Untreated Dogs Research Articles

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.

Effect of prior general anesthesia or sedation and antiseizure drugs on the diagnostic utility of wireless video electroencephalography in dogs.

BackgroundAmbulatory wireless video electroencephalography (AEEG) is the method of choice to discriminate epileptic seizures from other nonepileptic episodes. However, the influence of prior general anesthesia (GA), sedation, or antiseizure drug (ASD) on the diagnostic ability of AEEG is unknown.Hypothesis/ObjectivesThe use of sedation/GA or ASD treatment before AEEG recording may affect the diagnostic ability of AEEG and the time to first abnormality on AEEG.AnimalsA total of 108 client‐owned dogs undergoing ambulatory AEEG for paroxysmal episodes.MethodsRetrospective cohort study. Proportions of diagnostic AEEG and time to first abnormality were compared between dogs that received sedation/GA or neither for instrumentation as well as dogs receiving at least 1 ASD and untreated dogs.ResultsAmbulatory EEG was diagnostic in 60.2% of all dogs including 49% of the sedation/GA dogs and 68% of dogs that received neither (odds ratio [OR], 2.25; 95% confidence interval [CI], 1.02‐5.00; P = .05). The AEEG was diagnostic in 51% of dogs receiving at least 1 ASD and 66% of untreated dogs (OR, 1.95; 95% CI, 0.9‐4.3; P = .11). No difference was found in time to first abnormality between sedation/GA or neither or ASD‐treated or untreated dogs (P = .1 and P = .3 respectively). Ninety‐five percent of dogs had at least 1 abnormality within 277 minutes.Conclusion and Clinical ImportanceSedation/GA and concurrent ASD administration were not identified as confounding factors for decreasing AEEG diagnostic capability nor did they delay the time to first abnormality. A 4‐hour minimal recording period is recommended.

Urinary tract infections in dogs with spontaneous hypercortisolism - frequency, symptoms and involved pathogens.

Dogs with hypercortisolism are predisposed to developing bacteriuria associated either with clinical signs of cystitis or without clinical signs (subclinical bacteriuria). Based on current guidelines, dogs with subclinical bacteriuria should not be treated with antibiotics because there is no evidence that treatment improves outcome and because unnecessary treatments should be avoided. Before these guidelines were published in 2019, dogs with hypercortisolism and bacteriuria were commonly treated with antibiotics irrespective of clinical signs. Comprehensive data on the frequency of bacterial cystitis, subclinical bacteriuria and the outcome of antimicrobial treatment in dogs with hypercortisolism is sparse. The aims of this study were to investigate dogs with hypercortisolism for the presence of bacterial cystitis and subclinical bacteriuria, to address the pathogens involved, and to assess the outcome of antibiotic treatment. Dogs newly diagnosed with hypercortisolism between 2005 and 2015 from which a urine bacterial culture was available were included. Statistical analysis was performed with non-parametric tests. Of the 161client-owned dogs included, 29 (18%) showed bacteriuria, which was subclinical in 24 (83%) cases. Escherichia coli was the most commonly isolated pathogen (58%). Bacteriuria was not associated with sex or neutering status. In 14 dogs, follow-up data was available, of which 13 (93%) were treated with antimicrobials for 14 to 28 days. Follow-up bacterial culture (1 to 118 days after cessation of therapy) was negative in 10 (77%) treated dogs; a negative follow-up culture was not associated with gender, age or duration of treatment. Bacteriuria persisted in three treated dogs and the one untreated dog. The prevalence of positive bacterial urinary culture in dogs with hypercortisolism was lower than previously reported. In the majority of dogs, bacteriuria was subclinical. Most dogs had a negative bacterial culture result after antimicrobial treatment; however, more resistant bacteria were detected in persistently positive urine.

It is unclear what impact pheromonotherapy has on stress in dogs in shelter environments

PICO question
 In dogs in shelters does pheromonotherapy reduce stress compared to untreated dogs?
 
