Jérôme R.E. del Castillo

Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

By the end of 2004, the Canadian swine population had experienced a severe increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously unrecovered in North America. Thus, it became important to develop a diagnostic tool that could differentiate between the old and new circulating genotypes (PCV-2a and PCV-2b, respectively). Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A retrospective epidemiologic survey that used the mrtqPCR assay was performed to determine if cofactors could affect the risk of PCVAD. From 121 PCV-2-positive cases gathered for this study, 4.13%, 92.56%, and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, respectively. In a data analysis using univariate logistic regressions, the PCVAD-compatible (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by the PRRSV variables (P > 0.9), which suggests that mrtqPCR in tissue is a reliable alternative to IHC. Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical diseases.

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

IntroductionThe aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA).MethodsBaseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE2 and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05.ResultsA better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE2 (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone.ConclusionTiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from MARC-145 cell line

BackgroundAirborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM) and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro.ResultsThe SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFNα antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFNβ mRNA and no up regulation of IFNα mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFNγ and TNF-α mRNAs only in infected MARC-145 cells.ConclusionsIn conclusion, the SJPL cells are permissive to PRRSV. In addition, they are phenotypically different from MARC-145 cells and are an additional tool that could be used to study PRRSV pathogenesis mechanisms in vitro.

Validation of Orthopedic Postoperative Pain Assessment Methods for Dogs: A Prospective, Blinded, Randomized, Placebo-Controlled Study

In the context of translational research, there is growing interest in studying surgical orthopedic pain management approaches that are common to humans and dogs. The validity of postoperative pain assessment methods is uncertain with regards to responsiveness and the potential interference of analgesia. The hypothesis was that video analysis (as a reference), electrodermal activity, and two subjective pain scales (VAS and 4A-VET) would detect different levels of pain intensity in dogs after a standardized trochleoplasty procedure. In this prospective, blinded, randomized study, postoperative pain was assessed in 25 healthy dogs during a 48-hour time frame (T). Pain was managed with placebo (Group 1, n = 10), preemptive and multimodal analgesia (Group 2, n = 5), or preemptive analgesia consisting in oral tramadol (Group 3, n = 10). Changes over time among groups were analyzed using generalized estimating equations. Multivariate regression tested the significance of relationships between pain scales and video analysis. Video analysis identified that one orthopedic behavior, namely ‘Walking with full weight bearing’ of the operated leg, decreased more in Group 1 at T24 (indicative of pain), whereas three behaviors indicative of sedation decreased in Group 2 at T24 (all p<0.004). Electrodermal activity was higher in Group 1 than in Groups 2 and 3 until T1 (p<0.0003). The VAS was not responsive. 4A-VET showed divergent results as its orthopedic component (4A-VETleg) detected lower pain in Group 2 until T12 (p<0.0009), but its interactive component (4A-VETbeh) was increased in Group 2 from T12 to T48 (p<0.001). Concurrent validity established that 4A-VETleg scores the painful orthopedic condition accurately and that pain assessment through 4A-VETbeh and VAS was severely biased by the sedative side-effect of the analgesics. Finally, the video analysis offered a concise template for assessment in dogs with acute orthopedic pain. However, subjective pain quantification methods and electrodermal activity need further investigation.

Epidemiological, clinical and control investigations on field porcine coccidiosis: clinical, epidemiological and parasitological paradigms?

Abstract For several reasons, we are convinced that the parasitologist community must look again at porcine neonatal coccidiosis. We find it surprising that this disease is seldom addressed, and that assumptions are not always supported by clinico-epidemiological analysis. For example, the relationship between diarrhoea and the excretion of oocysts during experimental infection versus field infection is not questioned. Lindsay et al. review parasitological knowledge of this disease in the latest (1999) edition of Diseases of swine. Although this paper is divided into two parts, we have three distinct objectives: (1) to bring to the debate our experience in the medical control of this disease; (2) to propose a diagnostic methodology; (3) to raise some questions on various clinical, epidemiological and parasitological unknowns.

Evidence for non-linear pharmacokinetics of oxytocin in anesthetizetized rat.

