Detection of osteoarthritis in dogs by metabolic, pro-inflammatory and degenerative synovial fluid biomarkers and traditional radiographic screening: A pilot study.

Secondary osteoarthritis (OA) is a slow progressive, common disorder of synovial joints in dogs. It is characterized by a loss of balance between the synthesis and degeneration of articular cartilage components. Its diagnosis is currently based on the presence of clear radiographic changes, which only occur in the later stages of the disease. Hence, early diagnosis of OA remains a major problem. Therefore, interest in synovial fluid (SF) biomarkers has emerged. Besides pro-inflammatory and degenerative markers, i.e. tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), tenascin-c (TN-C) and matrix metalloproteinase-2 (MMP-2), metabolic parameters, i.e. pH, glucose and lactate, can potentially be used to detect OA. The current study demonstrated statistically significant differences in the SF levels of pH, glucose and lactate between OA-affected and normal joints. In addition, the in-house validated immuno-assays for TNF-alpha, IL-1beta, TN-C and MMP-2 allowed to demonstrate also statistically significant differences in the SF concentrations for all these biomarkers - except TNF-alpha - between OA-affected and normal joints. However, no correlation was found between any of these biomarkers and the currently used radiographic scoring system for OA in dogs. Future research is warranted to explore the potential of these biomarkers in the early detection of OA and in the severity characterization of this disease.

Serum hyaluronic acid, a marker for improved liver perfusion after gradual surgical attenuation of extrahepatic portosystemic shunt closure in dogs.

Current liver function tests used in dogs do not consistently normalise after successful surgical attenuation of portosystemic shunts (PSS). Serum hyaluronic acid (sHA) concentrations in dogs with PSS are reported to be higher at diagnosis than in healthy dogs. The objective of this study was to assess sHA as a marker of liver perfusion by measuring sHA concentrations in dogs before and after gradual surgical attenuation of extrahepatic (EH)PSS and by determining whether sHA concentrations could differentiate closed EHPSS from persistent shunting. Specificity of sHA was assessed by comparing sHA concentrations in dogs with EHPSS to those in dogs with other liver diseases. Twenty dogs with EHPSS had sHA concentrations measured at diagnosis, 1, 3, and 6 months postoperatively. In addition, sHA concentrations were determined in 10 dogs with other liver diseases. At EHPSS diagnosis, median sHA concentration was 335.6 ng/mL (43.0-790.7 ng/mL). All dogs had a significant decrease in sHA concentrations from 1 month postoperatively onwards (P < 0.05), regardless of surgical outcome. At all postoperative follow-up visits, there was a significant difference between the median sHA concentration in dogs with closed EHPSS vs. those with persistent shunting (P < 0.05). Median sHA concentration in dogs with other liver diseases was 89.8 ng/mL (22.9-160.0 ng/mL), which was significantly lower than dogs with EHPSS at diagnosis (P < 0.001). In conclusion, sHA is a promising non-invasive biomarker that can help to determine liver perfusion after surgical attenuation of EHPSS. In addition, sHA could potentially be used to differentiate dogs with EHPSS from dogs with other liver diseases.

Effect of subinhibitory exposure to quaternary ammonium compounds on the ciprofloxacin susceptibility of Escherichia coli strains in animal husbandry.

BACKGROUND: Quaternary ammonium compound based disinfectants are commonly used in pig and poultry husbandry to maintain farm hygiene. However, studies have shown that subinhibitory concentrations of these disinfectants may increase antibiotic resistance. Investigation of antibiotic susceptibility is usually assessed via the microbroth dilution method, although this conventional culture-based technique only provides information on the bacteriostatic activity of an antimicrobial agent. Therefore, experiments were performed to investigate the effect of prior benzalkonium chloride (BKC) exposure on the viability of subsequent ciprofloxacin (CIP) treated Escherichia coli. RESULTS: Following CIP treatment, bacterial cell counts were significantly higher after exposure to a subinhibitory BKC concentration than without BKC exposure. The flow cytometric results suggested a BKC-dependent onset of membrane damage and loss of membrane potential. CONCLUSION: Our results indicate a lower bactericidal effect of CIP treatment on BKC-exposed E. coli isolates compared to unexposed E. coli isolates.

Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids.

Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment.IMPORTANCE This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).

Multiplex analysis of pro-inflammatory cytokines in serum of Actinobacillus pleuropneumoniae-infected pigs.

Porcine pleuropneumonia is a severe respiratory disease caused by Actinobacillus (A.) pleuropneumoniae. The aim of the present study was to analyze serum samples of A. pleuropneumoniae-infected pigs for TNF-α, IL-1ß and IL-6 using a cytometric bead array (CBA) 3-plex assay and additionally for IL-6 using ELISA. The CBA 3-plex assay was successfully validated for use in serum. The limits of detection varied between 0.012 and 0.333 ng/mL, and the inter- and inter-assay coefficients of variation were <5% and <10%, respectively. Increased levels were observed for all 3 cytokines following experimental infection with A. pleuropneumoniae. Mean peak concentrations of TNF-α and IL-6 were recorded at 12h and at 10h p.i., respectively. For IL-6, similar concentration-time profiles were observed with CBA and ELISA. It is proposed that this immuno-assay can be applied for the screening of immunomodulatory properties of drugs and vaccine adjuvants in infection, inflammation and vaccination.

Secreted phospholipase A2 inhibitor modulates fatty acid composition and reduces obesity-induced inflammation in Beagle dogs.

