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BackgroundCytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development.MethodsRecent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model.ResultsProbing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis.ConclusionsProtein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Background: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. Objective: This study evaluated platelet function in HWI dogs. Methods: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. Results: Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P= .025, P= .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 10³/μL vs 0.267 ± 0.20 × 10³/μL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. Conclusions: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs. [ABSTRACT FROM AUTHOR]

Prior studies have identified high CD25 expression in canine diffuse large B‐cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B‐cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B‐cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196–348 days) and median PFS was 196 days (95% CI, 172–220 days). Higher percentage of B‐cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p =.02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed. [ABSTRACT FROM AUTHOR]

Background: The role of the renin‐angiotensin‐aldosterone system in cats with chronic kidney disease (CKD) is incompletely understood. Objective: To characterize components of the intrarenal renin‐angiotensin system (RAS) in cats with CKD. Animals: Eleven cats with naturally occurring CKD (CKD group) and 8 healthy control cats. Methods: Renal tissue samples were evaluated by reverse‐transcription polymerase chain reaction for renin, angiotensinogen, angiotensin‐converting enzyme (ACE), and angiotensin II type 1 receptor transcript levels, and by liquid chromatography‐mass spectrometry for quantification of angiotensin I, II, III, and IV concentrations. Linear mixed models were used to compare gene transcript levels and concentrations of angiotensin peptides between groups. Results: Cats of the CKD group were significantly older (P <.001) and more likely to be neutered (P =.007) than healthy control cats. Kidneys from cats with CKD had significantly higher transcript levels of angiotensinogen (P <.001) and lower transcript levels of ACE (P <.001) than those from control cats. Renal angiotensin I concentrations were increased in CKD compared with control kidneys (P =.001). No other significant differences in renal transcript levels or angiotensin peptide concentrations were noted between groups. Conclusion and Clinical Importance: The intrarenal RAS might be activated in cats with CKD. Small sample size and differences in age, neuter status, and dietary sodium intake between groups might have limited the ability to identify a significant difference in concentration of renal angiotensin II. [ABSTRACT FROM AUTHOR]

Background: Vincristine might increase circulating platelet numbers but the functional capacity of these newly released platelets is unknown. Objective: To evaluate and compare the functionality of mature and immature (reticulated) platelets after a single intravenous dose of vincristine in dogs. Animals: Ten healthy purpose‐bred dogs. Methods: Dogs prospectively received a single IV injection of 0.02 mg/kg vincristine or 0.9% saline. Before and after treatment on days 3, 5, and 7, platelets (resting and after thrombin stimulation) were assessed by flow cytometric determination of P‐selectin (CD62P) expression. Reticulated platelets were distinguished using thiazole orange (TO) staining. Results: Relative to saline, vincristine administration increased platelet count from day 0 to day 7 (225 ± 58 to 273 ± 65 × 103/μL, vs 299 ± 76.4 to 214 ± 20 × 103/μL, P =.01) and increased percentage of reticulated platelets from day 0 to day 5 (3.9 ± 1.5% to 6.1 ± 1.6%, P =.02). On all days, reticulated platelets had greater resting expression of CD62P than did mature platelets (49.6 ± 4% vs 10.2 ± 1%, P ≤.001). Across all days, CD62P expression by reticulated platelets in the vincristine and saline‐treated groups was not different when unstimulated (P =.7) or after thrombin stimulation (P =.33). Conclusions and Clinical Importance: Reticulated platelets released in response to vincristine administration function similarly to mature platelets. [ABSTRACT FROM AUTHOR]

Background: Increased gene transcription of hypoxia‐induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD). Objective: To characterize hypoxia‐induced profibrotic pathways in naturally occurring CKD in cats. Animals Twelve client‐owned cats with CKD and 8 healthy control cats. Methods: In this prospective, cross‐sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription‐quantitative PCR for characterization of transcript levels of hypoxia‐inducible factor‐1α (HIF1A), matrix metalloproteinases‐2 (MMP2), ‐7 (MMP7), and ‐9 (MMP9), tissue inhibitor of metalloproteinase‐1 (TIMP1), transforming growth factor‐β1 (TGFB1), and vascular endothelial growth factor‐A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys. Results: Kidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P <.001), and lower levels of VEGFA (P =.006) than those from control cats. Transcript levels of MMP7 (P =.05) and TIMP1 (P =.005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P =.002), MMP7 (P =.02), and TIMP1 (P =.02) were positively, whereas those of VEGFA (P =.003) were negatively, associated with histologic score severity. Conclusion and Clinical Significance: Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats. [ABSTRACT FROM AUTHOR]