 Clinical bottom line
 Category of research question
 Treatment
 The number and type of study designs reviewed
 Two studies were evaluated, one was a prospective blinded randomised placebo-controlled study (Tod et al., 2005) and the other was a prospective unblinded repeated measures study
 Strength of evidence
 The papers evaluated provide a weak-to-moderate strength of evidence, due to the limited sample size and short duration of the intervention
 Outcomes reported
 In both studies pheromonotherapy resulted in a statistically significant reduction in mean bark amplitude but not in duration or peak bark amplitude. Neither study found a significant change in expression of fear-related behaviours in response to a neutral stranger or a stressor
 Conclusion
 Based on th1e studies assessed in this Knowledge Summary it is not possible to determine if pheromonotherapy reduces stress in dogs in shelter environments. It cannot be determined whether the small absolute reduction in bark amplitude present in both studies is clinically or biologically significant. Additionally, barking is a non-specific behaviour (Protopopova, 2016; Pongráczet al., 2010; Taylor & Mills, 2007; and Yin, 2002), so these results cannot be interpreted as a reduction in stress without further study.
 It is beyond the scope of this Knowledge Summary to comment on any perceived or apparent difference in the effectiveness of pheromonotherapy in the shelter environment compared to the home environment
 
 How to apply this evidence in practice
 The application of evidence into practice should take into account multiple factors, not limited to: individual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where you work, the individual case in front of you, the availability of therapies and resources.
 Knowledge Summaries are a resource to help reinforce or inform decision-making. They do not override the responsibility or judgement of the practitioner to do what is best for the animal in their care.
 

Thalidomide Reduces Vascular Endothelial Growth Factor Immunostaining in Canine Splenic Hemangiosarcoma.

Hemangiosarcomas (HSA) are common neoplasms of dogs that often metastasize and are typically fatal. Recently it was demonstrated that thalidomide extends the survival time of dogs with HSA, potentially due to thalidomide-induced inhibition of vascular endothelial growth factor (VEGF) production by the neoplastic cells. To investigate this, immunostaining was used to evaluate VEGF within HSA metastases that developed after thalidomide treatment. The immunostaining was then compared to VEGF immunostaining in primary tumors from the same dogs prior to treatment with thalidomide and in metastatic tumors from untreated dogs with splenic HSA. Immunostaining was scored from 1 to 4 for each sample. Immunostaining in the metastatic lesions that had been treated with thalidomide had a mean immunostaining score of 1.4 which was significantly lower than the mean score in the corresponding primary splenic HSA (3.8, p = 0.02) and in metastases from untreated dogs (3.5, p = 0.02). This supports the hypothesis that thalidomide prolongs survival time in dogs with HSA due to inhibition of VEGF production by the neoplastic cells. As VEGF remained visible within HSAs exposed to thalidomide, additional treatments to inhibit VEGF production may further prolong survival times of dogs with these common canine neoplasms.

Spontaneous hemispheric ventricular collapse and subarachnoid haemorrhages in a dog with congenital hydrocephalus internus

BackgroundOverdrainage and collapse of the hemispheres is a potential severe complication after surgical treatment of internal hydrocephalus using ventriculoperitoneal shunts. Here we describe a case of a spontaneous hemispheric ventricular collapse in an untreated dog with congenital hydrocephalus internus.Case presentationA twelve-week-old, male, intact Golden Retriever was presented with a history of peracute obtundation, impaired vision, and progressive gait abnormalities of all limbs for three days. Neurological examination revealed a dome shaped skull, a broad-based stance and a moderate cerebellar ataxia. The postural responses were markedly delayed in all limbs. Moderate ventro-lateral strabismus, vertical nystagmus and absent menace response were observed bilaterally. Clinical signs indicated multifocal localisation (forebrain, cerebellum). Magnetic resonance imaging (MRI) showed dilation of all cerebral ventricles, irregular thinning of the periventricular white and grey matter, consistent with internal hydrocephalus. In addition, the hemispheres were collapsed at the right temporal and left frontal lobe with haemorrhage filling the adjacent subarachnoid space. The dog underwent left frontal and right temporal craniotomy for removal of the haemorrhage. The dog improved on all neurological signs and was discharged after seven days. A repeat MRI three months postsurgical intervention showed reexpansion of the cerebral hemispheres. Subarachnoid haemorrhages were markedly reduced.ConclusionsCollapse of the hemispheres can occur spontaneously in dogs with hydrocephalus internus. Removal of the haemorrhage can improve clinical signs.

