Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

A homozygous ADAMTS2 nonsense mutation in a Doberman Pinscher dog with Ehlers Danlos syndrome and extreme skin fragility.

An eight-week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper-mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper-elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole-genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C-to-T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Long-term evaluation of mesenchymal stem cell therapy in a feline model of chronic allergic asthma.

BACKGROUND: Mesenchymal stem cells (MSCs) decrease airway eosinophilia, airway hyperresponsiveness (AHR), and remodelling in murine models of acutely induced asthma. We hypothesized that MSCs would diminish these hallmark features in a chronic feline asthma model. OBJECTIVE: To document effects of allogeneic, adipose-derived MSCs on airway inflammation, AHR, and remodelling over time and investigate mechanisms by which MSCs alter local and systemic immunologic responses in chronic experimental feline allergic asthma. METHODS: Cats with chronic, experimentally induced asthma received six intravenous infusions of MSCs (0.36-2.5 × 10E7 MSCs/infusion) or placebo bimonthly at the time of study enrollment. Cats were evaluated at baseline and longitudinally for 1 year. Outcome measures included: bronchoalveolar lavage fluid cytology to assess airway eosinophilia, pulmonary mechanics and clinical scoring to assess AHR, and thoracic computed tomographic (CT) scans to assess structural changes (airway remodelling). CT scans were evaluated using a scoring system for lung attenuation (LA) and bronchial wall thickening (BWT). To assess mechanisms of MSC action, immunologic assays including allergen-specific IgE, cellular IL-10 production, and allergen-specific lymphocyte proliferation were performed. RESULTS: There were no differences between treatment groups or over time with respect to airway eosinophilia or AHR. However, significantly lower LA and BWT scores were noted in CT images of MSC-treated animals compared to placebo-treated cats at month 8 of the study (LA P = 0.0311; BWT P = 0.0489). No differences were noted between groups in the immunologic assays. CONCLUSIONS AND CLINICAL RELEVANCE: When administered after development of chronic allergic feline asthma, MSCs failed to reduce airway inflammation and AHR. However, repeated administration of MSCs at the start of study did reduce computed tomographic measures of airway remodelling by month 8, although the effect was not sustained at month 12. Further study of MSC therapy including repeated MSC administration is warranted to assess impact on remodelling in chronic asthma.

Neonatal aerosol exposure to Bermuda grass allergen prevents subsequent induction of experimental allergic feline asthma: evidence for establishing early immunologic tolerance.

Allergic asthma is increasing in industrialized countries, especially in children. Rodent and human studies suggest an opportunity to "prevent" asthma in the perinatal period. The aims of this study were to create a more "natural" model of feline asthma by exposing offspring of asthmatic queens to Bermuda grass allergen (BGA) by inhalation only, and to investigate maternal-fetal-infant interactions in the development of asthma. Kittens from asthmatic queens were divided into four groups: maternal exposure to aerosolized BGA during the third trimester, neonatal exposure to aerosolized BGA in the first three months of life, both maternal and neonatal exposure, or saline control. Kittens failing to achieve an asthmatic phenotype based on bronchoalveolar lavage fluid (BALF) analysis by 6 months underwent traditional sensitization: adjuvanted allergen injection, intranasal allergen, and aerosol challenges. BALF was collected at 3, 4 and 6 months, and after sensitization at 8 months, and analyzed for eosinophil counts and BGA-specific IgG and IgA. Intradermal testing (IDT) was performed at 6 and 7 months. At six months none of the kittens had airway eosinophilia, BGA-specific IgG or IgA, and were non-responsive to IDT. After sensitization, kittens receiving neonatal aerosolization failed to develop airway eosinophilia as seen in the controls. Kittens exposed to BGA aerosols, either in-utero or neonatally, continued to lack IDT response. Chronic exposure to BGA aerosols failed to induce asthma in kittens, and instead tolerized the kittens to BGA. This is the first evidence that neonatal intervention could potentially "prevent" allergic asthma in cats.