Background: Flow cytometry (FC) is used increasingly in veterinary medicine for further characterization of hematolymphoid cells. Guidelines for optimizing assay performance and interpretation of results are limited, and concordance of results across laboratories is unknown. Objectives: This study aimed to determine inter‐investigator agreement on the interpretation of FC results from split samples analyzed in different laboratories using various protocols, cytometers, and software; and on the interpretation of archived FC standard (FCS) data files contributed by the different investigators. Methods: This was a multicenter observational cross‐sectional study. Anticoagulated blood or lymph node aspirate samples from nine client‐owned dogs were aliquoted and shipped to participating laboratories. Samples were analyzed with individual laboratory‐developed protocols. In addition, FCS files from a set of separate samples from 11 client‐owned dogs were analyzed by participating investigators. A person not associated with the study tabulated the results and interpretations. Agreement of interpretations was assessed with Fleiss' kappa statistic. Results: Prolonged transit times affected sample quality for some laboratories. Overall agreement among investigators regarding the FC sample interpretation was strong (κ = 0.86 ± 0.19, P <.001), and for specific categories, ranged from moderate to perfect. Agreement of the lymphoproliferation or other leukocyte sample category from the analysis of the FCS files was weak (κ = 0.58 ± 0.05, P <.001). Conclusions: Lymphoproliferations were readily identified by FC, but identification of the categories of hematolymphoid neoplasia in fresh samples or archived files was variable. There is a need for a more standardized approach to maximize the enormous potential of FC in veterinary medicine. [ABSTRACT FROM AUTHOR]

Objective To describe the diagnosis, management, and outcome of pyothorax in a domestic ferret ( Mustela putorius furo). Case Summary A domestic ferret was evaluated for a history of lethargy, anorexia, and pyrexia. Pleural effusion was detected with radiography and ultrasonography, and a diagnosis of pyothorax was made following cytologic evaluation of pleural fluid. Bilateral thoracostomy tubes were placed for thoracic drainage and lavage, and the ferret was treated with intravenous crystalloid fluids, antimicrobials, and analgesics. Bacterial culture of the pleural fluid yielded Fusobacterium spp. and Actinomyces hordeovulneris. This treatment protocol resulted in resolution of pyothorax, and a positive clinical outcome. New or Unique Information Provided This is the first reported case of successful management of pyothorax caused by Fusobacterium spp. and A. hordeovulneris in a ferret. [ABSTRACT FROM AUTHOR]

The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group. [ABSTRACT FROM AUTHOR]

Background: Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well‐characterized. Hypothesis/Objectives: To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. Animals: Six bluetick coonhounds. Methods and materials: Lymphocyte‐enriched cells were incubated with or without the T‐cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 μM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 μM and Con A + ciclosporin. We assessed both T‐cell proliferation and the secretion of cytokines. Results: Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 μM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)‐2, IL‐15], pro‐inflammatory cytokines (interferon‐gamma (IFN‐γ), IL‐18) and the regulatory cytokine IL‐10; tumour necrosis factor alpha (TNF‐α) and IL‐6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co‐treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL‐2, IL‐10, IL‐15, IL‐18, IFN‐γ and TNF‐α. Surprisingly, oclacitinib at 1 μM (337 ng/mL, corresponding to the oral dosage of 0.4–0.6 mg/kg) did not significantly affect Con A‐stimulated T‐cell proliferation nor cytokine production (IL‐2, IL‐10, IL‐15, IL‐18, IFN‐γ and TNF‐α). Conclusions: Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs. Background – Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well‐characterized. Hypothesis/Objectives – To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. Conclusions – Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs. [ABSTRACT FROM AUTHOR]

The article presents a case study of an 8-year-old male, mixed-breed dog suffering from unilateral nasal discharge. The dog was treated with antibiotics and anti-inflammatory drugs. Mucus and nasopharyngeal tissue of the patient was obtained and submitted for fungal culture and histopathology, and smears were made for cytologic evaluation. The cytologic diagnosis revealed nasal capillariasis, an infection caused by Eucoleus boehmi, along with eosinophilic inflammation.

