Characterization of the intrarenal renin-angiotensin system in cats with naturally occurring chronic kidney disease.

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.

Analysis of genes associated with proinflammatory and profibrotic pathways upregulated in ischemia-induced chronic kidney disease in cats.

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.

Platelet number and function in response to a single intravenous dose of vincristine.

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.

Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease.

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-ß1 (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.

Profibrotic gene transcription in renal tissues from cats with ischemia-induced chronic kidney disease.

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 (RI group) and 8 healthy control cats. PROCEDURES: For cats of the RI group, both kidneys were harvested 6 months after ischemia was induced for 90 minutes in 1 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 factor-1α (HIF-1α), matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-ß1, and vascular endothelial growth factor A. Gene transcription and histologic lesions were compared among ischemic and contralateral kidneys of the RI group and control kidneys. RESULTS: Ischemic kidneys had greater transcript levels of MMP-7, MMP-9, and transforming growth factor-ß1 relative to control kidneys and of MMP-2 relative to contralateral kidneys. Transcription of TIMP-1 was upregulated and that of vascular endothelial growth factor A was downregulated in ischemic and contralateral kidneys relative to control kidneys. Transcription of HIF-1α did not differ among kidney groups. For ischemic kidneys, there were strong positive correlations between transcription of HIF-1α, MMP-2, MMP-7, and TIMP-1 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.

Limited genetic variability of Cytauxzoon felis apical membrane antigen-1 (ama1) from domestic cats and bobcats.

BACKGROUND: Cytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics. RESULTS: A 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans. CONCLUSIONS: In surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.

Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage.

Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99-100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis 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. METHODS: Recent 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. RESULTS: Probing 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. CONCLUSIONS: Protein 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.

Diagnosis and management of pyothorax in a domestic ferret (Mustela putorius furo).

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.

Extreme lymphocytosis with myelomonocytic morphology in a horse with diffuse large B-cell lymphoma.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /µL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny.

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.

PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).

Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.

Rapid High-Resolution Melt Analysis of Cytauxzoon felis Cytochrome b To Aid in the Prognosis of Cytauxzoonosis.

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.

A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

Cerebrospinal fluid from a dog with hind limb ataxia.

A 9-year-old spayed female German Shepherd dog with a history of orthopedic disease was presented to the North Carolina State University Veterinary Teaching Hospital for evaluation of recent, progressive, bilateral, hindlimb ataxia. Analysis of cisternal and lumbar cerebrospinal fluid (CSF) samples revealed normal total nucleated cell counts and a mild increase in protein concentration in the lumbar sample. In cytocentrifuged specimens of both CSF samples, aggregates of refractile, angular to irregular, pale blue to colorless, crystalline material were observed in the background. Some of the material appeared birefringent under polarized light. Differentials for the material included contrast agent, epidural anesthetics or other pharmacologic agents, or artifact introduced through sample processing, collection, or handling. Based on investigation of clinical and laboratory processes it was determined that tubes used to collect CSF in the hospital recently had been changed from additive-free glass tubes to silica-coated shatter-resistant plastic tubes (BD Vacutainer Plus serum tubes, silicone-coated, Becton Dickinson). A cytocentrifuged preparation of saline placed in a silica-coated tube contained crystalline material identical to that observed in the CSF samples; saline placed in an additive-free glass tube contained no material. In this case, we document the microscopic appearance of highly concentrated silica particles in cytocentrifuged preparations of CSF and underscore the importance of recognizing and identifying this artifact in cytologic preparations.