Comparison of direct and indirect bronchoprovocation testing using ventilator-acquired pulmonary mechanics in healthy cats and cats with experimental allergic asthma.

Airway hyperresponsiveness (AHR) is a key feature of asthma and can be measured using bronchoprovocation. Direct (methacholine, MCh) or indirect (adenosine-5-monophosphate, AMP; or mannitol) bronchoprovocants are used in human patients, the latter inducing AHR only with pre-existing airway inflammation. The present study compared the responses to direct (MCh) and indirect (mannitol, AMP) bronchoprovocation in healthy and asthmatic cats (n=6/group). The order of bronchoprovocant was randomized using a published table of random numbers and there was a 1-month washout before crossover to the next treatment. Pulmonary mechanics were measured in anesthetized and mechanically ventilated cats using a critical care ventilator. Saline at baseline and increasing doses of each bronchoprovocant were aerosolized for 30 s, followed by 4 min of data collection between doses. The endpoint for each bronchoprovocant was reached when airway resistance exceeded 200% of baseline values (EC200Raw). There was a significant difference (P<0.001) in the airway response of asthmatic vs. healthy cats over the range of MCh concentrations, despite there being no significant difference in the EC200Raw between the groups. Response to MCh was significantly greater (P<0.05) in asthmatic than in healthy cats at MCh concentrations as low as 0.0625 mg/mL. For AMP, a small subset of asthmatics (n=2/6) responded at low concentrations; four asthmatic cats and all healthy cats failed to respond even to the highest concentrations of AMP. One asthmatic cat but no healthy cats responded to mannitol. In conclusion, MCh discriminated asthmatic from healthy cats but neither AMP nor mannitol was an effective bronchoprovocant in this model.

Diminazene diaceturate for treatment of chronic Cytauxzoon felis parasitemia in naturally infected cats.

BACKGROUND: Cytauxzoon felis is a hemoprotozoal parasite that causes substantial morbidity and mortality during the acute phase of infection in cats. However, cats that survive the acute illness remain persistently infected and may serve as a reservoir for the tick-transmitted pathogen. OBJECTIVE: We investigated the ability of the antiprotozoal compound diminazene diaceturate to eliminate the pathogen from naturally infected C. felis carriers. ANIMALS: Seven healthy, chronically infected domestic cats housed in a research setting. METHODS: Prospective clinical trial. Cats were treated in a masked fashion with diminazene diaceturate (3 mg/kg) or placebo IM in a series of 2 injections 7 days apart. Clearance of the organism was assessed by light microscopy and real-time polymerase chain reaction (PCR) at 0, 3, 6, and 10 weeks. In addition, cats were monitored for behavioral changes or for changes on physical examination, CBC, plasma biochemical profile, and urinalysis periodically. Cats that remained parasitemic at the end of 10 weeks were switched to the alternative treatment and similarly monitored for an additional 10 weeks. RESULTS: Adverse events associated with treatment were limited to self-resolving hypersalivation and injection site soreness; the former was ameliorated by premedication with atropine. Parasite burden, as assayed by both light microscopy and real-time PCR, was similar between diminazene- and placebo-treated cats. CONCLUSIONS AND CLINICAL RELEVANCE: Diminazene diaceturate was unable to eliminate the pathogen or decrease parasite burden in healthy, chronically infected cats.

Efficacy of atovaquone and azithromycin or imidocarb dipropionate in cats with acute cytauxzoonosis.