Secreted phospholipase A2 inhibitor (sPLA2i) has been reported to have an anti-inflammatory function by blocking the production of inflammatory mediators. Obesity is characterized by low-grade inflammation and oxidative stress. The aim of this study was to investigate the effects of dietary supplementation of sPLA2i on inflammation, oxidative stress and serum fatty acid profile in dogs. Seven obese and seven lean Beagle dogs were used in a 28-day double blind cross-over design. Dogs were fed a control diet without supplemental sPLA2i or an sPLA2i supplemented diet. The sPLA2i diet decreased plasma fibrinogen levels and increased the protein:fibrinogen ratio in obese dogs to levels similar to those of lean dogs fed the same diet. Obese dogs had a higher plasma concentration of the lipophilic vitamin A with potential antioxidative capacity and a lower ratio of retinol binding protein 4:vitamin A compared to lean dogs, independent of the diets. A higher proportion of myristic acid (C14:0) and a lower proportion of linoleic acid (C18:2n-6) were observed in the dogs fed with the sPLA2i diet compared to dogs fed with the control diet. Furthermore, a higher ratio of n-6 to n-3, a lower proportion of n-3 polyunsaturated fatty acids and lower omega-3 index were observed in obese compared to lean dogs. The results indicate that obese dogs are characterized by a more 'proinflammatory' serum fatty acid profile and that diet inclusion of sPLA2i may reduce inflammation and alter fatty acid profile.

Dose-response effect of fish oil substitution in parturition feed on erythrocyte membrane characteristics and sow performance.

The present study aimed to investigate whether n-3 polyunsaturated fatty acids (PUFA) incorporate into erythrocyte membranes of peripartal sows in a dose-responsive manner and whether the altered fatty acid profile affects the cell membrane characteristics. At day 109 of gestation (day 0), 51 sows were divided into five treatment groups. Each group received a diet with a different ratio of fish oil to pork lard for nine consecutive days. Blood samples were taken at day 0 and 10 days later. The fatty acid profile of erythrocytes was determined, as well as the osmotic fragility and oxidative stability of erythrocytes. Thiobarbituric acid reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) were determined in plasma samples. Finally, reproductive and performance parameters of both sows and piglets were recorded until weaning. Supplementation of fish oil during the peripartal period changed the fatty acid profile of erythrocyte membranes in a dose-responsive manner. Although the n-3 PUFA content of erythrocyte membranes increased with increasing amounts of fish oil in the diet, no significant effect on erythrocyte osmotic fragility could be recorded. In contrast, oxidative stability of erythrocytes decreased linearly with increasing amounts of fish oil in the diet. Similarly, both TBARS and FRAP linearly increased with increasing percentages of fish oil in the diet. Neither piglet nor sow performance was influenced by dietary treatments, except for a decrease of both piglet survival and weaning weight with increasing quantities of fish oil supplemented. It is concluded that changes in dietary lipid sources can affect the membrane's fatty acid profile within days, and mainly influences oxidative stability of the cells.

Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression.

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation. In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1beta and interleukin-6 was subsequently optimized on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Heifer and quarter characteristics associated with periparturient blood and milk neutrophil apoptosis in healthy heifers and in heifers with subclinical mastitis.

Polymorphonuclear neutrophilic leukocytes (PMNL) play an important role in the first line cell-mediated immune defense of the body in general and of the mammary gland against mastitis pathogens in particular. Reduced viability of PMNL close to parturition may explain the high incidence of infectious diseases and the high prevalence of intramammary infections (IMI) in periparturient dairy heifers. Apoptosis of blood PMNL 1 wk before the expected calving date and of blood and milk PMNL at 1 to 4 d in milk was determined using flow cytometry. Information on heifer and gland characteristics was collected before calving and in early lactation. Data were analyzed using multivariable, multilevel regression analysis. Supplementation of a commercial mineral/vitamin mix before calving was associated with less blood (14.4 +/- 2.9 vs. 22.4 +/- 2.1%) and milk PMNL apoptosis (19.0 +/- 1.1 vs. 26.4 +/- 0.9%) near calving, presumably related to higher blood selenium concentrations. Both blood and milk PMNL apoptosis showed seasonal variation with the highest proportion of apoptotic cells between January and March (32.0 +/- 6.1 and 34.6 +/- 2.7%, respectively) and April and June (31.3 +/- 5.7 and 37.8 +/- 2.3%, respectively). Heifers losing 0.25 points or more of their body condition in the periparturient period had higher proportions of apoptotic blood PMNL in early lactation compared with heifers losing less than 0.25 points (24.0 +/- 2.8 vs. 16.6 +/- 1.7%). Milk PMNL apoptosis was less pronounced in quarters having teat orifices colonized with non-aureus staphylococci before calving (18.9 +/- 1.0 vs. 29.4 +/- 1.0%). The variation in blood PMNL apoptosis before and after calving mainly resided at the heifer level (71.4 and 98.4% of the total variation, respectively), whereas the variation in milk PMNL apoptosis mainly resided at the heifer (45.7% of the total variation) and quarter levels (45.5% of the total variation). These data imply that the impaired blood and milk PMNL viability in periparturient heifers can be reduced by optimization of certain heifer management practices such as supplementation of minerals/vitamins, and pasture and feeding strategies.

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability.

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.

In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability.

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625 mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2 mM) was associated with a dramatic increase of phagocytosis-associated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the relationships between intensity of lipomobilization, plasma NEFA, and bovine PMN functions.