OBJECTIVE: To use RNA sequencing (RNAseq) to characterize renal transcriptional activities of genes associated with proinflammatory and profibrotic pathways in ischemia-induced chronic kidney disease (CKD) in cats. SAMPLES: Banked renal tissues from 6 cats with experimentally induced CKD (renal ischemia [RI] group) and 9 healthy cats (control group). PROCEDURES: Transcriptome analysis with RNAseq, followed by gene ontology and cluster analyses, were performed on banked tissue samples of the right kidneys (control kidneys) from cats in the control group and of both kidneys from cats in the RI group, in which unilateral (right) RI had been induced 6 months before the cats were euthanized and the ischemic kidneys (IKs) and contralateral nonischemic kidneys (CNIKs) were harvested. Results for the IKs, CNIKs, and control kidneys were compared to identify potential differentially expressed genes and overrepresented proinflammatory and profibrotic pathways. RESULTS: Genes from the gene ontology pathways of collagen binding (eg, transforming growth factor-ß1), metalloendopeptidase activity (eg, metalloproteinase [MMP]-7, MMP-9, MMP-11, MMP-13, MMP-16, MMP-23B, and MMP-28), chemokine activity, and T-cell migration were overrepresented as upregulated in tissue samples of the IKs versus control kidneys. Genes associated with the extracellular matrix (eg, TIMP-1, fibulin-1, secreted phosphoprotein-1, matrix Gla protein, and connective tissue growth factor) were upregulated in tissue samples from both the IKs and CNIKs, compared with tissues from the control kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: Unilateral ischemic injury differentially altered gene expression in both kidneys, compared with control kidneys. Fibulin-1, secreted phosphoprotein-1, and matrix Gla protein may be candidate biomarkers of active kidney injury in cats. [ABSTRACT FROM AUTHOR]

OBJECTIVE To characterize transcription of profibrotic mediators in renal tissues of cats with ischemia-induced chronic kidney disease (CKD). SAMPLE Banked renal tissues from 6 cats with experimentally induced CKD (Rl group) and 8 healthy control cats. PROCEDURES For cats of the Rl group, both kidneys were harvested 6 months after ischemia was induced for 90 minutes in I kidney. For control cats, the right kidney was evaluated. All kidney specimens were histologically examined for fibrosis, inflammation, and tubular atrophy. Renal tissue homogenates underwent reverse transcription quantitative PCR assay evaluation to characterize gene transcription of hypoxia-inducible fa c to r-la (H IF - la), matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase- l (TIMP-I), transforming growth factor-|3l, and vascular endothelial growth factor A. Gene transcription and histologic lesions were compared among ischemic and contralateral kidneys of the Rl group and control kidneys. RESULTS Ischemic kidneys had greater transcript levels of MMP-7, MMP-9, and transforming growth factor-pi relative to control kidneys and of MMP-2 relative to contralateral kidneys. Transcription of TIMP-I was upregulated and that of vascular endothelial growth factor A was downregulated in ischemic and contralateral kidneys relative to control kidneys. Transcription of H IF - la did not differ among kidney groups. For ischemic kidneys, there were strong positive correlations between transcription of H IF - lα, MMP-2, MMP-7, and TIMP-I and severity of fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE Transcription of genes involved in profibrotic pathways remained altered in both kidneys 6 months after transient renal ischemia. This suggested that a single unilateral renal insult can have lasting effects on both kidneys. [ABSTRACT FROM AUTHOR]

Cytauxzoon felis is a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targets C. felis cytochrome b (cytb), of which 30 unique genotypes have been identified. The C. felis cytb genotype cytb1 is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguish C. felis cytb1 from other cytochrome b genotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in the C. felis cytb gene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguish cytb1 from other cytb genotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which the C. felis cytb genotypes had been characterized previously. The PCR panel identified C. felis cytb1 with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cytauxzoon felis, an emerging virulent protozoan parasite that infects domestic cats, is treated with atovaquone and azithromycin (A&A). Atovaquone targets parasite cytochrome b. We characterized the C. felis cytochrome b gene (cytb) in cats with cytauxzoonosis and found a cytb genotype that was associated with survival in A&A-treated cats.