaherpesviruses in the gastric mucosa of horses and warrants further investigation to determine the contribution of these viruses to the development of EGUS and/or other gastrointestinal diseases.
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http://dx.doi.org/10.1016/j.virusres.2017.09.002DOI Listing
October 2017

Histopathology case definition of naturally acquired Salmonella enterica serovar Dublin infection in young Holstein cattle in the northeastern United States.

J Vet Diagn Invest 2017 Nov 9;29(6):860-864. Epub 2017 Jun 9.

Departments of Biomedical Sciences and Population Medicine and Diagnostic Sciences, and New York Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY.

Salmonella enterica subsp. enterica serovar Dublin ( Salmonella Dublin) is a host-adapted bacterium that causes high morbidity and mortality in dairy cattle worldwide. A retrospective search of archives at the New York Animal Health Diagnostic Center revealed 57 culture-confirmed Salmonella Dublin cases from New York and Pennsylvania in which detailed histology of multiple tissues was available. Tissues routinely submitted by referring veterinarians for histologic evaluation included sections of heart, lungs, liver, spleen, and lymph nodes. Of the 57 S almonella Dublin-positive cases, all were Holstein breed, 53 were female (93%), and 49 (86%) were <6 mo of age. Specifically, in calves <6 mo of age, >90% (45 of 49) of lungs, 90% (28 of 31) of livers, 50% (11 of 22) of spleens, and 62% (18 of 29) of lymph nodes examined had moderate-to-severe inflammation with or without necrosis. Inconstant lesions were seen in 48% (10 of 21) of hearts examined, and consisted of variable inflammatory infiltrates and rare areas of necrosis. We propose a histopathology case definition of Salmonella Dublin in <6-mo-old Holstein cattle that includes a combination of pulmonary alveolar capillary neutrophilia with or without hepatocellular necrosis and paratyphoid granulomas, splenitis, and lymphadenitis. These findings will assist in the development of improved protocols for the diagnosis of infectious diseases of dairy cattle.
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http://dx.doi.org/10.1177/1040638717712757DOI Listing
November 2017

Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

Mol Biol Cell 2016 Feb 2;27(3):483-90. Epub 2015 Dec 2.

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 Graduate Program in Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853

Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn's disease (CD). Consistent with this observation, laboratory mice with enterocyte-specific Sel1L deficiency (Sel1L(ΔIEC)) develop spontaneous enteritis and have increased susceptibility to Toxoplasma gondii-induced ileitis. This is associated with profound defects in Paneth cells and a disproportionate increase of Ruminococcus gnavus, a mucolytic bacterium with known association with CD. Surprisingly, whereas both ER stress sensor IRE1α and effector CHOP are activated in the small intestine of Sel1L(ΔIEC) mice, they are not solely responsible for ERAD deficiency-associated lesions seen in the small intestine. Thus our study points to a constitutive role of Sel1L-Hrd1 ERAD in epithelial cell biology and the pathogenesis of intestinal inflammation in CD.
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http://dx.doi.org/10.1091/mbc.E15-10-0724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751599PMC
February 2016

IRE1α is an endogenous substrate of endoplasmic-reticulum-associated degradation.

Nat Cell Biol 2015 Dec 9;17(12):1546-55. Epub 2015 Nov 9.

Graduate Program in Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1L(ΔIEC)) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.
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http://dx.doi.org/10.1038/ncb3266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670240PMC
December 2015

Characterization of the cytolethal distending toxin (typhoid toxin) in non-typhoidal Salmonella serovars.

Gut Pathog 2015 24;7:19. Epub 2015 Jul 24.

Department of Food Science, College of Agriculture and Life Sciences, Cornell University, 347 Stocking Hall, Ithaca, NY 14853 USA.

Background: For many putative Salmonella enterica subsp. enterica virulence genes, functional characterization across serovars has been limited. Cytolethal distending toxin B (CdtB) is an incompletely characterized virulence factor that is found not only in Salmonella enterica subsp. enterica serovar Typhi (Salmonella Typhi) and dozens of Gram negative bacterial pathogens, but also in non-typhoidal Salmonella (NTS) serovars.

