Dirofilaria repens in a dog imported to Denmark: A potential for emerging zoonotic disease.

Dirofilarosis is spreading among dogs and humans in Europe with infections being established in many countries. Here, we describe the first molecular biologically confirmed case of D. repens infection in an imported dog in Denmark and highlight the potential zoonotic aspects from this emerging zoonotic parasite in central and northern Europe as at least one to two generations of Dirofilaria spp. can occur per year in Denmark.

Active and Passive Immunization Against Staphylococcus aureus Periprosthetic Osteomyelitis in Rats.

BACKGROUND/AIM: Staphylococcus aureus infection associated with orthopedic implants cannot always be controlled. We used a knee prosthesis model with implant-related osteomyelitis in rats to explore induction of an effective immune response with active and passive immunization. MATERIALS AND METHODS: Fifty-two Sprague-Dawley rats were divided into active (N=28) and passive immunization groups (N=24). A bacterial inoculum of 103 S. aureus MN8 was injected into the tibia and the femur marrow before insertion of a non-constrained knee prosthesis in each rat. The active-immunization group received a synthetic oligosaccharide of polysaccharide poly-N-acetylglucosamine (PNAG), 9G1cNH2 and the passive-immunization group received immunization with immunoglobulin from rabbits infected with S. aureus. RESULTS/CONCLUSION: Active immunization against PNAG significantly reduced the consequences of osteomyelitis infection from PNAG-producing intercellular adhesion (ica+) but not ica- S. aureus. Passive immunization resulted in better clinical assessments in animals challenged with either ica+ or ica- S. aureus, suggesting a lack of specificity in this antiserum.

Prevalence of feline haemoplasma in cats in Denmark.

BACKGROUND: Infections with the three feline haemotropic mycoplasmas Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis cause feline infectious anemia. The purpose of this study was to investigate the prevalence of carriage of feline haemoplasma in Danish cats in different age groups. The presence was detected by a conventional polymerase chain reaction (PCR) assay on blood samples as well as by real-time PCR (RT-PCR). RESULTS: The study revealed a prevalence of 14.9% Candidatus Mycoplasma haemominutum positive cats and 1.5% Mycoplasma haemofelis positive cats. No cats were found positive for Candidatus Mycoplasma turicensis. The results showed a statistically significant higher prevalence in older (>8 years) cats compared to younger cats and a higher prevalence among domestic cats compared to purebred cats. As part of this study, we developed a cloning strategy to obtain Danish positive controls of haemoplasma 16S rRNA. CONCLUSION: From convenience-sampled cats in Denmark, we found that 16.4% were carriers of feline haemotropic mycoplasmas. Haemoplasma was mostly found in older and domestic cats. The prevalence found in Denmark is similar to that found in several other European countries.

A novel knee prosthesis model of implant-related osteomyelitis in rats.

BACKGROUND AND PURPOSE: There have been numerous reports of animal models of osteomyelitis. Very few of these have been prosthesis models that imitate human conditions. We have developed a new rat model of implant-related osteomyelitis that mimics human osteomyelitis, to investigate the pathology of infection after orthopedic implant surgery. METHODS: 2 wild-type strains of Staphylococcus aureus, MN8 and UAMS-1, and their corresponding mutants that are unable to produce poly-N-acetyl glucosamine (PNAG) (ica::tet) were injected into the medullary canals of the femur and tibia at 3 different doses: 10(2), 10(3), and > 10(4) CFU/rat. We measured clinical signs, inflammatory markers, radiographic signs, histopathology, and bacteriology in the infected animals. RESULTS: An inoculum of at least 10(4) cfu of either wild-type bacterial strain resulted in histological, bacteriological, and radiographic signs of osteomyelitis with loosening of the prosthesis. An inoculum of 10(3) CFU gave signs of osteomyelitis but the prosthesis remained in situ. Bacterial inocula of 10(2) cfu gave no signs of osteolysis. INTERPRETATION: We have established a new knee prosthesis model that is suitable for reliable induction of experimental implant-associated osteomyelitis with the prosthesis in situ, using a small inoculum of S. aureus. At a dose of 10(3) CFU/rat, bacteria unable to produce PNAG (ica::tet) had only minor defects in their virulence.

Emotions in veterinary surgical students: a qualitative study.