PURPOSE Because oxytocin (OT) is potentially useful in cardiovascular therapy but has hormonal roles on the cardiovascular and renal systems, we characterized its pharmacokinetic (PK) properties as a function of dose. METHODS A single intravenous bolus of OT was given at doses of 200, 300, 500, 1000, 3000, 5000 and 10000 ng/kg to anesthetized male rats (n >= 4 per dose). Blood samples (6) were taken over 72 min to 150 min, depending on dose. The individual time-courses of plasma OT concentrations were analyzed with a one- or an open two-compartment PK model. Kruskal-Wallis tests (alpha=0.05) were used to compare the PK parameters among groups. RESULTS At doses up to 500 ng/kg, OT showed a higher median systemic clearance (CLT = 0.0624 L/(min*kg); 0.0622 +/- 0.0228 as mean +/- SD value), a higher median central compartment volume of distribution (VC = 0.7906 L/kg; 0.6961 +/- 0.1754), and a lower median elimination half life (t(1/2)(lambdaz) 7.94 min; 9.08 +/- 4.3) with respect to the higher doses (CLT = 0.0266 L/(min*kg); 0.0284 +/- 0.0098, VC = 0.2213 L/kg; 0.2227 +/- 0.1142, and t(1/2)(lambdaz) 21.09 min; 28.36 +/- 21.8), all differences being significant (p 0.0008). Minimal differences were found for the estimates of these PK parameters among the 4 higher OT doses. CONCLUSION The PK properties and persistence of exogenous OT are not proportional to dose, therefore this must be accounted for in dosing regimen design for potential cardiovascular therapy.

Involvement of the nitric oxide-soluble guanylyl cyclase pathway in the oxytocin-mediated differentiation of porcine bone marrow stem cells into cardiomyocytes

Bone marrow stem cells (BMSCs) express cardiac markers in vitro and in vivo upon induction. Cardiomyogenic differentiation of embryonic stem cells induced by oxytocin (OT) involves the nitric oxide (NO)-soluble guanylyl cyclase (sGC) pathway. Also, OT improved cardiomyogenic differentiation of porcine BMSCs (pBMSCs). Here, we document the role of NO pathway in OT-mediated cardiomyogenic differentiation of pBMSCs obtained from bone marrow aspirates of juvenile pigs. Cells were exposed (OT cells) or not (control cells) to OT, in presence or absence of a NO synthase inhibitor (L-NAME) and a sGC inhibitor (ODQ). Gene (RT-PCR) and protein expression (immunocytochemistry) of NOS was up-regulated after OT induction. Exposure of OT cells to L-NAME, ODQ, or both, leaded to a significant reduction in cardiac troponin I transcripts, and protein (Western Blot) expression. For the latter, ODQ looked more performing in inhibition than L-NAME. Expression of cardiac troponin T and myosin heavy chain (immunocytochemistry) was less abundant in OT cells exposed to inhibitors without apparent synergic effect between L-NAME and ODQ. In control cells, protein expression remained low. Moreover, OT-induced cell proliferation, and this effect was counteracted by NOS/sGC inhibitors. Inhibiting NO production and NO effector, sGC, affected the OT-mediated differentiation of pBMSCs, because abundance of cardiac proteins was reduced to levels similar to those observed in control cells. We propose that following treatment with OT, activation of NO pathway directs pBMSCs to a preferential cardiomyogenic phenotype and stimulates cell proliferation.

Haemodynamic interactions of medetomidine and the peripheral alpha-2 antagonist MK-467 during step infusions in isoflurane-anaesthetised dogs

The haemodynamic interactions of a step infusion with medetomidine (MED) and the peripherally acting alpha-2 antagonist MK-467 (MK) were compared with MED infused alone in isoflurane-anaesthetised dogs. Eight purposely-bred Beagles were used in a randomised crossover study. Anaesthesia was induced with propofol intravenously (IV) and maintained with isoflurane in oxygen. Dogs received 1.25 µg/kg MED as a 1 min loading dose IV, along with a step-down MED infusion at rates of 8.0 µg/kg/h (step 1: 0-20 min), 5.5 µg/kg/h (step 2: 20-40 min) and 4.0 µg/kg/h (step 3: 40-95 min). Five minutes after starting the MED infusion, the dogs received MK-467 in a step-up infusion at rates of 100 µg/kg/h (step 1: 5-35 min), 200 µg/kg/h (step 2: 35-65 min) and 500 µg/kg/h (step 3: 65-95 min). Heart rate (HR), systolic (SAP) and mean arterial (MAP) blood pressures and arteriovenous oxygen content differences (a-vO2 diff) were calculated. Plasma drug concentrations were analysed. Repeated-measures general linear mixed models with Bonferroni correction were used for statistical analyses. MED infusion alone increased SAP maximally by 24.9%, MAP by 34.7% and a-vO2 diff by 222.5%, and reduced HR by 32.3%, but these changes were significantly attenuated by MK-467. Most MED effects returned to baseline during step 2 of MK-467 infusion and step 3 of MED infusion (MED/MK-467 ratio 1:18 to 1:50). Plasma concentrations of MED tended to be lower with the addition of MK-467. The use of step infusions helped to narrow down the therapeutic range for the MED/MK-467 infusion dose ratio during isoflurane anaesthesia in dogs.