Methods: A comparative genomics approach was performed to characterize sequence conservation of the typhoid toxin (TT), encoded in the CdtB-islet, between Salmonella Typhi and NTS serovars. The cytotoxic activity of representative Salmonella enterica subsp. enterica serovars Javiana, Montevideo and Schwarzengrund strains and their respective isogenic cdtB mutants was determined in human intestinal epithelial Henle-407 cells by assessment of cell cycle progression of infected cells using fluorescence-activated cell sorting (FACS). Two-way analysis of variance (ANOVA) was used to determine whether cdtB deletion had a significant (p < 0.05) effect on the percentage of Henle-407 cells at each stage of the cell cycle.

Results: Here we show that a CdtB-islet encoding the cytolethal distending toxin B (CdtB), pertussis-like toxin A (PltA), and pertussis-like toxin B (PltB) is present in a dozen NTS serovars and that these proteins have a high level of sequence conservation and each form monophyletic clades with corresponding Salmonella Typhi genes. Human epithelial Henle-407 cells infected with three representative CdtB-encoding NTS serovars displayed G2/M phase cell cycle arrest that was absent in cells infected with corresponding isogenic cdtB null mutants (p < 0.0001 for the factor ∆cdtB deletion).

Conclusion: Our results show that CdtB encoded by NTS serovars has a genomic organization, amino acid sequence conservation and biological activity similar to the TT, and thus, may contribute to disease pathogenesis.
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http://dx.doi.org/10.1186/s13099-015-0065-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511993PMC
July 2015

Genotypic characterization of canine coronaviruses associated with fatal canine neonatal enteritis in the United States.

J Clin Microbiol 2014 Dec 24;52(12):4230-8. Epub 2014 Sep 24.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

Emerging canine coronavirus (CCoV) variants that are associated with systemic infections have been reported in the European Union; however, CCoV-associated disease in the United States is incompletely characterized. The purpose of this study was to correlate the clinicopathological findings and viral antigen distribution with the genotypic characteristics of CCoV in 11 puppies from nine premises in five states that were submitted for diagnostic investigation at Cornell University between 2008 and 2013. CCoV antigen was found in epithelial cells of small intestinal villi in all puppies and the colon in 2 of the 10 puppies where colon specimens were available. No evidence of systemic CCoV infection was found. Comparative sequence analyses of viral RNA extracted from intestinal tissues revealed CCoV-II genotype in 9 out of 11 puppies. Of the nine CCoV-IIs, five were subtyped as group IIa and one as IIb, while three CCoVs could not be subtyped. One of the CCoV-IIa variants was isolated in cell culture. Infection with CCoV alone was found in five puppies, of which two also had small intestinal intussusception. Concurrent infections with either parvovirus (n = 1), attaching-effacing Escherichia coli (n = 4), or protozoan parasites (n = 3) were found in the other six puppies. CCoV is an important differential diagnosis in outbreaks of severe enterocolitis among puppies between 4 days and 21 weeks of age that are housed at high population density. These findings will assist with the rapid laboratory diagnosis of enteritis in puppies and highlight the need for continued surveillance for CCoV variants and intestinal viral diseases of global significance.
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http://dx.doi.org/10.1128/JCM.02158-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313292PMC
December 2014

Canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins.

Viruses 2014 Aug 22;6(8):3363-76. Epub 2014 Aug 22.

Department of Microbiology & Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA.

Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.
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http://dx.doi.org/10.3390/v6083363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147700PMC
August 2014

Diet-induced alterations in gut microflora contribute to lethal pulmonary damage in TLR2/TLR4-deficient mice.

Cell Rep 2014 Jul 19;8(1):137-49. Epub 2014 Jun 19.

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA; Graduate Program in Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA; Graduate Program in Genetics, Genomics and Development, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Chronic intake of Western diet has driven an epidemic of obesity and metabolic syndrome, but how it induces mortality remains unclear. Here, we show that chronic intake of a high-fat diet (HFD), not a low-fat diet, leads to severe pulmonary damage and mortality in mice deficient in Toll-like receptors 2 and 4 (DKO). Diet-induced pulmonary lesions are blocked by antibiotic treatment and are transmissible to wild-type mice upon either cohousing or fecal transplantation, pointing to the existence of bacterial pathogens. Indeed, diet and innate deficiency exert significant impact on gut microbiota composition. Thus, chronic intake of HFD promotes severe pulmonary damage and mortality in DKO mice in part via gut dysbiosis, a finding that may be important for immunodeficient patients, particularly those on chemotherapy or radiotherapy, where gut-microbiota-caused conditions are often life threatening.
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http://dx.doi.org/10.1016/j.celrep.2014.05.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103790PMC
July 2014

Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis.