Cognitive Abilities of Dogs with Mucopolysaccharidosis I: Learning and Memory

Simple SummaryLysosomes are cell organelles that contain enzymes that break down large molecules to be recycled or discarded. When lysosomal enzymes fail to perform this function, molecules become trapped and cause cellular destruction. Mucopolysaccharidosis I (MPS I) is a rare disease that occurs in dogs and humans due to a deficiency of the lysosomal enzyme, alpha-L-iduronidase. Humans affected with MPS I experience mild to severe clinical signs in facial features, skeletal changes, cognitive decline, and heart, liver, and respiratory disease. Similarly, MPS I in dogs also cause facial changes, musculoskeletal degeneration, spinal cord compression, and heart and liver disease. However, the cognitive ability in dogs affected with MPS I has not been investigated. The purpose of this pilot study was to determine the feasibility of conducting cognitive tests on MPS I affected dogs and their cognitive abilities. Three groups of dogs were tested: MPS I untreated, MPS I treated, and clinically normal. Dogs were successfully trained to perform the cognitive tests. Differences in their ability to reach the criterion was evident in attention oddity and scent discrimination tests. This study found cognition testing of dogs affected with MPS I to be feasible and recommend future studies focus on a single cognitive domain at a time.Mucopolysaccharidosis I (MPS I) results from a deficiency of a lysosomal enzyme, alpha-L-iduronidase (IDUA). IDUA deficiency leads to glycosaminoglycan (GAG) accumulation resulting in cellular degeneration and multi-organ dysfunction. The primary aims of this pilot study were to determine the feasibility of cognitive testing MPS I affected dogs and to determine their non-social cognitive abilities with and without gene therapy. Fourteen dogs were tested: 5 MPS I untreated, 5 MPS I treated, and 4 clinically normal. The treated group received intrathecal gene therapy as neonates to replace the IDUA gene. Cognitive tests included delayed non-match to position (DNMP), two-object visual discrimination (VD), reversal learning (RL), attention oddity (AO), and two-scent discrimination (SD). Responses were recorded as correct, incorrect, or no response, and analyzed using mixed effect logistic regression analysis. Significant differences were not observed among the three groups for DNMP, VD, RL, or AO. The MPS I untreated dogs were excluded from AO testing due to failing to pass acquisition of the task, potentially representing a learning or executive function deficit. The MPS I affected group (treated and untreated) was significantly more likely to discriminate between scents than the normal group, which may be due to an age effect. The normal group was comprised of the oldest dogs, and a mixed effect logistic model indicated that older dogs were more likely to respond incorrectly on scent discrimination. Overall, this study found that cognition testing of MPS I affected dogs to be feasible. This work provides a framework to refine future cognition studies of dogs affected with diseases, including MPS I, in order to assess therapies in a more comprehensive manner.

Genome analysis of enterobacteriaceae with non-wild type susceptibility to third-generation cephalosporins recovered from diseased dogs and cats in Europe

