Comparative study of immunohematological tests with canine blood samples submitted for a direct antiglobulin (Coombs') test.

BACKGROUND: A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. MATERIAL AND METHODS: Anticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics; neutral gel column cards, microtiter plates (at 4°, 22°, and 37°C), capillary tubes, and flow cytometry used in laboratories. RESULTS: Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1-24 weeks after initial assessment. CONCLUSIONS: Based upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable.

Signalment and Clinical Data of Cats with Exocrine Pancreatic Insufficiency Diagnosed Using Feline Trypsin-like Immunoreactivity in Routine Diagnostics.

Serum feline trypsin-like immunoreactivity (fTLI) is commonly used to diagnose feline exocrine pancreatic insufficiency (EPI). This study aimed to describe signalment and clinical data of cats with EPI. Determination of TLI was performed using an in-house ELISA; the reference interval was defined using a Reference Limit Estimator. Groups were formed from 4813 cats (2019-2020), based on their fTLI concentration: 1 (<8 µg/L; decreased; n = 275), 2 (8-88 µg/L; reference interval; n = 4256), and 3 (>88 µg/L; increased; n = 282). Males and Domestic Shorthairs were most common in all groups. Group 3 had the highest (13 years), and group 1 had the lowest (9 years), median age. Clinical information was available for 200 cats (decreased fTLI: n = 87, lower reference interval (8-12 µg/L): n = 113). Treatment response was observed in 83% (decreased fTLI) and 66% (lower reference interval). EPI cats displayed weight loss (69%), diarrhoea (68%), vomiting (41%), anorexia (39%), poor hair coat (35%), lethargy (33%), and/or polyphagia (21%). The lower the serum fTLI concentration, the more often good treatment response was reported (p = 0.022) but there were no statistically significant clinical signs. In conclusion, fTLI is a helpful parameter to diagnose EPI but predicting treatment response based on signalment or clinical signs is not possible.

Histopathological findings and canine pancreatic lipase immunoreactivity in normal dogs and dogs with inflammatory and neoplastic diseases of the pancreas.

BACKGROUND: Diagnosis of pancreatic diseases in dogs is still challenging because of variable clinical signs, which do not always correspond with clinical pathology and histopathological findings. OBJECTIVES: To characterize inflammatory and neoplastic pancreatic diseases of dogs and to correlate these findings with clinical findings and canine pancreatic lipase immunoreactivity (cPLI) results. ANIMALS: Tissue specimens and corresponding blood samples from 72 dogs submitted for routine diagnostic testing. METHODS: Four groups were defined histologically: (1) normal pancreas (n = 40), (2) mild pancreatitis (n = 8), (3) moderate or severe pancreatitis (acute, n = 11; chronic, n = 1), and (4) pancreatic neoplasms (n = 12). An in-house cPLI ELISA (<180 µg/L, normal; >310 µg/L, pancreatitis) was performed. RESULTS: In dogs with normal pancreas, 92.5% of serum cPLI results were within the reference range and significantly lower than in dogs with mild acute pancreatitis, moderate or severe acute pancreatitis and pancreatic tumors. In dogs with moderate or severe acute pancreatitis, cPLI sensitivity was 90.9% (95% confidence interval [CI], 58.7%-99.8%). Most dogs (9/12) with pancreatic tumors (group 4) had additional pancreatic inflammation and cPLI results were increased in 10 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: High cPLI indicates serious acute pancreatitis but underlying pancreatic neoplasms should also be taken into consideration. This study confirms the relevance of histopathology in the diagnostic evaluation of pancreatic diseases.

Accuracy of a diagnostic model based on serum biochemical parameters in detecting cows at an increased risk of chronic fascioliasis.