PLoS One 2014 14;9(1):e85579. Epub 2014 Jan 14.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 quarters and received 4 doses of 300 mg cefapirin in one quarter and 4 doses of 300 mg cefapirin together with 20 mg prednisolone in another quarter. At 24 h (n = 3) or 48 h (n = 3) post-challenge, tissue samples from control and treated quarters were collected for microarray analysis. Gene expression analysis of challenged, un-treated quarters revealed an up-regulation of transcripts associated with immune response functions compared to un-challenged quarters. Both treatments resulted in down-regulation of these transcripts compared to challenged, un-treated quarters most prominently for genes representing Chemokine and TLR-signaling pathways. Gene expression of Lipopolysaccharide Binding Protein (LBP), CCL2 and CXCL2 were only significantly down-regulated in cefapirin-prednisolone-treated quarters compared to un-treated controls. Down-regulation of chemokines was further confirmed on the basis of protein levels in milk whey for CXCL1, CXCL2 and CXCL8 in both treatments with a greater decrease in cefapirin-prednisolone-treated quarters. The data reveal a significant effect of treatment on cell recruitment with a more pronounced effect in cefapirin-prednisolone treated quarters. Provided a rapid bacteriological clearance, combination therapy may prevent neutrophil-induced tissue damage and promote recovery of the gland.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085579PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891811PMC
September 2014

Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival.

Proc Natl Acad Sci U S A 2014 Feb 22;111(5):E582-91. Epub 2014 Jan 22.

Graduate Program in Biochemistry, Molecular and Cell Biology, Division of Nutritional Sciences, and Department of Animal Science, Cornell University, Ithaca, NY 14853.

Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L's physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we show that Sel1L is indispensable for Hrd1 stability, ER homeostasis, and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 wk with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation, and promotes cell death. Serendipitously, using a biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of the mammalian Hrd1 ERAD complex and ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival.
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http://dx.doi.org/10.1073/pnas.1318114111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918815PMC
February 2014

Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

Helicobacter 2013 Dec 29;18(6):433-43. Epub 2013 Jul 29.

School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA; Vaccine Branch, National Cancer Institute, Bethesda, MD, 20892-5065, USA.

Background: Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated.

Materials And Methods: The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry.

Results: Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death.

Conclusion: All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.
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http://dx.doi.org/10.1111/hel.12084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808484PMC
December 2013

Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.

Emerg Infect Dis 2013 Jul;19(7):1066-73

Cornell University College of Veterinary Medicine,Ithaca, New York 14853, USA.

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.
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http://dx.doi.org/10.3201/eid1907.121094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713968PMC
July 2013

Prednisolone and cefapirin act synergistically in resolving experimental Escherichia coli mastitis.

J Dairy Sci 2013 Jul 16;96(7):4406-18. Epub 2013 May 16.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Mastitis in dairy cows is typically treated with intramammary antibiotics. The combination of antibiotics with corticosteroids tends to have a large market share where these products are registered. Our objective was to investigate the effect of prednisolone in combination with cefapirin on the inflammatory response of experimentally induced Escherichia coli mastitis. Six midlactating Holstein-Friesian cows were challenged in 3 quarters with E. coli and treated at 4, 12, 24, and 36 h postinfection with 300 mg of cefapirin in 1 quarter and a combination of 300 mg of cefapirin and 20mg of prednisolone in another quarter. At 24h (n=3) or 48 h (n=3) postinfection cows were euthanized for tissue sampling. Clinical scores, somatic cell count, and California mastitis test scores, as well as IL-1β, IFN-γ, IL-4, and IL-10 levels and bacterial growth in milk, were measured every 6h. Experimental inoculation caused a moderate clinical mastitis in all cows in challenged, untreated quarters. The E. coli challenge strain was recovered from all infected quarters and confirmed by PCR-based fingerprinting. Challenged, untreated control quarters showed increased concentrations of all measured cytokines together with recruitment of polymorphonuclear neutrophilic leukocytes at 24 and 48 h postchallenge. Both treatments reduced udder swelling and sensitivity with no statistically significant difference between treatment groups. Administration of cefapirin alone or in combination with prednisolone resulted in significantly lower concentrations of IFN-γ, IL-1β, and IL-10 compared with challenged, untreated quarters. Treated quarters did show IL-4 production, but concentrations were significantly decreased compared with untreated, challenged quarters. Quarters treated with the combination of cefapirin and prednisolone showed a significantly lower concentration of IL-4 compared with cefapirin-only treatment. At both 24 and 48 h postinoculation, the level of polymorphonuclear neutrophilic leukocyte recruitment was lowest in challenged quarters treated with a combination of cefapirin and prednisolone, followed by cefapirin alone. Taken together, treatment with cefapirin alone inhibited bacterial growth in milk and reduced the host inflammatory responses. Addition of prednisolone to cefapirin had a synergistic effect, resulting in a lower density of leukocytes in tissue and milk and a quicker restoration of milk quality.
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http://dx.doi.org/10.3168/jds.2012-6455DOI Listing
July 2013

