Andreas Moritz

Differential Responses of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

ABSTRACT Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johnes disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-γ and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-γ (6 h), and TNF-α (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-α (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.

Management of Advanced Tracheal Collapse in Dogs Using Intraluminal Self-Expanding Biliary Wallstents

Twenty-four client-owned dogs with tracheal collapse refractory to conventional treatment underwent management with an intraluminal self-expanding stainless-steel endoprosthesis (Wallstent). Initial improvement of clinical signs was observed in 95.8% of the dogs. Two dogs (8.3%) died within a median interval of 6 days after stent implantation due to incorrect placement and size of the stent and emphysema, respectively. A dry cough occurred temporarily in most of the patients. One dog each (4.1%) suffered mild transient tracheal hemorrhage and pneumomediastinum. The results showed that the initial survival rate of intraluminal stabilization was comparable with surgical implantation of extratracheal prostheses. Clinical reevaluation was performed in 18 dogs within a median interval of 68 days after treatment. Of the dogs treated, 30.4% were reported to be asymptomatic after stent implantation, 60.9% improved markedly, and 4.3% remained symptomatic. In all patients undergoing endoscopy, the Wallstents were almost completely covered with tracheal epithelium. A median shortening of 27.3% of the endoprosthesis within a median interval of 175 days after stent implantation in 15 of 18 dogs was noted. The shortening was associated with clinical signs in 2 patients. In 5 dogs, steroid-responsive granuloma formation resulted in a severe reduction of the tracheal lumen in 3 patients. The results suggest that implantation of Wallstents was minimally invasive and provided stabilization of collapsed thoracic tracheal portions in addition to the cervical part of the trachea. This minimally invasive method for the management of severe tracheal collapse therefore provides an attractive alternative to surgery.

Establishment of reference intervals for kaolin-activated thromboelastography in dogs including an assessment of the effects of sex and anticoagulant use.

Tissue factor (TF)- and kaolin-activated thromboelastography (TEG) have been performed in a small number of healthy dogs, but reference intervals have not been assessed in a larger number of dogs. The goal of the current study was to establish reference intervals and assess intra-assay repeatability for kaolin-activated TEG in dogs. Additionally, the impact of sex and the influence of anticoagulant (native blood vs. recalcified citrate anticoagulated blood) were evaluated. Thromboelastography analyses were performed in 56 healthy dogs including German Shepherd Dogs (n = 19), Beagles (n = 15), and others (n = 22). Median age was 2 years (range: 1–6 years) and sex was evenly distributed (31 males and 25 females). To establish reference intervals, citrated whole-blood samples were collected, and TEG was performed 1 hr after sampling. Five TEG variables (R = reaction time; K = clot formation time; α = angle α; MA = maximal amplitude; G-value reflecting clot stability) were evaluated, and reference intervals were defined as the mean + 1.96-fold standard deviation. Intra-assay repeatability was assessed by calculating the pooled variance estimate in duplicate measurements of 6 healthy dogs. The effect of anticoagulant was assessed in 17 specimens. Reference intervals were as follows: R = 1.8–8.6 min; angle α = 36.9–74.6 degrees; K = 1.3–5.7 min; MA = 42.9–67.9 mm, and G = 3.2–9.6 Kdyn/cm 2 . Coefficients of variation for R, K, angle α, MA, and G were 7.6%, 17.7%, 7.4%, 2.9%, and 6.6%, respectively. There was no significant impact of sex or anticoagulant on results. Interindividual variation was higher in native samples than in citrated whole blood. A limitation of the current study was that most of the samples were obtained from Beagles and German Shepherd Dogs. This study provides useful reference intervals for kaolin-activated TEG.

Prevalence of feline haemotropic mycoplasmas in convenience samples of cats in Germany.

The aim of this prospective study was to evaluate the prevalence of feline haemotropic mycoplasmas in Germany, to determine probable risk factors for these infections and to compare the diagnostic value of microscopic examination of blood smears to polymerase chain reaction (PCR). For the prevalence study, convenience samples (Ethylene diamine-tetraacetic acid (EDTA) blood) from 262 (64.5% male and 35.5% female) cats were included. A PCR for the detection of Mycoplasma haemofelis (MHF) and ‘Candidatus Mycoplasma haemominutum’ (CMH) as well as a feline leukaemia virus (FeLV)/feline immunodeficiency virus (FIV) enzyme-linked immunoassay was performed. Blood smears from 224 cats were examined and the sensitivity and specificity of the microscopic diagnosis were determined. The prevalence of CMH, MHF, and CMH/MHF co-infection was 22.5%, 4.5%, and 0.8%, respectively. CMH was significantly associated with male gender (P=0.047), older age (P=0.0015) and both FeLV (P=0.002) and FIV infections (P<0.0001). However, there was no association between the presence of anaemia and CMH/MHF infection. The respective sensitivity and specificity of the microscopic diagnosis were 10.3% and 87.1% for a CMH infection and 0.0% and 98.0% for MHF infection.