BackgroundExtended-spectrum-β-lactamases (ESBL) and plasmid-mediated cephalosporinases (pAmpC)-producing Enterobacteriaceae isolates are now reported worldwide in humans, animals, and in the environment. We identified the determinants of resistance to β-lactams and associated resistance genes as well as phylogenetic diversity of 53 ESBL- or pAmpC-producing Enterobacteriaceae isolated from dogs and cats in Europe. Materials/methodsOf a collection of 842 Enterobacteriaceae isolates that were recovered in 2013 and 2014 from 842 diseased and untreated dogs and cats, for 242 ampicillin or amoxicillin resistant isolates (MIC ≥ 16 mg/L), cefotaxime (CTX) and ceftazidime (CAZ) MICs were determined. Isolates with CTX and/or CAZ MIC ≥ 1 mg/L (n = 63) were selected, and their genomes were fully sequenced using Illumina Technology. Genomic data were explored to identify the resistance determinants, the plasmid incompatibility groups, and the sequence types (STs). Plasmid location of blaESBL and blaAmpC was evaluated for all isolates based on the co-localization of resistance and plasmid incompatibility group genes on the same contig. Phylogenetic trees were constructed using core-genome MLST. ResultsOf the 63 sequenced isolates, 53 isolates harbored a blaESBL or blaAmpC gene. Ten CTX and/or CAZ non-wild type isolates had neither blaESBL nor blaAmpC. Among the 63 isolates, 44 (69.8 %) were Escherichia coli, 11 (17.5 %) were Klebsiella pneumoniae, and 8 (12.7 %) were Proteus mirabilis. Fifty-one (80.9 %) isolates originated from dogs and 12 (19.1 %) from cats. Isolates were sampled from urinary tract (n = 36), skin and soft tissue (n = 22) and respiratory tract infections (n = 5). Thirty-two isolates (32/53, 60.4 %) carried blaESBL genes, including blaCTX-M-15 (n = 12), blaCTX-M-14 (n = 6), blaCTX-M-1 (n = 5), blaCTX-M-2 (n = 3), blaCTX-M-27 (n = 3), blaSHV-28 (n = 4), blaSHV-12 (n = 2), and blaVEB-6 (n = 1). Four isolates of K. pneumoniae had both blaCTX-M-15 and blaSHV-28. Twenty-one isolates (21/53, 39.6 %) carried genes encoding pAmpC, including blaCMY-2 (n = 19) and blaDHA-1 (n = 2). Thirteen E. coli isolates harbored both blaESBL or blaAmpC genes and plasmids of incompatibility groups IncIB (9/13), IncI1 (8/13), and IncFII (6/13). In addition to the reduced susceptibility to CTX and/or CAZ, reduced susceptibility or evidence of acquired resistance to at least one other relevant class of antibiotics was observed for all 63 isolates. E. coliisolates clustered in 23 STs, including B2 virulent clones from humans such as ST131 (n = 5), K. pneumoniae isolates mostly clustered in 3 STs: ST11 (n = 4), ST307 (n = 3), and ST16 (n = 2). Phylogenetic analysis identified the spread of E. coli ST131 blaCTX-M-27, and of K. pneumoniae ST307 harboring blaCTX-M-15 and blaSHV-28 or ST11 blaCTX-M-15. ConclusionsWe report here a 6.3 % prevalence of ESBL/pAmpC producing Enterobacteriaceae in diseased dogs and cats. This EU survey confirms that dogs and cats can be infected with epidemic multidrug resistant clones that may also spread in humans.

Comparison of Serum Trace Nutrient Concentrations in Epileptics Compared to Healthy Dogs.

Idiopathic epilepsy (IE) is a common cause of seizures in dogs. There are several investigations regarding serum concentrations of trace nutrients, including copper, selenium, zinc, manganese, and iron in human epileptics and animal models. However, research of this nature in dogs with epilepsy is lacking. The purpose of this prospective study was to compare serum concentrations of several trace nutrients in healthy dogs to dogs with idiopathic epilepsy. Healthy client-owned dogs (n = 50) and dogs with IE (n = 92) were enrolled and blood samples were collected for trace nutrient analysis. Epileptics were subdivided into three groups: controlled: n = 27, uncontrolled: n = 42, and untreated: n = 23. Serum was evaluated for concentrations of copper, selenium, zinc, cobalt, manganese, molybdenum, and iron using inductively coupled plasma mass spectroscopy. Uncontrolled epileptics had significantly higher manganese concentrations compared to normal dogs (p = 0.007). Untreated epileptics had higher iron levels than the other three groups (p = 0.04). Significantly higher levels of copper (p < 0.0001) were found in controlled and uncontrolled epileptics compared to normal or untreated dogs. Significantly higher levels of molybdenum (p = 0.01) were found in controlled epileptics compared to normal or untreated epileptics. Uncontrolled and controlled epileptics had significantly higher levels of selenium (p = 0.0003) vs. normal dogs, and uncontrolled epileptics had higher levels of zinc (p = 0.0002) than normal and untreated dogs. The significant difference in serum concentrations of several trace nutrients (manganese, selenium, and zinc) may suggest a role for these nutrients in the pathophysiology and/or treatment of epilepsy. Additionally, these results suggest that anti-convulsant therapy may affect copper and molybdenum metabolism.

Oxidative-antioxidative status and hepatic and renal factors following melatonin administration in castrated and intact dogs

BackgroundsMelatonin has significant antioxidant and hepatoprotective effects in normal and oxidative stress conditions. The aim of the present study was to assess the effects of melatonin on antioxidant, hepatic, and renal factors in intact and castrated dogs. Twenty male mixed-breed adult dogs were aligned in an experimental randomized and controlled trial. The dogs were randomly divided into four equal groups: melatonin, castrated, castrated and melatonin, and control. They were treated with melatonin (0.3 mg/Kg, once daily, orally) immediately after the castration for 1 month and their blood samples were collected weekly from 2 days after treatment with melatonin.ResultsTreating castrated dogs with melatonin increased the level of glutathione peroxidase, superoxide dismutase, and catalase compared with that of the control and castrated groups. The malondialdehyde level increased significantly following castration. Melatonin treatment decreased malondialdehyde concentration in the castrated dogs. Castration increased the level of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase significantly in comparison with that of the control group. Treating the castrated dogs with melatonin decreased significantly liver enzymes compared with those of the castrated dogs. Blood urea nitrogen and creatinine levels increased in the castrated dogs in comparison with that of the control group.ConclusionsThe administration of melatonin in castrated dogs increased antioxidant activity and decreased oxidation products, compared with those of the castrated and untreated dogs, without adverse effects on liver enzymes and kidney function.

