Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia

Background Canine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity—and thus disease—in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. Methods Blood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott’s test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. Results A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9–15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen’s kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26–13.51). All samples positive for the modified Knott’s test were also positive for D. immitis DNA by PCR. Conclusions This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity. Graphical Abstract

Effect of Leukoreduction by Pre-Storage Filtration on Coagulation Activity of Canine Plasma Collected for Transfusion

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions and is extensively used in human, but rarely in veterinary patients. The concentration of some coagulation proteins can be affected by the processing steps used for the preparation of leuko-reduced plasma units. In this study, we assessed the effect of leukoreduction on coagulation activity of canine plasma collected for transfusion. Ten plasma units, five obtained from non-leuko-reduced (non-LR) whole blood (WB) units and five from leuko-reduced (LR) WB units were evaluated. Prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factor activities of factors (F) V, VIII, X, XI, and von Willebrand (vWF), fibrinogen and D-dimers content were assessed at collection (baseline value, D0) and after 7 days of frozen storage at −18 °C (D7). Compared to non-LR plasma units, LR units showed a statistically significant prolonged aPTT and reduced FXI activity. Filtration had no significant effect on the other factors and parameters evaluated. Filtration-dependent changes appear to have no impact on the therapeutic quality of plasma obtained from leuko-reduced whole blood, other than for FXI activity. Further studies on a larger sample size comparing the same unit before and after leukoreduction are needed to confirm these findings.

Alteration of the Canine Metabolome After a 3-Week Supplementation of Cannabidiol (CBD) Containing Treats: An Exploratory Study of Healthy Animals

Despite the increased interest and widespread use of cannabidiol (CBD) in humans and companion animals, much remains to be learned about its effects on health and physiology. Metabolomics is a useful tool to evaluate changes in the health status of animals and to analyze metabolic alterations caused by diet, disease, or other factors. Thus, the purpose of this investigation was to evaluate the impact of CBD supplementation on the canine plasma metabolome. Sixteen dogs (18.2 ± 3.4 kg BW) were utilized in a completely randomized design with treatments consisting of control and 4.5 mg CBD/kg BW/d. After 21 d of treatment, blood was collected ~2 h after treat consumption. Plasma collected from samples was analyzed using CIL/LC-MS-based untargeted metabolomics to analyze amine/phenol- and carbonyl-containing metabolites. Metabolites that differed — fold change (FC) ≥ 1.2 or ≤ 0.83 and false discovery ratio (FDR) ≤ 0.05 — between the two treatments were identified using a volcano plot. Biomarker analysis based on receiver operating characteristic (ROC) curves was performed to identify biomarker candidates (area under ROC ≥ 0.90) of the effects of CBD supplementation. Volcano plot analysis revealed that 32 amine/phenol-containing metabolites and five carbonyl-containing metabolites were differentially altered (FC ≥ 1.2 or ≤ 0.83, FDR ≤ 0.05) by CBD; these metabolites are involved in the metabolism of amino acids, glucose, vitamins, nucleotides, and hydroxycinnamic acid derivatives. Biomarker analysis identified 24 amine/phenol-containing metabolites and 1 carbonyl-containing metabolite as candidate biomarkers of the effects of CBD (area under ROC ≥ 0.90; P &lt; 0.01). Results of this study indicate that 3 weeks of 4.5 mg CBD/kg BW/d supplementation altered the canine metabolome. Additional work is warranted to investigate the physiological relevance of these changes.

Spatial analysis of canine leishmaniasis in an area of transmission of the semi-arid region of the State of Paraíba, Brazil

Visceral leishmaniasis is a widely distributed zoonosis and canine infection is an important indicator of risk for the occurrence of the disease in humans. The goal of this analysis was to study the spatial clustering of canine leishmaniasis (CL) in the municipality of Santa Luzia, state of Paraíba. For this, 749 samples of canine plasma were tested using three serological tests. The dog was considered positive if it reacted in two serological tests. The location of the residences was performed with a Global Positioning System receiver (GPS Garmin® eTrex 30), and used to perform georeferencing and spatial analysis. The prevalence of CL was 15.49% and it was observed that most cases of the urban area were concentrated in the Frei Damião neighborhood, on the outskirts of the city, where a high-risk cluster for the occurrence of the disease was formed (p = 0.02; RR = 2.48). No statistically significant cluster was observed in rural areas. CL is widely distributed in the municipality of Santa Luzia in a heterogeneous manner and with a tendency to urbanization. The areas identified with high prevalence and highest risk should be prioritized to maximize the efficiency of the Visceral Leishmaniasis Surveillance and Control Program and minimize the chance of new canine and human cases.

P6342Subcutaneous delivery of NPA7, first-in-class novel bispecific designer peptide: enhances cardiorenal function and suppresses renin and aldosterone in vivo and in vitro

Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P<0.05 vs. BL. Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.