Efficacy of a continuous, multiagent chemotherapeutic protocol versus a short-term single-agent protocol in dogs with lymphoma

OBJECTIVE To compare response rates and remission and survival times in dogs with lymphoma treated with a continuous, multiagent, doxorubicin-based chemotherapeutic protocol or with a short-term single-agent protocol incorporating doxorubicin. DESIGN Nonrandomized controlled clinical trial. ANIMALS 114 dogs with lymphoma. PROCEDURES Dogs were treated with a chemotherapeutic protocol consisting of L-asparaginase, vincristine, cyclophosphamide, doxorubicin, methotrexate, and prednisolone (n=87) or doxorubicin alone (27). RESULTS 63 of 86 (73%) dogs treated with the multiagent protocol (data on response was unavailable for 1 dog) and 14 of 27 (52%) dogs treated with the single-agent protocol had a complete remission. Dogs with lymphoma classified as substage<or=and dogs with a high BUN concentration at the time of initial diagnosis were significantly less likely to have a complete remission. No significant difference in remission or survival time could be demonstrated between treatment groups. Incidence of hematologic and gastrointestinal tract toxicoses did not differ between treatment groups, with the exception that vomiting was more common among dogs treated with the multiagent protocol. CONCLUSIONS AND CLINICAL RELEVANCE In this population of dogs, we were not able to identify any significant difference in remission or survival times between dogs with lymphoma treated with a continuous, multiagent chemotherapeutic protocol and dogs treated with a short-term single-agent protocol involving doxorubicin.

Comparative clinical study of canine and feline total blood cell count results with seven in-clinic and two commercial laboratory hematology analyzers

BACKGROUND A CBC is an integral part of the assessment of health and disease in companion animals. While in the past newer technologies for CBC analysis were limited to large clinical pathology laboratories, several smaller and affordable automated hematology analyzers have been developed for in-clinic use. OBJECTIVES The purpose of this study was to compare CBC results generated by 7 in-clinic laser- and impedance-based hematology instruments and 2 commercial laboratory analyzers. METHODS Over a 3-month period, fresh EDTA-anticoagulated blood samples from healthy and diseased dogs (n=260) and cats (n=110) were analyzed on the LaserCyte, ForCyte, MS45, Heska CBC, Scil Vet ABC, VetScan HMT, QBC Vet Autoread, CELL-DYN 3500, and ADVIA 120 analyzers. Results were compared by regression correlation (linear, Deming, Passing-Bablok) and Bland-Altman bias plots using the ADVIA as the criterion standard for all analytes except HCT, which was compared with manual PCV. Precision, linearity, and carryover also were evaluated. RESULTS For most analytes, the in-clinic analyzers and the CELL-DYN performed similarly and correlated well with the ADVIA. The biases ranged from -0.6 to 2.4 x 10(9)/L for WBC count, 0 to 0.9 x 10(12)/L for RBC count, -1.5 to 0.7 g/dL for hemoglobin concentration, -4.3 to 8.3 fL for MCV, and -69.3 to 77.2 x 10(9)/L for platelet count. Compared with PCV, the HCT on most analyzers had a bias from 0.1% to 7.2%. Canine reticulocyte counts on the LaserCyte and ForCyte correlated but had a negative bias compared with those on the ADVIA. Precision, linearity, and carryover results were excellent for most analyzers. CONCLUSIONS Total WBC and RBC counts were acceptable on all in-clinic hematology instruments studied, with limitations for some RBC parameters and platelet counts. Together with evaluation of a blood film, these in-clinic instruments can provide useful information on canine and feline patients in veterinary practices.

Evaluation of the automated hematology analyzer Sysmex XT-2000iV ™ compared to the ADVIA ® 2120 for its use in dogs, cats, and horses. Part II: Accuracy of leukocyte differential and reticulocyte count, impact of anticoagulant and sample aging.