A surgical educational environment is potentially stressful and can negatively affect students' learning. The aim of the present study was to investigate the emotions experienced by veterinary students in relation to their first encounter with live-animal surgery and to identify possible sources of positive and negative emotions, respectively. During a Basic Surgical Skills course, 155 veterinary fourth-year students completed a survey. Of these, 26 students additionally participated in individual semi-structured interviews. The results of the study show that students often experienced a combination of emotions; 63% of students experienced negative emotions, while 58% experienced positive ones. In addition, 61% of students reported feeling excited or tense. Students' statements reveal that anxiety is perceived as counterproductive to learning, while excitement seems to enhance students' focus and engagement. Our study identified the most common sources of positive and negative emotions to be "being able to prepare well" and "lack of self-confidence," respectively. Our findings suggest that there are factors that we can influence in the surgical learning environment to minimize negative emotions and enhance positive emotions and engagement, thereby improving students' learning.

Anxiety in veterinary surgical students: a quantitative study.

The surgical educational environment is potentially stressful and this can negatively affect students' learning. The aim of this study was to investigate whether veterinary students' level of anxiety is higher in a surgical course than in a non-surgical course and if pre-surgical training in a Surgical Skills Lab (SSL) has an anxiety reducing effect. Investigations were carried out as a comparative study and a parallel group study. Potential participants were fourth-year veterinary students who attended a surgical course (Basic Surgical Skills) and a non-surgical course (Clinical Examination Skills); both courses were offered in multiple classes (with a total of 171 students in 2009 and 156 students in 2010). All classes in 2009 participated in the SSL stage of the Basic Surgical Skills course before performing live-animal surgery, and one class (28 students) in 2010 did not. Two validated anxiety questionnaires (Spielberger's state-trait anxiety inventory and Cox and Kenardy's performance anxiety questionnaire) were used. Anxiety levels were measured before the non-surgical course (111 students from 2009) and before live-animal surgery during the surgical course (153 students from 2009 and 28 students from 2010). Our results show that anxiety levels in veterinary students are significantly higher in a surgical course than in a non-surgical course (p<.001), and that practicing in a SSL helps reduce anxiety before live-animal surgery (p<.005).

Breed-specific variation of hematologic and biochemical analytes in healthy adult Bernese Mountain dogs.

BACKGROUND: Hematology and serum biochemistry reference intervals in dogs may be affected by internal factors, such as breed and age, and external factors, such as the environment, diet, and lifestyle. In humans, it is well established that geographic origin and age may have an impact on reference intervals and, therefore, more specific reference intervals are sought for subpopulations. OBJECTIVE: The objective of this study was to validate and transfer standard laboratory reference intervals for healthy Bernese Mountain dogs and to create new intervals for analytes where the established laboratory reference intervals were rejected. METHODS: The procedure was performed using the human Clinical and Laboratory Standards Institute-approved model modified for veterinary use. Thirty-two dogs were included in the study using a direct a priori method, as recommended. RESULTS: While 23 of the standard laboratory reference intervals were readily validated, 7 of the analytes (eosinophils, MCHC, alkaline phosphatase [ALP], gamma-glutamyltransferase, total bilirubin, amylase, and cholesterol) required new reference intervals according to the standard. These were calculated using the robust method. In particular, the new reference range for ALP was wide compared with the established laboratory reference interval. No clinical causes were found for differences in the results of these analytes. CONCLUSION: We found significant differences in 7 hematologic and serum biochemical analytes for which a breed-specific variation appears to be the most plausible explanation. Breed-specific reference intervals for Bernese Mountain dogs will help avoid misinterpretation of laboratory results in the diagnostic process.

A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5'-diphosphocholine coupled dendrimers.

C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosphocholine. In this design synthetic globular polymers (dendrimers) are used as scaffolds for the multivalent display of phosphocholine molecules. CRP present in a sample binds to the phosphocholine moiety presented at high density in the coating layer and is detectable by specific antibodies. The ELISA was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature and the assay showed high intra- and interassay reproducibility. Results were compared with an immunoturbidimetric method and with a commercial ELISA kit and there was very good agreement with the immunoturbidimetric method, however not with the commercial assay, probably due to a calibration discrepancy. The assay is applicable to other species by providing an adequate detection antibody having the desired species specificity.

Study on biological variation of haemostatic parameters in clinically healthy dogs.