Hemophilia A Dogs Tolerant to Human Factor VIII Provide a Unique Model to Determine Efficacy and Safety of AAV Delivery of Novel Factor VIII Variants

The hemophilia dog models are valuable for evaluating the efficacy of novel hemophilia therapeutics. The hemophilia B dog was predictive of the therapeutic dose of adeno-associated viral (AAV) vector delivery of human factor IX in clinical trials. In addition, it provides an opportunity to study the long-term efficacy and safety after AAV administration. However, there are several challenges in using the hemophilia A (HA) dog model for gene therapy studies. First, canine factor VIII (cFVIII) has higher specific activity and increased rate of secretion compared to human FVIII (hFVIII). This significant difference between cFVIII and hFVIII prevents the use of a species-specific transgene to predict the efficacy of AAV-hFVIII. Second, the HA dogs are immunocompetent and develop an immune response to the xenoprotein, hFVIII, that precludes the ability to measure transgene expression. Therefore, in order to employ this valuable model for gene therapy studies, we generated a unique cohort of HA dogs that are tolerant to B-domain deleted (BDD) hFVIII. We hypothesized that tolerizing dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response to a novel hFVIII variant. To tolerize the dogs to hFVIII, neonatal HA dogs were treated with a retrovirus (RV-hAAT-hFVIII-BDD-WPRE, 3x109 TU/kg) (n=5) on day 2 of life. The hFVIII expression was between 0.3%-6% at 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. To determine if the dogs were tolerant to hFVIII-BDD, the dogs were challenged with hFVIII-BDD protein at 5-6 months post-RV administration (Xyntha, 25IU/kg per wk x 6 wks, I.V.). Anti-hFVIII antibodies were monitored closely throughout the challenge and up to 8 weeks after the last challenge. In 4 out of 5 dogs, no anti-hFVIII immune response was observed based on IgG1, IgG2, total IgG or Bethesda titer. In contrast, naïve HA dogs (n=2) developed high level anti-hFVIII IgG2 (1.2-3.2 μg/mL), total IgG (3.4-5.0 μg/mL), and Bethesda titer (4.1-67.8 BU/mL) after the same challenge regimen. Interestingly, the hFVIII activity in one RV-treated dog (V06) was undetectable at 6 months post-RV administration. After the challenge, V06 had anti-hFVIII IgG2 (1.7 μg/mL), total IgG (2.6 μg/mL), and a Bethesda titer (9.5 BU/mL), suggesting that FVIII must be maintained to achieve tolerance. These dogs were used to evaluate the efficacy of AAV serotype 8 (AAV8) delivery of a hFVIII-BDD codon-optimized sequence driven by a hepatocyte promoter, modified transthyretin promoter (TTRm). S29 was delivered AAV8-TTRm-hFVIII-CO (2x1012 vg/kg). Prior to AAV delivery, the levels of hFVIII activity were 0.5-1% from the tolerization with the RV. After AAV administration the hFVIII activity was 3.8% at d168 and 4.7% at d387, resulting in a 4% increase in hFVIII expression. No anti-hFVIII antibodies were detected. The annual bleeding rate (ABR) for S29 post-RV delivery was 5 and after AAV delivery was 0, showing an improvement in the bleeding phenotype in contrast to untreated HA dogs (ABR=13, n=11). A hFVIII-tolerized littermate, S28, was recently treated with a hFVIII variant, AAV8-TTRm-hFVIII-CO-Δ3-SP/DE (2x1012 vg/kg). The hFVIII-Δ3-SP/DE variant has a deletion of the furin site (1645-47) and replaces residues SD at 1657-58 with PE. This variant showed higher specific activity (2-fold) in vitro and increased secretion (4-fold) compared to wild type hFVIII-BDD in the setting of AAV delivery in HA mice. Based on the results in S28, we will determine the dosing of V26 and V27. These studies demonstrate that sustained low level hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII, while allowing for the subsequent assessment of AAV efficacy. A clinically relevant dose of AAV8-TTRm-hFVIII-CO resulted in therapeutic levels of hFVIII expression while ongoing studies will allow investigation of the efficacy of the hFVIII-BDD variant, Δ3-SP/DE, in the setting of AAV administration in a large animal model. Overall, these studies demonstrate that RV-targeting of hFVIII-BDD expression to the liver in neonatal HA dogs leads to tolerance to this xenoprotein and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of novel FVIII variants. Disclosures Sabatino: Spark Therapeutics: Patents &amp; Royalties.