BACKGROUND: Imidocarb or a combination of atovaquone and azithromycin (A&A) has been suggested for treatment of cats with cytauxzoonosis, but neither has been prospectively evaluated for efficacy. HYPOTHESIS/OBJECTIVES: That survival to hospital discharge is improved by treatment with A&A as compared with imidocarb. ANIMALS: Eighty acutely ill cats with Cytauxzoon felis infection treated at one of 18 veterinary clinics in 5 states. METHODS: An open-label, randomized prospective study compared survival in cats treated with atovaquone (15 mg/kg p.o. q8h) and azithromycin (10 mg/kg p.o. q24h) or imidocarb (3.5 mg/kg i.m.). All received heparin, fluids, and supportive care. Clinical and clinicopathologic data from initial presentation were collated. Parasitemia was quantified (n = 79) and pathogens genotyped (n = 60). Logistic regression was used to determine the impact of treatment group on the primary endpoint, survival to hospital discharge or death. Covariants were analyzed by rank-sum testing. RESULTS: Of 53 cats treated with A&A, 32 (60%) survived to discharge while only 7 of 27 cats (26%) treated with imidocarb survived (P = .0036; odds ratio 7.2, 95% CI 2.2, 24). Cats with a lower parasitemia were more likely to survive, as were cats with higher white blood cell counts and lower total bilirubin. Unique pathogen genotypes were identified from 15 cats, while genotype isolated from 21 cats had been described previously. There were multiple pathogen genotypes identified in 24 cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Survival to discharge was more likely in cats treated with A&A as compared with imidocarb, although case fatality rate remained high.

Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in three populations.

BACKGROUND: A higher prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization is reported in healthcare workers compared with nonhealthcare workers. HYPOTHESIS: The prevalence of MRSA colonization differed in people and pets in households with healthcare workers as compared with households without healthcare workers. SUBJECTS: A person and 1 dog or cat from 586 households defined as either a nonhealthcare (n = 213), veterinary healthcare (n = 211), or human healthcare (n = 162) worker household. METHODS: Prospective cross-sectional study. Samples from humans and pets were cultured in vitro. Staphylococcus aureus was identified as methicillin sensitive (MSSA) or MRSA with mecA polymerase chain reaction. Pulsed-field gel electrophoresis and spa-typing were used to characterize relatedness of S. aureus and MRSA and assign USA types. RESULTS: The prevalence of MSSA and MRSA in humans was 21.5% (126/586) and 5.63% (33/586), respectively, and 7.85% (46/586) and 3.41% (20/586), respectively, in pets. There were no differences in prevalences of either MSSA or MRSA between household types. The proportion of MRSA among all S. aureus isolates in humans and pets was 20.8% (33/159) and 30.3% (20/66), respectively. In < 1.0% (4/586) of households, the same strain of MRSA was found in both a person and a pet. CONCLUSIONS AND CLINICAL IMPORTANCE: There were no differences in the prevalences of MSSA or MRSA between healthcare worker and nonhealthcare worker households. Pets and people colonized with S. aureus were as likely to be colonized with MRSA. Colonization of a person and their pet with the same strain of MRSA was rare.

Endocrine and immunologic effects of inhaled fluticasone propionate in healthy dogs.

BACKGROUND: Inhaled glucocorticoids reduce airway inflammation while minimizing systemic effects in several species. HYPOTHESIS: Inhaled fluticasone suppresses the hypothalamic-pituitary-adrenal axis (HPAA), modifies immune function, and induces clinical signs to a lesser extent than PO-administered prednisone in dogs. ANIMALS: Seven healthy adult pet dogs. METHODS: Dogs were randomized to 1 of 3 treatment groups in a crossover design: fluticasone propionate (220 mug actuation of a metered dose inhaler delivered via a spacer and mask, q12h), placebo (spacer and mask alone, q12h), or prednisone (1 mg/kg PO q24h). Each treatment was administered for 3 weeks followed by a 4-week washout. Appetite, attitude, and water consumption were recorded during the last week of each treatment period. Urine cortisol : creatinine ratios, ACTH stimulation tests, white blood cell counts, lymphocyte phenotype, and serum IgM and IgA concentrations were recorded at each baseline and after the last day of each treatment. Clinical observations were expressed descriptively. Friedman's test was applied to all data comparisons. Pairwise comparisons were made with a mixed model analysis when data were normally distributed, whereas signed rank tests were used otherwise (significance P-value <.01). RESULTS: Appetite and water consumption increased during prednisone treatment. Peak serum cortisol concentrations post-ACTH were significantly decreased in prednisone- and fluticasone-treated dogs compared with placebo (prednisone > fluticasone). Serum IgM concentrations were significantly decreased in dogs treated with prednisone. CONCLUSIONS AND CLINICAL IMPORTANCE: As used, fluticasone suppresses the HPAA to a lesser extent than prednisone and may avert systemic signs associated with PO-administered glucocorticoids in dogs.