Thromboelastography (TEG) may be a valuable supplement to the coagulation assays activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen, antithrombin (AT) and D-Dimer currently used in most clinical pathology laboratories. Allowable imprecision and bias reference limits for analytical tests can be calculated based on measurements of biological variation. No studies to date have examined the effect of biological variation on these haemostasis parameters in the same group of dogs. Plasma samples were collected after a set protocol once weekly for five consecutive weeks from eight healthy dogs (four males and four females) and stored at -80 degrees C until analysis. Randomized duplicate coagulation tests and TEG analyses were performed on all plasma samples within one run. The data were analyzed for outliers and subsequently subjected to nested analysis of variance to obtain the coefficient of analytical, intra-individual and inter-individual variation. From these objective analytical performance standards for imprecision, critical difference, total error and the index of individuality were calculated to assess the utility of conventional population-based reference ranges. All the clotting times (aPTT, PT and TT), fibrinogen, AT and D-Dimer showed a degree of individuality, which may make the use of population-based reference ranges alone an insensitive interpretation criterion, whereas a population-based reference interval seems to be sensitive for interpreting all TEG parameters. Analytical performance standards for imprecision were only met for one of the coagulation assays, whereas all TEG parameters except the alpha angle, alpha achieved this analytical goal.

Comparative analysis of haematological, haemostatic, and inflammatory parameters in canine venous and arterial blood samples.

The objective of the present study was to validate the use of blood collected from an indwelling arterial catheter for analysis of haematological, coagulation and inflammatory parameters in canines compared to venous blood collected directly from the jugular vein. Blood samples were collected from 11 dogs. Agreement between sampling methods was found for neutrophil and monocyte counts, prothrombin time, activated partial thromboplastin time, antithrombin, protein C, factor VIII and C-reactive protein, whereas a statistically significant difference was found for white blood cells, lymphocyte, erythrocyte and platelet counts, haemoglobin, haematocrit, fibrinogen and thrombin time (TT). In conclusion, it is necessary to be aware that results from a complete blood count obtained from canine venous and arterial blood samples may not be comparable. Values for haemostatic parameters from arterial and venous blood samples, with the exception of fibrinogen and TT, were however statistically identical.

Use of serum C-reactive protein as an early marker of inflammatory activity in canine type II immune-mediated polyarthritis: case report.

BACKGROUND: Monitoring systemic inflammatory activity during steroid therapy of canine immune-mediated polyarthritis (IMPA) is difficult and mainly relies on clinical signs. CASE PRESENTATION: Canine serum C-reactive protein (CRP) was measured serially and blinded during a 27-week follow-up period of a case of Anaplasma phagocytophilia induced type II immune-mediated polyarthritis. CONCLUSION: WBC was, as expected, observed not to reflect the inflammatory activity during steroid treatment in a clinical useful manner, whereas, CRP is suggested a valuable unbiased marker of inflammatory activity during steroid treatment in this case.

Method comparison in the clinical laboratory.

Studies comparing a new method with an established method, to assess whether the new measurements are comparable with existing ones, are frequently conducted in clinical pathology laboratories. Assessment usually involves statistical analysis of paired results from the 2 methods to objectively investigate sources of analytical error (total, random, and systematic). In this review article, the types of errors that can be assessed in performing this task are described, and a general protocol for comparison of quantitative methods is recommended. The typical protocol has 9 steps: 1) state the purpose of the experiment, 2) establish a theoretical basis for the method comparison experiment, 3) become familiar with the new method, 4) obtain estimates of random error for both methods, 5) estimate the number of samples to be included in the method comparison experiment, 6) define acceptable difference between the 2 methods, 7) measure the patient samples, 8) analyze the data, and 9) judge acceptability. The protocol includes the essential investigations and decisions needed to objectively assess the overall analytical performance of a new method compared to a reference or established method. The choice of statistical methods and recommendations of decision criteria within the stages are discussed. Use of the protocol for decision-making is exemplified by the comparison of 2 methods for measuring alanine aminotransferase activity in serum from dogs. Finally, a protocol for comparing simpler semiquantitative methods with established methods that measure on a continuous scale is suggested.

Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs.