Assessment of the efficacy of firocoxib (Previcox\xae) and grapiprant (Galliprant\xae) in an induced model of acute arthritis in dogs

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are an important tool in the management of canine osteoarthritis, with the most recent introduction into the category being grapiprant, a piprant that selectively targets the EP4 prostaglandin receptor. To date there have been no efficacy studies comparing grapiprant with other NSAIDs. A randomized, two-sequence, assessor-blinded study involving two separate experiments was undertaken to measure the potency and persistence of acute pain control over 24 h resulting from a single oral dose of either firocoxib (Previcox®) or grapiprant (Galliprant®) in an acute arthritis model.ResultsForce-plate derived lameness ratios (0, no force recorded on the plate; 1, normal force) for the untreated group remained at 0 for most post-arthritis induction (PAI) assessments in both experiments. Throughout Experiment 1, mean PAI lameness ratios of the firocoxib-treated group remained at or above 0.80. In the grapiprant-treated group, ratios were 0 at 5 and 7 h PAI (7 and 9 h post-treatment), and 0.16 at 10 h PAI (12 h post-treatment). For lameness ratios, relative to the firocoxib group, the control and grapiprant group ratios were significantly lower at each PAI assessment (p ≤ 0.026 and p < 0.001, respectively), except at 1.5 h PAI at which acute pain was still not installed in untreated control dogs. In Experiment 2 the mean lameness ratios for the control group were 0 at 3, 5 and 7 h PAI, and in the grapiprant group at 5, 7 and 10 h PAI (i.e., 19, 21, and 24 h post-treatment). In the firocoxib group the lowest mean lameness ratio of 0.36 occurred at 3 h PAI (i.e. 17 h post-treatment). Except at 1.5 and 3 h PAI (i.e. 15.5 and 17 h post-treatment), due to the needed time for pain to install in the untreated control dogs, the lameness ratio differences between the firocoxib and both the control and grapiprant groups were significant at all assessments (p ≤ 0.033 for both groups). No significant differences were detected between the grapiprant and control groups in either experiment.ConclusionsFirocoxib treatment prior to induction of arthritis in dogs resulted in a high level of analgesia from the first post-treatment assessment at 1.5 h through 24 h post-treatment. The reduction in lameness provided by firocoxib was consistently superior to that provided by grapiprant, which was not significantly different from untreated controls.

PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting Trypanosoma cruzi kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect T. cruzi in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown T. cruzi infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; p = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; p > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized T. cruzi DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

Effects of a Nutritional Supplement on Cognitive Function in Aged Dogs and on Synaptic Function of Primary Cultured Neurons.

Simple SummaryWe tested the effects of a nutraceutical product, DiSeniorTM, by spatial navigation test and by in vitro and in vivo experiments. Results showed that DiSeniorTM was safe and able to ameliorate cognitive functions in aged dogs, as demonstrated by the better performances in the treated with respect the untreated groups. The increase of cFOS, a functional marker of activity in cultured neurons, indicated a positive effect of the substance on neuronal functions. The study suggests that DiSeniorTM can improve the quality of life of elderly dogs and may slow the onset of cognitive dysfunction symptoms associated with aging.The objective of this research was to investigate the efficacy of DìSeniorTM, a nutraceutical formulated to improve cognitive functions in elderly dogs. To this purpose, some clinical and metabolic investigations and a spatial navigation test were performed in treated and untreated dogs. Moreover, the nutraceutical was also tested on primary hippocampal neuron cultures. Results showed no adverse effects on the dogs’ health and a positive effect on learning. In vitro effects on neuron cultures showed an increase in the level of cFOS in treated neurons compared with the vehicle, suggesting that DiSeniorTM has also a positive effect on neuronal functions. Overall, this study suggests that DiSeniorTM can exert a beneficial effect on aged dogs by preventing the negative effects of aging on cognition. Further studies are needed to assess the mechanisms by which it acts on neurons and the specific effect of the different components alone or combined.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms

BackgroundAngiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells.MethodsThe expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls.ResultsFirst, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections.ConclusionsThe ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Evaluation of Intraperitoneal and Incisional Bupivacaine or Levobupivacaine for Postoperative Analgesia in Ovariohysterectomized Dogs

Background: Ovariohysterectomy (OHE) is the most commonly performed elective surgical procedure in companion animals. OHE offers benefits of control of population and decreased risk of potentially life-threatening diseases such as mammarian tumours and pyometra. Traditional OHE intervention causes inflammation and pain due to trauma during organ manipulation. The purpose of this study was to compare the effect of intraperitoneal and incisional administration of bupivacaine (BP) or levobupivacaine (LP) on postoperative analgesia in dogs undergoing the OHE procedure.Materials, Methods &amp; Results: A total of 24 mix-breed bitches aged between 1 - 3 years and weighed 19 - 20 kg were used in this study. The animals were divided into three groups as control (n = 8), BP (n = 8) and LP group (n = 8). The animals were kept under surveillance at the hospitalisation unit of the animal hospital for one day before the elective OHE. The dogs were fasted for 12 h before the surgery, with adlib water consumption. Atropine sulphate 0.045 mg/kg was administered subcutaneously approximately 30 min before general anaesthesia. Midazolam (0.3 mg/kg) was intravenously injected into all dogs for pre-anaesthetic medication. After sedation, anaesthesia was induced with propofol (4 mg/kg, IV) and then the dogs were orotracheally intubated using cuffed endotracheal tubes. General anaesthesia was maintained by administration of 2% isoflurane. The ventral abdomen was prepared aseptically for OHE following the general anaesthesia. All animals were operated on by the same surgeon. During surgery, sprayed bupivacaine 0.5% (4.4 mg/kg) with an equal volume of saline in BP group, levobupivacaine 0.5% (4.4 mg/kg) with an equal volume of saline in LP were then applied over the ovaries, uterine broad ligaments and cervix uteri. After removal of the uterine body, either LP or BP was sprayed to left and right, or cranial and caudal parts of the abdominal cavity. Finally, the incision line was sprayed before closing the skin. The control group received only sprayed saline. The pain evaluation was carried out with modified Melbourne pain scoring scale. To determine cortisol concentrations, blood samples were taken before anaesthesia induction (baseline) and postoperative 0, 0.5, 1, 2, 4, 6, 12 and 24 h. Postoperative pain scores were higher in the control group than BP and LP groups following surgery at 30 min, 1st, 4th, and 6th h. In all groups, cortisol levels increased postoperatively, whereas they decreased more rapidly in BP and LP groups. There was a steady increase at postoperative 0 min, 30 min, 1st, 2nd, 4th and 6th h in the control group and at postoperative 0 min, 30 min and 1 h in BP and LP groups (P &lt; 0.05).Discussion: Intraperitoneal and incisional bupivacaine shows significantly superior postoperative pain management benefits after the closure of skin, compared to untreated dogs and it is also superior to lidocaine treatment at 0.5 h following the visual analogy pain assessment system. In the present study, the observation of lower pain scores in BP group at 0.5, 1, 4 and 6 h as compared to the control group was similar to findings of other studies. LP group also showed decreased pain scores at above-mentioned h without any significant difference. However, only in LP group, pain scores at 24 h were lower than postoperative pain scores. In conclusion, it is suggested that sprayed intraperitoneal and incisional BP and LP are very effective for preventing postoperative pain ovariohysterectomized dogs.