Local photodynamic therapy for equine squamous cell carcinoma: evaluation of a novel treatment method in a murine model.

The objective of this study was to evaluate the effect of local photodynamic therapy (PDT) with verteporfin on tumor growth inhibition of squamous cell carcinoma (SCC) in a murine model. SCC was implanted in 85 nude mice by subcutaneous injection of A-431 SCC cells. Treatment groups (10 mice/group) received an intra-tumoral injection of verteporfin dissolved in dimethyl sulfoxide (DMSO) or 5% dextrose solution at a dose of 0.01 or 0.1mg/cm3. Controls received only solvent, or no injectate. All groups received identical light illumination (100J/cm2). Relative change in tumor volume (RCTV) at day 30 was compared between groups using the Wilcoxon rank sum test (P< 0.05). Local PDT with verteporfin at a dose of 0.1mg/cm3 resulted in significantly lower RCTV at day 30 compared to controls. Choice of solvent (DMSO versus D5W) did not affect the results. Local PDT may be an effective adjunctive therapy for the treatment of periocular equine SCC.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

Polymerase chain reaction detection of Cytauxzoon felis from field-collected ticks and sequence analysis of the small subunit and internal transcribed spacer 1 region of the ribosomal RNA gene.

Cytauxzoon felis produces a disease in domestic cats in the Midwest (U.S.A.), which often leads to a fatal outcome. Although the clinical disease process is well described, there are still many unanswered questions about this organism. For example, it is unknown whether species of ticks other than Dermacentor variabilis can serve as vectors for transmission. With recent reports of surviving cats from limited geographic areas, another relevant question is the potential for genetically less virulent organism strains. This study evaluated 352 individual or pooled tick samples (1,362 total ticks) for the presence of C. felis small subunit ribosomal RNA and internal transcribed spacer 1 (ITS-1) region genes using a polymerase chain reaction (PCR). These ticks were collected from dogs and cats in several Missouri counties, including 10 from cats diagnosed with cytauxzoonosis. Only 3 positive C. felis samples were identified in Amblyomma americanum nymphs, and there was very limited genetic variation noted in both genes. The small number of positive samples did not allow the study to determine which PCR analysis was more sensitive. This is the first known report of ITS-1 gene identification and sequencing for C. felis. It is also the first published investigation of genetic variation in C. felis.

CYP3A inductive potential of the rifamycins, rifabutin and rifampin, in the rabbit.

Rifabutin is effective in the treatment and prevention of Mycobacterium avium infection in people with HIV infection. Rifabutin is structurally related to another rifamycin, rifampin, a well-known inducer of the human P-450 isoform 3A. The rabbit isoform CYP3A6 and the human isoform CYP3A4 have similar P-450 predominance and substrate specificity and are both induced by rifampin. Our goal was to predict the CYP3A induction capacity of rifabutin and to determine if ex vivo CYP3A induction potential of rifamycins is predictive of that obtained in vivo. We determined the in vivo and ex vivo CYP3A6 induction by 4 days of treatment with rifabutin (100 mg/kg), rifampin (100 mg/kg), or vehicle (DMSO) in the rabbit. The ex vivo measures were CYP3A6 activity (N-demethylation of erythromycin and hydroxylation of triazolam) and CYP3A content in rabbit hepatic microsomes preparations. The in vivo measures were oral clearance of triazolam and its formation clearance to its hydroxylated metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam. Rifampin increased CYP3A6 activity by 2- to 3-fold in hepatic microsomes compared to vehicle. Rifabutin increased CYP3A content 1.7-fold, but did not significantly increase microsomal CYP3A6 activity. Oral triazolam clearance and formation clearances to the two hydroxylated metabolites were 2- to 3-fold greater in rabbits treated with rifampin. These clearances were unaffected by rifabutin administration. Ex vivo enzyme activities correlated with in vivo changes in clearance of triazolam and the formation clearance to its hydroxylated metabolites. Rifabutin is a weaker inducer of CYP3A6 than rifampin. These data suggest that ex vivo enzyme activity is a viable approach to predict in vivo inductive potential of CYP3A inducers.