BACKGROUND: Thromboelastography (TEG) is an analytical method that enables global assessment of hemostatic function in whole blood (WB) with evaluation of both plasma and cellular components of hemostasis. TEG has a largely unused potential in the diagnostic workup and monitoring of dogs with hemostatic disorders and it may be a valuable supplement to traditional coagulation parameters. OBJECTIVES: The objective of this study was to establish a clinically applicable reference interval for a TEG assay using recombinant human tissue factor (TF) as the activator on citrated WB from clinically healthy dogs and to evaluate the stability of citrated WB stored for 30 minutes (T30) and 120 minutes (T120) at room temperature (RT). Additionally, we evaluated the analytical variation in reaction time (R), clotting time (K), angle (alpha), and maximum amplitude (MA). METHODS: Blood was collected from 18 clinically healthy dogs. Duplicate TEG analyses with TF as the activator at a concentration of 1:50,000 were performed on canine citrated WB at T30 and T120. R, K, a, and MAwere analyzed. RESULTS: Mean TEG values at T30/T120 were R = 5.61/4.91 minutes, K = 4.20/3.34 minutes, alpha = 45.33/50.90 degrees , and MA = 47.96/50.19 mm. Significant differences in these values were observed after storage for T30 and T120 at RT, with a tendency towards hypercoagulability at T120. The mean coefficients of variation were low. CONCLUSIONS: Canine citrated WB can be used for TEG analysis with human recombinant TF as the activator when stored at RT for T30 or T120. At both time points, the analytical variation was low, suggesting that TEG analysis may be of value in evaluating dogs with hemostatic disorders. A fixed time point should be chosen for serial measurements.

Internal quality control of a turbidimetric immunoassay for canine serum C-reactive protein based on pooled patient samples.

BACKGROUND: Optimized internal quality control (IQC) procedures are important to ensure that only results without medically important errors are used for medical decision-making and to ensure that unnecessary rejection of valid analytical runs is avoided. Additionally, estimates of the analytical performance can be derived from IQC data. In the absence of available species-specific standards of a compound, the use of alternative control materials based on patient samples is a possibility, although investigations on the suitability of this approach are needed. OBJECTIVES: The objective of the study was to plan and implement a simple IQC procedure with control material based on pooled canine serum samples for a turbidimetric immunoassay (TIA) for the determination of human C-reactive protein (CRP) that recently was validated for the determination of canine serum CRP, and to assess the clinical analytical performance of the assay. METHODS: Proposed guidelines for the planning and implementation of IQC procedures were followed by using 2 control materials. Quality requirements of the assay were defined objectively by means of available data on biological variation, and goals for IQC performance were defined according to recommendations (probability of error detection [P(ed)] >.90 and of false rejection [P(fr)] <.05). Analytical performance was evaluated by means of medical decision charts. RESULTS: The control rule of 1(2.5s) (ie, rejection of the analytical run if at least 1 of 2 control materials deviates from the mean by more than 2.5 SD) fulfilled the criteria of predicted IQC performance (P(ed) =.94-1.00, P(fr) =.03). The IQC method was successfully implemented over a 14-week period. The observed coefficient of variation in the period of monitoring was 3.8% (low) and 2.9% (high), which equals excellent analytical performance. CONCLUSIONS: It was possible to plan and implement a simple IQC procedure for the CRP-TIA with control materials based on canine serum samples that fulfilled the criteria of high error detection and low false rejection of valid analytical runs. The assay showed excellent long-term analytical performance over a 14-week period.

Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration.

BACKGROUND: Serum C-reactive protein (CRP) is an acute phase marker in dogs that is useful for the diagnosis and monitoring of inflammatory disease. Rapid, reliable, and automated assays are preferable for routine evaluation of canine serum CRP concentration. OBJECTIVE: The aim of this study was to evaluate whether canine serum CRP concentration could be measured reliably using an automated turbidometric immunoassay (TIA) designed for use with human serum. METHODS: A commercially available TIA for human serum CRP (Bayer, Newbury, UK) was used to measure canine serum CRP concentration. Cross-reactivity of antigen was evaluated by the Ouchterlony procedure. Intra- and interassay imprecision was investigated by multiple measurements on canine serum samples and serum pools, respectively. Assay inaccuracy was investigated by linearity under dilution and comparison of methodologies (canine CRP ELISA, Tridelta Development Ltd, Kildare, UK). Then the assay was applied to serum samples from 14 clinically healthy dogs, 11 dogs with neoplasia, 13 with infections, 8 with endocrine or metabolic diseases, and 10 with miscellaneous diseases. RESULTS: Cross-reactivity between canine serum CRP and the anti-human CRP antibody was found. Intra- and interassay imprecision ranged from 5.2% to 10.8% and 3.0% to 10.2%, respectively. Serum CRP concentration was measured in a linear and proportional manner. There was no significant disagreement and there was linear correlation of the results in the comparison of methodologies, except for a slight proportional discrepancy at low CRP concentrations (<10 microg/mL). Dogs with infections had a significantly higher concentration of serum CRP than did all other dogs, and dogs with neoplasia had a significantly higher concentration of serum CRP than did clinically healthy dogs. CONCLUSIONS: Canine serum CRP concentration can be measured reliably using the commercially available TIA designed for human CRP.