In adult cattle Fasciola hepatica infection usually follows a chronic subclinical course, and reduces both the milk yield and milk quality, resulting in considerable financial losses. Effective control of the disease is based on reliable identification of asymptomatically infected individuals, which now requires special parasitological or serological diagnostic tests. It is also known that F. hepatica infection induces alterations in some serum biochemical parameters. Therefore, the study was conducted to develop a model based on serum biochemical parameters allowing to identify cows at higher risk of chronic fascioliasis. Six hundred sixty eight adult dairy cows from 97 herds located in central and northeastern Poland were clinically examined, and blood and fecal samples from them were collected for a routine monitoring of fascioliasis and metabolic profile. Using the combination of fecal sedimentation test and indirect ELISA based on excretory/secretory products 203 cows, apparently healthy in clinical examination, were definitively classed as affected by (47 cows, 23.2%) or free from (156 cows, 76.8%) chronic fascioliasis. Their serum was screened for the activity of 4 enzymes (γ-glutamyl transpeptidase, glutamate dehydrogenase, aspartate aminotransferase, creatine kinase) and concentration of 18 other components (total bilirubin, total protein, albumin, globulin, urea, glucose, non-esterified fatty acids, ß-hydroxybutyrate, sodium, potassium, chloride, calcium, magnesium, copper, zinc, iron, selenium, and haptoglobin). Logistic regression analysis was used to build 4 multivariable models allowing for identification of cows at risk of chronic fascioliasis. Then, the accuracy was compared between the models using the area under the receiver operating characteristic curve (AUROC), and an optimal cut-off value was determined for the most accurate model using Youden J index. The most accurate proved to be the model based on glutamate dehydrogenase activity and globulin, urea (all three positively linked with risk of chronic fascioliasis), and selenium concentration (negatively linked) adjusted by the access to pasture and cow's age. At the optimal cut-off of 0.37 this model had sensitivity of 85.1% (CI 95%:72.3%, 92.6%), specificity of 90.4% (CI 95%:84.7%, 94.1%), positive likelihood ratio of 8.8 (CI 95%:5.4, 14.5), and negative likelihood ratio of 0.16 (CI 95%:0.08, 0.33). This model performed significantly better than model including only γ-glutamyl transpeptidase or model including both γ-glutamyl transpeptidase and aspartate aminotransferase (both also adjusted by the access to pasture and cow's age), and was the only model which performed significantly better than the basic model based solely on the access to pasture and cow's age.

Herd-level seroprevalence of Fasciola hepatica and Ostertagia ostertagi infection in dairy cattle population in the central and northeastern Poland.

BACKGROUND: Fasciola hepatica and Ostertagia ostertagi infections are widespread in cattle population of Europe, however data on their prevalence in Poland are only fragmentary. Therefore, the cross-sectional study was carried out to determine the herd-level seroprevalence of F. hepatica and O. ostertagi infection in dairy cattle population in the central and north-eastern provinces Poland, and to identify basic local risk factors for these infections. In total, 598 herds were enrolled, 394 (65.9%) in the north-eastern province and 204 (34.1%) in the central province. In each herd the questionnaire survey was conducted and bulk-tank milk (BTM) sample was collected and screened using two indirect immunoenzymatic tests. Optical density ratio (ODR) was regarded as the quantitative proxy of exposure to either of the two parasites. RESULTS: Both Fasciola and Ostertagia ELISA ODR in the north-eastern province was significantly higher than ODR in the central province. At the cut-off value of ODR = 0.27 the true herd-level seroprevalence of F. hepatica was 79.6% (95% CI: 74.0%, 84.3%) in the north-eastern province and 13.0% (95% CI: 5.3%, 21.7%) in the central province. At the cut-off of ODR = 0.50151 of 188 herds (80.3%, 95% CI: 74.1%, 85.4%) were seropositive for O. ostertagi in the north-eastern province and only 70 of 136 herds (51.5%, 95% CI: 43.1%, 59.7%) were seropositive in the central province. Location of a herd in the north-eastern province, longer grazing period practiced in a herd and > 50%-share of grazing grass in roughage were all positively related to the increase in exposure to both parasites. Moreover, the use of hay or haylage as main roughage proved to be positively related to the increase in exposure to F. hepatica. CONCLUSIONS: F. hepatica and O. ostertagi are widespread in cattle population in Poland, however their occurrence at a herd-level varies between different regions of Poland. This diversity can only partly be explained by different herd management, and appears linked to environmental and climate conditions typical for these regions.

Herd-level seroprevalence of Neospora caninum infection in dairy cattle in central and northeastern Poland.

