Amy L. Warren

Invasion, establishment, and range expansion of two parasitic nematodes in the Canadian Arctic

Climate warming is occurring at an unprecedented rate in the Arctic and is having profound effects on host-parasite interactions, including range expansion. Recently, two species of protostrongylid nematodes have emerged for the first time in muskoxen and caribou on Victoria Island in the western Canadian Arctic Archipelago. Umingmakstrongylus pallikuukensis, the muskox lungworm, was detected for the first time in 2008 in muskoxen at a community hunt on the southwest corner of the island and by 2012, it was found several hundred kilometers east in commercially harvested muskoxen near the town of Ikaluktutiak. In 2010, Varestrongylus sp., a recently discovered lungworm of caribou and muskoxen was found in muskoxen near Ikaluktutiak and has been found annually in this area since then. Whereas invasion of the island by U. pallikuukensis appears to have been mediated by stochastic movement of muskoxen from the mainland to the southwest corner of the island, Varestrongylus has likely been introduced at several times and locations by the seasonal migration of caribou between the island and the mainland. A newly permissive climate, now suitable for completion of the parasite life cycles in a single summer, likely facilitated the initial establishment and now drives range expansion for both parasites.

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow–derived macrophages, but not dendritic cells, to process and present the I-Ab–immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-Ab binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-Ab mice that were deficient in the p47phox or gp91phox subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4+ T cells in the CNS, despite eliciting a normal primary CD4+ T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4+ T cell–driven autoimmune disease processes.

Occurrence, diagnosis, and strain typing of Mycobacterium avium subspecies paratuberculosis infection in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in southwestern Alberta.

The role that wildlife may play in the transmission of Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne’s disease (JD), and the potential consequences of infection in these populations are being given increasing consideration. A yearling male Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from southwestern Alberta, Canada, was found infected with Map in August 2009. Clinical signs of emaciation and diarrhea and histologic findings of diffuse granulomatous enteritis of the distal ileum, lymphadenitis of the mesenteric lymph nodes, and lymphangitis of the ileum were similar to previously described cases of JD in bighorn sheep. Infection with Map was confirmed by bacterial isolation through fecal culture, acid-fast staining, and polymerase chain reaction (PCR) of IS900. The Map1506 gene was sequenced, and the isolate was identified as a Cattle (Type II) strain. In a follow-up herd-level survey, three of 44 fecal samples (7%) from individual bighorn sheep from the same herd as the index case were PCR-positive and identified as Type II Map strains. Twenty-five samples from a distant bighorn population were negative. Additional strain typing of the isolates from the index case and the positive fecal samples was done by sequencing three discriminatory short sequence repeat (SSR) regions. All four SSR profiles differed from one another, suggesting multiple introductions or a long-existing circulation of Map within this bighorn population. Detailed molecular analyses are essential for understanding and managing diseases at the wildlife-livestock interface.

Reliability of 400-cell and 5-field leukocyte differential counts for equine bronchoalveolar lavage fluid

BACKGROUND Reliable enumeration of mast cells and eosinophils in equine bronchoalveolar lavage (BAL) fluid is important because small increases in the percentages of these cells support the clinical diagnosis of inflammatory airway disease (IAD). Increases in BAL neutrophils also occur with IAD but are not specific due to overlap between IAD and recurrent airway obstruction (RAO). OBJECTIVES The objectives of this study were to evaluate the reliability of a standard 400-cell leukocyte differential count and an alternate method evaluating 5 microscopic fields at 500× magnification in equine BAL fluid cytocentrifuged preparations. METHODS BAL samples from 60 horses with and without pulmonary inflammation were evaluated using 400-cell and 5-field leukocyte differential counting methods. Reliability of enumeration of each leukocyte type was assessed by calculating and comparing intraclass correlation coefficients (ICC). Reliability of mast cell enumeration was further evaluated by comparing ICCs of slides with different cell densities. RESULTS Reliability was higher for all cell types with the 5-field method; however, overall the difference between methods was not statistically significant. Neutrophil reliability was high (ICC > 0.90) with both methods. Adequate reliability (ICC > 0.85) for mast cells was achieved only with the 5-field method on slides with higher cell density. CONCLUSION Enumeration of mast cells is unreliable when the standard 400-cell differential counting method is used, whereas the 5-field method on slides with higher cell density reached acceptable reproducibility. Neutrophil percentages were highly reliable with both methods.

What is your diagnosis? Biopsy impression smear of a hepatic mass in a yearling Thoroughbred filly.

A 1-year-old Thoroughbred filly was presented to the Cornell University Hospital for Animals with a 10-day history of fever, diarrhea, inappetance, and hypodipsia. Clinical pathology abnormalities found by the referring veterinarian included erythrocytosis, hyperproteinemia, and increased serum gamma-glutamyltransferase and lactate dehydrogenase activities. At Cornell University, the laboratory abnormalities were confirmed and also included thrombocytosis and hypoglycemia. Erythrocytosis persisted despite vigorous fluid therapy. Ultrasound examination revealed an extremely enlarged liver with abnormal echogenicity and a 21 x 25-cm hepatic mass with varied echogenicity. Imprints of an ultrasound-guided biopsy of the mass revealed a neoplastic epithelial population of uncertain origin, although the cells did not resemble hepatocytes. Together with the presenting signs, signalment, ultrasonographic findings, and persistent erythrocytosis, the cytologic findings were considered to be most consistent with hepatoblastoma. Histopathologic examination of the mass at necropsy confirmed the diagnosis and findings also included bone marrow erythroid hyperplasia. Serum erythropoietin concentration was 28.0 mU/mL (reference interval 1.0-11.8 mU/mL), supporting erythropoietin production by the tumor and secondary inappropriate erythrocytosis. To our knowledge, this report is the first to document secondary erythrocytosis with increased erythropoietin concentration in a horse with hepatoblastoma, and also the first to describe the cytopathologic features of this rare tumor.

Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii.

The function of mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK)-2 in resistance and pathology during infection has not been greatly studied. Here, we employed Jnk2(-/-) mice to investigate the role of JNK2 in resistance and immunity during oral infection with the protozoan pathogen Toxoplasma gondii. We found increased host resistance in the absence of JNK2 as determined by lower parasite burden and increased host survival. Lack of JNK2 also correlated with decreased neutrophil recruitment to the intestinal mucosa and less pathology in the small intestine. In the absence of JNK2, IL-12 production was slightly but significantly increased in restimulated splenocyte populations as well as in purified splenic dendritic cell cultures. These results provide evidence that expression of JNK2 plays a role in T. gondii-induced immunopathology, at the same time in promoting susceptibility to this parasitic pathogen.

A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis

BackgroundHypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established.MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1β and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student’s t test. EAE clinical scoring was analyzed using the Mann–Whitney U test.ResultsWe showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1β during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1β, which in turn reduced the ability of mice to generate Th17 responses—critical steps in the pathogenesis of EAE and MS.ConclusionTogether, these data support a novel role for cathepsin Z in the propagation of IL-1β-driven neuroinflammation.

Oslerus osleri (Metastrongyloidea; Filaroididae) in Gray Wolves (Canis lupus) from Banff National Park, Alberta, Canada

Oslerus osleri is a filaroid parasite of the respiratory tract of canids. In North America, it is most commonly reported from coyotes (Canis latrans) and domestic dogs (Canis lupus familiaris), but reports in gray wolves (Canis lupus) are infrequent. We report a new geographic record for O. osleri in four gray wolves from Banff National Park, Alberta, Canada. Adult nematodes found in nodules in the submucosa of the trachea and bronchi were identified as O. osleri based on morphometry of spicules of males. We sequenced two segments of the genome of adult nematodes: a 1,111– base pair (bp) segment of the 18S region that was 100% identical to the 18S region of O. osleri from a coyote in California and a 537-bp segment that included the ITS-2 region and partial 5.8S and 28S genes. Histopathologically, there were submucosal nodules of adult nematodes surrounded by fibrosis and lymphoplasmacytic inflammation. These findings are consistent with O. osleri infections in other canids. The importance of this parasite in wild canid populations is unknown, but prevalence may be underestimated because many studies have focused on gastrointestinal parasites or used routine fecal flotation that has low sensitivity for this parasite. The ecology and population genetics of this parasite where multiple species of canids are sympatric warrant closer investigation to determine potential for interspecies transmission.

Sheep-Associated Malignant Catarrhal Fever–Like Skin Disease in a Free-Ranging Bighorn Sheep (Ovis canadensis), Alberta, Canada

Abstract Malignant catarrhal fever–like clinical disease was diagnosed in a free-ranging bighorn sheep (Ovis canadensis) from Alberta, Canada, in June 2015. Antemortem and gross pathology findings included muscle atrophy, marked weight loss, and bilaterally symmetric alopecia with hyperpigmentation and crusting over the face, medial surfaces of the pinnae, dorsal trunk, distal limbs, perineal area, and tail. Histologically, the skin lesions were characterized by granulomatous mural folliculitis with numerous multinucleated giant cells and fewer lymphocytes and eosinophils consistent with previous reports of chronic ovine herpesvirus-2 (OvHV-2) infection. Multiple skin samples were positive for OvHV-2 DNA on PCR, and on partial sequencing of the viral DNA, there was 94% homology with reference GenBank OvHV-2. Quantitative PCR confirmed an increased level of OvHV-2 DNA in the lesional skin tissues. Based on exclusion of other disease processes, gross and histological lesions, PCR, and viral DNA sequencing results, a diagnosis of OvHV-2–mediated malignant catarrhal fever–like dermatitis was made.

The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response

The synthetic supercooling drug, icilin, and its primary receptor target, the cation channel transient receptor potential (TRP) melastatin‐8 (TRPM8), have been described as potent negative regulators of inflammation in the colon. The aim of this study was to determine whether the anti‐inflammatory action of icilin could potentially be used to treat autoimmune neuroinflammatory disorders, such as multiple sclerosis (MS). During experimental autoimmune encephalomyelitis (EAE)—a CD4+ T cell‐driven murine model of MS—we found that both wild‐type (WT) and TRPM8‐deficient EAE mice were protected from disease progression during icilin treatment, as evidenced by delays in clinical onset and reductions in neuroinflammation. In vitro, icilin potently inhibited the proliferation of murine and human CD4+ T cells, with the peripheral expansion of autoantigen‐restricted T cells similarly diminished by the administration of icilin in mice. Attenuation of both TRPM8–/– and TRP ankyrin‐1–/– T‐cell proliferation by icilin was consistent with the WT phenotype, which suggests a mechanism that is independent of these channels. In addition, icilin treatment altered the expressional profile of activated CD4+ T cells to one that was indicative of restricted effector function and limited neuroinflammatory potential. These findings identify a potent antiinflammatory role for icilin in lymphocyte‐mediated neuroinflammation and highlight clear pleiotropic effects of the compound beyond classic TRP channel activation.—Ewanchuk, B. W., Allan, E. R. O., Warren, A. L., Ramachandran, R., Yates, R. M. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T‐cell response. FASEB J. 32,1236‐1249 (2018). www.fasebj.org