Mammary duct ectasia in dogs: 51 cases (1992-1999).

OBJECTIVE: To evaluate the clinical and pathologic characteristics of mammary duct ectasia in dogs. DESIGN: Retrospective study. ANIMALS: 51 dogs with mammary duct ectasia. PROCEDURE: Information regarding body condition, history, number and location of affected mammary glands, appearance of lesions, surgical treatment, nonsurgical treatment, and evidence of recurrence or development of mammary neoplasia was obtained from surveys sent to referring veterinarians. Results of information from examination of histologic sections and referring veterinarians were evaluated for all mammary duct ectasia biopsies performed between 1992 and 1999. RESULTS: Duct ectasia was the primary diagnosis in 51 of 1,825 (2.8%) mammary biopsy specimens and comprised 48% of nonneoplastic mammary diseases. Affected dogs were evenly distributed over a range of 1 to 13 years of age, with a mean age at the time of diagnosis of 6.1 +/- 3.1 years. All dogs were female (31 sexually intact, 20 spayed); 10 of 26 had whelped. Duct ectasia was described as nodular (26 dogs), cystic (13), and multiglandular (11) and located in caudal (31) more often than cranial (14) or middle glands (10). Ectasia recurred in 3 dogs. One dog had a history of previously excised mammary adenocarcinoma; another subsequently developed mammary carcinoma. CONCLUSIONS AND CLINICAL RELEVANCE: Duct ectasia affected mature, sexually intact and spayed female dogs over a wide age range. Certain breeds were affected more commonly than expected. Increased risk for mammary neoplasia was not evident. Duct ectasia should be considered as a cause for mammary enlargement, especially in young dogs or when its cystic nature is evident. Mastectomy is usually curative, and neoplasia should be ruled out in dogs with ectasia.

Alveolar mucosal approach to the canine nasal cavity.

A nine-year-old, intact female Afghan hound was presented for evaluation of an intermittent, mucopurulent, unilateral nasal discharge with a three-year duration. Radiographs showed the ipsilateral canine tooth within the rostral nasal cavity. The tooth was removed through an alveolar mucosal rhinotomy. There has been no recurrence of the nasal discharge or complications associated with the surgical procedure during the 20-month follow-up period.

Pseudomonas aeruginosa infection of respiratory epithelium in a cystic fibrosis xenograft model.

Pulmonary infection with Pseudomonas aeruginosa in patients with cystic fibrosis (CF) causes a chronic destructive bronchitis. A xenograft model was used to study the susceptibility of the CF respiratory epithelium to P. aeruginosa strain PAK and the virulence of certain mutants. Despite an early trend toward increased susceptibility, colonization of CF xenografts (ID(95), 62 colony-forming units [cfu]) was not statistically different (P=.5) than in xenografts with normal respiratory cells (ID(95), 1.2x10(3) cfu). Infection severity in 12 CF xenografts (mean polymorphonuclear leukocyte [PMNL] density, 1.88x10(6)+/-1.75x10(6)/xenograft) was similar to that in 16 non-CF xenografts (3.19x10(6)+/-2.45x10(6) PMNL/xenograft; P=.38), despite slightly greater bacterial density in the CF xenografts (mean, 1.57+/-2.73x10(6) cfu/xenograft) versus xenografts with normal epithelium (mean, 1.03+/-1.3x10(6) cfu/xenograft). P. aeruginosa mutants pilA and fliF, but not rpoN, colonized normal respiratory xenografts, indicating that colonization and infection in this model depend on an uncharacterized RpoN-controlled gene. This model appears to be suitable for genetic study of P. aeruginosa virulence but not of the CF respiratory tract's unique susceptibility.