A serosurvey was carried out to estimate the herd-level seroprevalence of Neospora caninum infection in cattle in central and northeastern Poland. Ninety seven dairy cattle herds from 2 provinces of Poland (Podlaskie, 47 herds and Lodzkie, 50 herds) were randomly enrolled in the study using two-stage cluster method. A simple random selection was applied within each herd to select a sample of adult cows (≥18 month-old). A total number of 734 cows were enrolled in the study. The animals were screened with a commercial competitive ELISA (Bio-X Diagnostics, Belgium). To calculate true herd-level seroprevalence test sensitivity and specificity were adjusted from an individual- to a herd-level using FreeCalc method. The true overall herd-level seroprevalence of N. caninum infection was 56.7% (95% CI: 47.5%, 65.9%). The true herd-level seroprevalence in Podlaskie was 63.3% (95% CI: 43.0%, 83.6%) and 50.5% (95% CI: 32.8%, 68.2%) in Lodzkie province and these figures did not differ significantly between the two provinces (chi2 test p = 0.238). One hundred forty three of 734 cows (19.5%) were seropositive which gave the true overall individual-level seroprevalence of 20.1% (95% CI: 17.4%, 23.2%). Percentage of seropositive cows in each herd varied from 6% to 80%. This study is the first epidemiological investigation of herd-level seroprevalence of N. caninum infection in Polish dairy cattle population. In conclusion, the result of the study confirmed previous data that N. caninum infection is widespread in the Polish cattle population and thus should be considered as a potential cause of spontaneous abortions.

The performance of ice-free cryopreserved heart valve allografts in an orthotopic pulmonary sheep model.

Transplantation of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. In previous studies we demonstrated that conventional frozen cryopreservation (FC) is accompanied by serious alterations of extracellular matrix (ECM) structures. As the main culprit of the observed damages ice crystal formation was identified. Objective of this study was the application principles of cryoprotection as observed in nature, occurring in animals or plants, for ice-free cryopreservation (IFC) of heart valves. Using IFC, valves were processed and stored above the glass transition temperature of the cryoprotectant formulation (-124 degrees C) at -80 degrees C to avoid any ice formation, tissue-glass cracking and preserving ECM. After implantation in the orthotopic pulmonary position in sheep, we demonstrate that IFC resulted in cell free matrices, while maintaining crucial ECM-components such as elastin and collagen, translating into superior hemodynamics. In contrast, we reveal that FC valves showed ECM damage that was not restored in vivo, and T-cell inflammation of the stroma with significant leaflet thickening. Compared to currently applied FC practice IFC also reduced infrastructural needs for preservation, storage and shipping. These results have important implications for clinical valve transplantation including the promise of better long-term function and lower costs.

Cytokine gene expression profiles of bovine dendritic cells after interaction with Mycobacterium avium ssp. paratuberculosis (M.a.p.), Escherichia coli (E. coli) or recombinant M.a.p. heat shock protein 70.

Mycobacterium avium paratuberculosis (M.a.p.) resides and replicates in macrophages. Many of the of immune mechanisms aiding M.a.p. survival in the host's cells are known. However, little is known about interactions of M.a.p. with dendritic cells (DC). As DC are important for the induction of protective immunity against infectious diseases, we investigated the interaction of M.a.p. with these cells. Quantitative real-time PCR (RT-PCR) was used to analyse differential expression of cytokine genes after 6 h and 24 h of incubation by immature DC that phagocytosed either M.a.p. or Escherichia coli (E. coli). We hypothesized that phagocytosis of E. coli would induce pro-inflammatory cytokines due to abundant presence of lipopolysaccharide (LPS) and that the cytokine expression profile induced by phagocytosis of live M.a.p. would differ. In addition we hypothesized that incubation of immature DC with rHsp70, an immunodominant antigen of M.a.p., would induce a similar profile of cytokine gene expression as phagocytosis of intact M.a.p. However, phagocytosis of both E. coli and M.a.p. resulted in a cytokine gene expression pattern representative of a (pro-)inflammatory reaction, dominated by strong induction of IL-12 gene expression, that was higher after 24 h than after 6 h of incubation, although the response to M.a.p. was less vigorous than to E. coli. Incubation with rHsp70 resulted in a more inhibitory type of cytokine gene expression, with delayed IL-12 gene expression and downregulation of the genes for IL-1beta and IL-6 after 24 h of incubation. We conclude that bovine DC produce an immuno-stimulatory, anti-mycobacterial response to infection with M.a.p., while Hsp70 potentially contributes to pathogen virulence by allowing the bacteria to invade the host cell.