Prenatal Treatment of X-Linked Hypohidrotic Ectodermal Dysplasia using Recombinant Ectodysplasin in a Canine Model.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is caused by defects in the EDA gene that inactivate the function of ectodysplasin A1 (EDA1). This leads to abnormal development of eccrine glands, hair follicles, and teeth, and to frequent respiratory infections. Previous studies in the naturally occurring dog model demonstrated partial prevention of the XLHED phenotype by postnatal administration of recombinant EDA1. The results suggested that a single or two temporally spaced injections of EDI200 prenatally might improve the clinical outcome in the dog model. Fetuses received ultrasound-guided EDI200 intra-amniotically at gestational days 32 and 45, or 45 or 55 alone (of a 65-day pregnancy). Growth rates, lacrimation, hair growth, meibomian glands, sweating, dentition, and mucociliary clearance were compared in treated and untreated XLHED-affected dogs, and in heterozygous and wild-type control dogs. Improved phenotypic outcomes were noted in the earlier and more frequently treated animals. All animals treated prenatally showed positive responses compared with untreated dogs with XLHED, most notably in the transfer of moisture through paw pads, suggesting improved onset of sweating ability and restored meibomian gland development. These results exemplify the feasibility of ultrasound-guided intra-amniotic injections for the treatment of developmental disorders, with improved formation of specific EDA1-dependent structures in dogs with XLHED.

AST-120, an Adsorbent of Uremic Toxins, Improves the Pathophysiology of Heart Failure in Conscious Dogs.

Several lines of evidence suggest that renal dysfunction is associated with cardiovascular toxicity through the action of uremic toxins. The levels of those uremic toxins can be reportedly reduced by the spherical carbon adsorbent AST-120. Because heart failure (HF) causes renal dysfunction by low cardiac output and renal edema, the removal of uremic toxins could be cardioprotective. To determine whether blood levels of the uremic toxin indoxyl sulfate (IS) increase in HF and whether AST-120 can reduce those levels and improve HF. We induced HF in 12 beagle dogs by 6weeks of rapid right ventricular pacing at 230 beats per min. We treated six dogs with a 1-g/kg/day oral dosage of AST-120 for 14days from week 4 after the start of rapid ventricular pacing. The other six dogs did not receive any treatment (control group). In the untreated dogs, IS levels increased as cardiac function deteriorated. In contrast, plasma IS levels in the treated dogs decreased to baseline levels, with both left ventricular fractional shortening and pulmonary capillary wedge pressure also improving when compared with untreated dogs. Finally, AST-120 treatment was shown to reduce both myocardial apoptosis and fibrosis along with decreases in extracellular signal-regulated kinase phosphorylation, the Bax/Bcl-2 ratio, and TGF-β1 expression and increases in AKT phosphorylation. IS levels are increased in HF. AST-120 treatment reduces the levels of IS and improves the pathophysiology of HF in a canine model. AST-120 could be a novel candidate for the treatment of HF.

Use of recombinant canine granulocyte-colony stimulating factor to increase leukocyte count in dogs naturally infected by canine parvovirus

Canine parvovirus (CPV) is one of the most important cause of mortality in young dogs and no specific treatment exists. Since prolonged leukopenia greatly increases the risk of death in infected pups, strategies to counteract this decline were investigated. The outcomes of CPV naturally infected pups treated with the recombinant canine granulocyte-colony stimulating factor (rcG-CSF), in combination with the routine therapy, were compared with similarly-managed infected pups not treated with rcG-CSF. A non-randomized prospective clinical trial was performed on 62 CPV infected pups with WBC counts <3000 cells/μL and two different groups were selected based on a non-randomized approach. Group A dogs (31/62) received 5 μg/Kg of rcG-CSF daily from the hospitalization day until WBC reached the reference range (3–5 days) and group B (31/62) received 1 ml of placebo injection. All dogs in group A recovered, while five dogs in group B died. The rcG-CSF treatment demonstrated a statistically significant effect on WBC counts (p < 0.0001) and, surprisingly, also on lymphocytes and monocytes counts (p < 0.0001). There was no significant effect of treatment on neutrophil count (p = 0.5502). Although lymphocytes and monocytes are not a specific target for rcG-CSF, our study highlights that rcG-CSF is able to improve haematological parameters compared to untreated dogs and a clear increase in their number was detected, as previously described for humans treated with the homologous molecule.

Evaluation of ions and metals in the blood of GRMD dogs submitted to hASCs therapy by NAA and XRF techniques

The elements Br, Ca, Cl, Cr, Fe, K, Mg, Na, P, Rb, S, and Zn were investigated in the whole blood samples of Golden Retriever dogs submitted to cell therapy (hASCs). These analyses were performed over 2 years using Neutron Activation Analysis and X-Ray Fluorescence techniques. The results were compared with control and untreated dog's. A significant increase was observed in K blood levels. There was also variation in blood levels of Br, Cr, Fe, Rb, S, and Zn.