Complete Genome Sequence of the Porcine Strain Brachyspira pilosicoli P43/6/78(T.).

Genome Announc 2013 Jan 21;1(1). Epub 2013 Feb 21.

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.

Reported herein is the complete genome sequence of strain P43/6/78, isolated from a pig with clinical disease. This sequence will aid in the study of genome-wide comparison among species.
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http://dx.doi.org/10.1128/genomeA.00215-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587939PMC
January 2013

Pathology in practice: myocarditis attributable to PCV-2 infection in a pig fetus.

J Am Vet Med Assoc 2013 Feb;242(3):317-9

Clemson Veterinary Diagnostic Center, Clemson University, Columbia, SC 29229, USA.

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http://dx.doi.org/10.2460/javma.242.3.317DOI Listing
February 2013

Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps.

Infect Immun 2012 Feb 21;80(2):768-77. Epub 2011 Nov 21.

Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA.

Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.
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http://dx.doi.org/10.1128/IAI.05730-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264325PMC
February 2012

Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages.

Microbiology (Reading) 2011 Jul 12;157(Pt 7):1851-1875. Epub 2011 May 12.

Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.

Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.
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http://dx.doi.org/10.1099/mic.0.049536-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167888PMC
July 2011

Helicobacter hepaticus cytolethal distending toxin causes cell death in intestinal epithelial cells via mitochondrial apoptotic pathway.

Helicobacter 2010 Apr;15(2):98-107

School of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA.

Background: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells.

Materials And Methods: INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed.

Results: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells.

Conclusion: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.
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http://dx.doi.org/10.1111/j.1523-5378.2010.00749.xDOI Listing
April 2010

Perturbation of the small intestine microbial ecology by streptomycin alters pathology in a Salmonella enterica serovar typhimurium murine model of infection.

Infect Immun 2009 Jul 11;77(7):2691-702. Epub 2009 May 11.

Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853, USA.

The small intestine is an important site of infection for many enteric bacterial pathogens, and murine models, including the streptomycin-treated mouse model of infection, are frequently used to study these infections. The environment of the mouse small intestine and the microbiota with which enteric pathogens are likely to interact, however, have not been well described. Therefore, we compared the microbiota and the concentrations of short-chain fatty acids (SCFAs) present in the ileum and cecum of streptomycin-treated mice and untreated controls. We found that the microbiota in the ileum of untreated mice differed greatly from that of the cecum of the same mice, primarily among families of the phylum Firmicutes. Upon treatment with streptomycin, substantial changes in the microbial composition occurred, with a marked loss of population complexity. Characterization of the metabolic products of the microbiota, the SCFAs, showed that formate was present in the ileum but low or not detectable in the cecum while butyrate was present in the cecum but not the ileum. Treatment with streptomycin altered the SCFAs in the cecum, significantly decreasing the concentration of acetate, propionate, and butyrate. In this work, we also characterized the pathology of Salmonella infection in the ileum. Infection of streptomycin-treated mice with Salmonella was characterized by a significant increase in the relative and absolute levels of the pathogen and was associated with more severe ileal inflammation and pathology. Together these results provide a better understanding of the ileal environment in the mouse and the changes that occur upon streptomycin treatment.
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http://dx.doi.org/10.1128/IAI.01570-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708583PMC
July 2009
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