Rivaroxaban demonstrates in vitro anticoagulant effects in canine plasma.

Rivaroxaban is an oral direct factor X inhibitor used in human thrombotic disorders and its oral administration makes it an attractive potential anticoagulant for dogs. The objective of this study was to evaluate the in vitro anticoagulant effect of rivaroxaban on canine pooled platelet-poor plasma (PPP). Pooled PPP was collected from 20 healthy adult Beagle dogs. Aliquots of pooled citrated PPP were treated in vitro with DMSO solutions of rivaroxaban (98% purity) to obtain 19 final concentrations ranging from 0 to 1000 mg/L of drug. Samples were immediately submitted for the following coagulation assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), tissue factor-induced thrombin generation and anti-factor Xa activity. Concentration-effect data were analyzed with various nonlinear regression models for stimulatory or inhibitory effects. Rivaroxaban caused a concentration-dependent prolongation of all coagulation parameters. Rivaroxaban concentration for 50% baseline inhibition of the propagation phase of thrombin (rate index) was 0.024 mg/L, and for 50% baseline inhibition of the optical density in the anti-factor Xa activity assay was 0.053 mg/L. At these concentrations, PT and aPTT remained within the reference range. Two-fold prolongation from baseline of PT and aPTT was achieved with higher concentrations, i.e. 1.24 and 1.69 mg/L, respectively. Thrombin generation was completely suppressed by concentrations ≥0.8 mg/L. In conclusion, rivaroxaban showed an in vitro concentration-dependent anticoagulant effect on canine plasma. Thrombin generation and anti-factor Xa activity were more sensitive and accurate than PT and aPTT in detecting the anticoagulant effect of rivaroxaban.

Determination of glomerular filtration rate in anesthetized pigs by use of three-phase whole-kidney computed tomography and Patlak plot analysis

OBJECTIVE To develop a whole-kidney computed tomography (CT) technique that would allow 3-point Patlak plot determination of glomular filtration rate (GFR) and assess the correlation of GFR determined via CT (CT-GFR) with GFR determined via renal plasma clearance of inulin (Inu-GFR) in pigs. ANIMALS 6 healthy anesthetized pigs. PROCEDURES Each pig underwent 3-phase whole-kidney helical CT (arterial, early, and late parenchymal phases) before and after contrast medium administration. After contrast medium administration, corrected Hounsfield unit values were determined for each kidney and the aorta. A 3-point Patlak plot for each kidney was generated, and plasma clearance per unit volume was multiplied by renal volume to obtain whole-animal CT-GFR. Correlations of mean Inu-GFR for the left and right kidneys (and combined [total] values) with the corresponding CT-GFRs were assessed via linear regression and Bland-Altman analyses. RESULTS Left kidney, right kidney, and total CT-GFRs were good predictors of the respective Inu-GFR values (r(2) = 92.3%, r(2) = 85.5%, and r(2) = 93.7%, respectively). For the left kidney, no significant bias between Inu-GFR and CT-GFR was detected. Right kidney and total CT-GFRs underestimated the corresponding Inu-GFRs (mean underestimation, -8.4 mL*min(1) and -12.6 mL*min(1), respectively). CONCLUSIONS AND CLINICAL RELEVANCE Three-phase whole-kidney CT with Patlak plot analysis of GFR may underestimate right kidney and total Inu-GFRs in pigs. The Patlak plot generated may be sensitive to nonlinearity caused by temporal variation in GFR. Nonetheless, the 3-phase CT approach offers some practical advantages for simultaneous evaluation of renal morphology and measurement of GFR.