The exon-skipping oligonucleotide, KitStop, depletes tissue-resident mast cells in vivo to ameliorate anaphylaxis.

Introduction: Anaphylaxis represents the most extreme and life-threatening form of allergic disease and is considered a medical emergency requiring immediate intervention. Additionally, some people with mastocytosis experience recurrent episodes of anaphylaxis during normal daily activities without exposure to known triggers. While acute therapy consists primarily of epinephrine and supportive care, chronic therapy relies mostly on desensitization and immunotherapy against the offending allergen, which is a time-consuming and sometimes unsuccessful process. These treatments also necessitate identification of the triggering allergen which is not always possible, and thus highlighting a need for alternative treatments for mast cell-mediated diseases. Methods: The exon-skipping oligonucleotide KitStop was administered to mice intradermally, intraperitoneally, or systemically at a dose of 12.5 mg/kg. Local mast cell numbers were enumerated via peritoneal lavage or skin histology, and passive systemic anaphylaxis was induced to evaluate KitStop's global systemic effect. A complete blood count and biochemistry panel were performed to assess the risk of acute toxicity following KitStop administration. Results: Here, we report the use of an exon-skipping oligonucleotide, which we have previously termed KitStop, to safely reduce the severity and duration of the anaphylactic response via mast cell depopulation in tissues. KitStop administration results in the integration of a premature stop codon within the mRNA transcript of the KIT receptor-a receptor tyrosine kinase found primarily on mast cells and whose gain-of-function mutation can lead to systemic mastocytosis. Following either local or systemic KitStop treatment, mice had significantly reduced mast cell numbers in the skin and peritoneum. In addition, KitStop-treated mice experienced a significantly diminished anaphylactic response using a model of passive systemic anaphylaxis when compared with control mice. Discussion: KitStop treatment results in a significant reduction in systemic mast cell responses, thus offering the potential to serve as a powerful additional treatment modality for patients that suffer from anaphylaxis.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness.

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.

A novel missense mutation of the NAT10 gene in a juvenile Schnauzer dog with chronic respiratory tract infections.

An 18-month-old intact male Schnauzer dog was evaluated for chronic, lifelong respiratory tract infections that were unresponsive to administration of a variety of antibiotics and corticosteroids. The dog developed persistent vomiting and diarrhea around 1 year of age that was minimally responsive to diet change, antibiotics, and corticosteroids. Despite supportive care, the dog was ultimately euthanized at 20 months of age due to persistent respiratory and gastrointestinal disease. Whole genome sequencing discovered a deleterious missense A/C mutation within the NAT10 gene, a gene essential for microtubule acetylation, appropriate ciliary development, and cytokinesis. Pipeline analysis of the genomes of 579 dogs from 55 breeds did not detect this mutation. Though never described in veterinary medicine, NAT10 mutation occurs in humans with ciliary aplasia, suggesting a pathophysiological mechanism for this dog and highlighting an associated mutation or possible novel genetic cause of chronic respiratory infections in dogs.

Dysbiosis of fecal microbiota in cats with naturally occurring and experimentally induced Tritrichomonas foetus infection.

The protozoal pathogen Tritrichomonas foetus infects the colon of domestic cats and is a major cause of chronic colitis and diarrhea. Treatment failure is common, but antibiotics may improve clinical signs in a subset of cats, leading researchers to question involvement of the colonic microbiota in disease pathogenesis. Studies performed in women with venereal Trichomonas vaginalis infections have revealed that dysbiosis of host microbiota contributes to pathogenicity with similar findings also found in mice with intestinal Tritrichomonas musculis The aim of this study was to characterize differences in the fecal microbiota of cats with and without naturally occurring T. foetus infection and in a group of kittens prior to and after experimentally induced infection. Archived fecal DNA from cats undergoing testing for T. foetus infection (n = 89) and experimentally infected kittens (n = 4; at pre-, 2 weeks, and 9 weeks post-infection) were analyzed by sequencing of 16S rRNA genes. Amongst the naturally infected population, the genera Megamonas and Helicobacter were significantly increased in prevalence and abundance in cats testing positive for T. foetus infection. In the group of four experimentally infected kittens, fecal samples post-infection had significantly lower abundance of genus Dialister and Megamonas and greater abundance of the class Betaproteobacteria and family Succinivibrionaceae. We hypothesize that T. foetus promotes dysbiosis by competition for fermentable substrates used by these bacteria and that metabolic byproducts may contribute to the pathogenesis of colonic inflammation and diarrhea. Future studies are warranted for the measurement of fecal concentrations of microbial and protozoal metabolites in cats with T. foetus infection for the identification of potential therapeutic targets.

Reversible myelosuppression suspected to be secondary to linezolid in a cat with infected subcutaneous ureteral bypass systems.

CASE SUMMARY: A 5-year-old castrated male domestic shorthair cat was presented for a multidrug-resistant Enterococcus faecium urinary tract infection within its bilateral subcutaneous ureteral bypass systems. After considerable consultation, the cat was treated with oral linezolid (10 mg/kg q12h) for two separate 2-week courses over 5 weeks. Over this time period, the cat became progressively neutropenic and thrombocytopenic, but was otherwise clinically stable. Upon cessation of the linezolid, the bicytopenia resolved within 12 days. RELEVANCE AND NOVEL INFORMATION: The reversible myelosuppression in this case is suspected to be secondary to linezolid administration. While previously reported in people, this effect has not been reported at therapeutic doses in veterinary species. This report demonstrates the potential for adverse drug reaction development in cats treated with prolonged linezolid therapy and highlights the need for extreme caution when utilizing linezolid in patients with renal insufficiency. Linezolid is the only drug currently approved by the Food and Drug Administration to treat vancomycin-resistant enterococci infections in people; however, resistance to this antibiotic appears to be increasing. Multidrug-resistant organisms continue to be a real global public health threat in both human and veterinary medicine. Third-tier antibiotics should only be considered under extreme circumstances and after considerable consultation with a specialist. Please note that the authors of this manuscript followed American Veterinary Medical Association policies on stewardship and International Society for Companion Animal Infectious Diseases guidelines, and do not promote or encourage the use in daily practice.

Association of fecal sample collection technique and treatment history with Tritrichomonas foetus polymerase chain reaction test results in 1717 cats.

BACKGROUND: Fecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs. OBJECTIVES: To establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus. ANIMALS: Fecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017. METHODS: This study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form. RESULTS: Positive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31-3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%-39.4%) cats treated with ronidazole had a second positive test result. CONCLUSIONS AND CLINICAL IMPORTANCE: Results of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all-potential-causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug.

Experimental infection of New Zealand Merino sheep with a suspension of Mycobacterium avium subspecies paratuberculosis (Map) strain Telford: Kinetics of the immune response, histopathology and Map culture.

A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johne's disease in NZ Merino lambs.