The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K3–EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact of anticoagulant and sample storage was assessed in specimens obtained from an additional 9 cats, 10 dogs, and 10 horses. Agreement between both analyzers was excellent to good except for monocytes and canine reticulocytes. Spearman rank correlation coefficients (rs) between Sysmex XT-2000iV and manual differential were good to fair and ranged from 0.91 (cat lymphocytes) to 0.44 (cat monocytes). Hematocrit value (Hct), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC; all: P < 0.001), and mean corpuscular volume (MCV; P < 0.01) were higher in canine citrated blood compared to heparin and EDTA. In cats, lymphocytes and monocytes were lower in heparinized blood compared to EDTA (P < 0.05), whereas in horses no significant effect was seen. Regarding storage time and temperature, white and red blood cell counts, hemoglobin, and MCH were stable. Hct, MCV, and MCHC were influenced by erythrocyte swelling. Differential count remained stable for 24 hr (22°C) and nearly 72 hr (4°C) except for monocytes. The overall performance of the Sysmex XT-2000iV was excellent and compared favorably with that of the ADVIA 2120. A special strength was the excellent detection of feline eosinophils.

The speed of kill of fluralaner (Bravecto™) against Ixodes ricinus ticks on dogs

BackgroundPathogens that are transmitted by ticks to dogs, such as Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi sensu latu, and Ehrlichia canis, are an increasing problem in the world. One method to prevent pathogen transmission to dogs is to kill the ticks before transmission occurs. Fluralaner (Bravecto™) is a novel isoxazoline insecticide and acaricide that provides long persistent antiparasitic activity following systemic administration. This study investigated the speed of kill of fluralaner against Ixodes ricinus ticks on dogs.MethodsA total of 48 dogs were randomized to 8 groups of 6 dogs and each dog was infested with 50 female and 10 male I. ricinus ticks. Two days later (day 0), 4 groups received a single treatment of 25 mg fluralaner/kg body weight as Bravecto™ chewable tablets; the dogs in the other 4 groups were left untreated. Separate control and treatment groups were paired at each time point (4, 8, 12, or 24 hours after treatment) for assessment of tick-killing efficacy. At 4, 8, and 12 weeks after treatment, all dogs were re-infested with 50 female I. ricinus ticks and subsequently assessed for live or dead ticks at either 4, 8, 12, or 24 hours after re-infestation. Efficacy was calculated for each assessment time point by comparison of the treatment group with the respective control group.ResultsTick-killing efficacy was 89.6% at 4 hours, 97.9% at 8 hours, and 100% at 12 and 24 hours after treatment. Eight hours after re-infestation, efficacy was 96.8%, 83.5%, and 45.8% at 4, 8, and 12 weeks after treatment, respectively. At least 98.1% tick-killing efficacy was demonstrated 12 and 24 hours after re-infestation over the entire 12 week study period.ConclusionsFluralaner kills ticks rapidly after treatment at 4 hours, and over its entire 12-week period of efficacy, it achieves an almost complete killing effect within 12 hours after tick infestation. The rapid tick-killing effect together with the long duration of efficacy enables fluralaner to aid in the prevention of tick borne diseases.

Evaluation of three automated human immunoturbidimetric assays for the detection of C-reactive protein in dogs

C-reactive protein (CRP) is a major, acute-phase protein in dogs; however, there is a need for automated assays to ensure in-time patient monitoring. Three automated immunoturbidimetric assays (Randox, Thermo, and Wako) developed for human beings were evaluated for their ability to detect canine CRP, including method validation, evaluation of diagnostic use, and establishment of exploratory reference intervals. Sera from 36 healthy dogs and 82 diseased dogs were included for method comparison with the enzyme-linked immunosorbent assay (ELISA; Tridelta) serving as the reference method. A nonparametric estimate of the 1-sided 95% reference interval was established (n = 36). Precision study revealed good intraassay coefficients of variation (CVs) of 1–10%, 0–9%, and 2–13% for the Randox, Thermo, and Wako assays, respectively. Interassay CVs were 18%, 24%, and 19% respectively. Because of a low linear range, the Thermo test was considered unsuitable for use with canine specimens. No significant differences were present between the results obtained with the Randox and Wako assays with CRP concentrations less than 15 mg/l; however, median CRP results differed significantly between the Thermo test and the ELISA (P = 0.03). Bland–Altman analysis detected a proportional bias of 0.28, −0.59, and 0.61 mg/l for the Randox, Thermo, and Wako assays, respectively. For all tests, median CRP values were significantly different between healthy dogs and dogs with neoplasia. The upper limit of the reference intervals were 8.2 and 9.9 mg/l for the Randox and Wako assays, respectively. In contrast to the Thermo test, the Randox and Wako assays were suitable for detection of abnormally high canine CRP concentrations; however, improvement of assay precision and evaluation of accuracy are warranted before their clinical use with canine specimens.

Genetic relatedness of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from a dog and the dog owner.

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n=3) and the anterior nares of the dog owner (n=1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.