Serum concentration of homocysteine in spontaneous feline chronic kidney disease.

Serum homocysteine (Hcy) increases in people and dogs with chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy) has also been associated with CKD-related hypertension and proteinuria. The aims of this study were to: (1) validate an enzymatic method for quantification of Hcy in feline serum; (2) evaluate whether HHcy was associated with the presence and severity of CKD, proteinuria or hypertension; and (3) determine whether HHcy could predict disease progression. The intra- and inter-assay coefficients of variation (CVs) and the recovery rates of linearity under dilution and spiking recovery tests of the enzymatic method were 3.1-6.7%, 11.6-12.5%, 96.9±5.4% and 96.9±5.4%, respectively. Healthy cats at risk of CKD (n=17) and cats with CKD (n=19) were sampled over a 6-month period (63 samples in total). Cats with CKD had significantly higher Hcy concentrations (P=0.005) than cats at risk. The concentration of Hcy was higher (P=0.002) in moderate-severe CKD than in mild CKD and correlated moderately with serum creatinine (P<0.0001; r=0.51). The concentration of Hcy increased with the magnitude of proteinuria and correlated weakly with urinary protein to creatinine ratio (P=0.045; r=0.26). HHcy was not associated with hypertension. At the time of enrollment, Hcy concentration was significantly higher (P=0.046) in cats that developed CKD compared to cats that remained stable. The enzymatic method for Hcy measurement in feline serum was precise and accurate. HHcy was relatively common in cats with advanced CKD and seemed to predict disease progression, but further studies are warranted.

Detecting total immunoglobulins in diverse animal species with a novel split enzymatic assay.

BACKGROUND: Total immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized. RESULTS: IgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments. CONCLUSIONS: STIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.

Bayesian evaluation of three serological tests for the diagnosis of bovine brucellosis in Bangladesh.

We evaluated the performance of three serological tests - an immunoglobulin G indirect enzyme linked immunosorbent assay (iELISA), a Rose Bengal test and a slow agglutination test (SAT) - for the diagnosis of bovine brucellosis in Bangladesh. Cattle sera (n = 1360) sourced from Mymensingh district (MD) and a Government owned dairy farm (GF) were tested in parallel. We used a Bayesian latent class model that adjusted for the conditional dependence among the three tests and assumed constant diagnostic accuracy of the three tests in both populations. The sensitivity and specificity of the three tests varied from 84.6% to 93.7%, respectively. The true prevalences of bovine brucellosis in MD and the GF were 0.6% and 20.4%, respectively. Parallel interpretation of iELISA and SAT yielded the highest negative predictive values: 99.9% in MD and 99.6% in the GF; whereas serial interpretation of both iELISA and SAT produced the highest positive predictive value (PPV): 99.9% in the GF and also high PPV (98.9%) in MD. We recommend the use of both iELISA and SAT together and serial interpretation for culling and parallel interpretation for import decisions. Removal of brucellosis positive cattle will contribute to the control of brucellosis as a public health risk in Bangladesh.

Use of two commercial caprine arthritis-encephalitis immunoenzymatic assays for screening of arthritic goats.

Roughly one-fourth of goats infected with small ruminant lentivirus (SRLV) develop caprine arthritis-encephalitis (CAE). We compared the profile of antibody response to surface glycoprotein (SU), and combined transmembrane glycoprotein and capsid protein (TM/CA) in SRLV-infected arthritic and asymptomatic goats, and determined the ability of 2 commercial ELISAs to distinguish between arthritic and asymptomatic goats. We used sera from 312 SRLV-seropositive dairy goats in a whole-virus ELISA; 222 were collected from arthritic goats and 90 from apparently healthy goats. Sera were screened with a competitive inhibition ELISA based on SU antigen (SU-ELISA) and an indirect ELISA based on TM and CA antigens (TM/CA-ELISA). Receiver operating characteristic (ROC) curves were prepared for both ELISAs, and areas under the ROC curves (AUC) were compared. The proportion of goats with antibody response stronger to SU antigen than to TM/CA antigen was significantly higher among arthritic than asymptomatic goats (58.1% vs. 28.9%; p < 0.001). Antibody response to SU antigen was a good predictor of the arthritic form of CAE: AUC for SU-ELISA was 89.7% (95% CI: 85.2%, 94.2%), compared to 59.3% (95% CI: 51.9%, 66.8%) for TM/CA-ELISA ( p < 0.001). With the cutoff set at percentage of inhibition of 56%, SU-ELISA had sensitivity of 86.9% (95% CI: 81.9%, 90.7%) and specificity of 84.4% (95% CI: 75.6%, 90.5%) in discriminating between arthritic and asymptomatic goats.

Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160µg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%.

Plasma hormones, metabolites, milk production, and cholesterol levels in Murrah buffaloes fed with Asparagus racemosus in transition and postpartum period.

Ten dry and pregnant Murrah buffaloes were selected to investigate the effect of Asparagus racemosus feeding on hormones, metabolites, milk yield, and plasma cholesterol levels. The treatment groups of buffaloes were fed with A. racemosus (shatavari) @ 150 g/day/animal during prepartum and @ 300 g/day/animal during the postpartum period. Blood samples collected on -6, -4, -2-week, day of parturition (0), and +2, +4, and +6-week postpartum were analyzed for plasma total cholesterol, triglycerides, HDL, low-density lipoproteins (LDL), prolactin, cortisol, and blood metabolites. Milk samples collected at weekly intervals (+1, +3, +5, and 7 weeks) were analyzed for total milk fat cholesterol. Prepartum plasma cholesterol concentrations were significantly higher in treatment group over the control (P < 0.05). Mean plasma triglycerides, LDL cholesterol, HDL cholesterol, glucose, and nonesterified fatty acid (NEFA) levels varied nonsignificantly between groups. Plasma prolactin and cortisol concentrations were significantly (P < 0.01) more in treatment group than in control group. On day of parturition, plasma prolactin, cortisol, LDL, and plasma total cholesterol were higher (P < 0.01) in treatment group buffaloes in comparison to control group. A. racemosus feeding significantly (P < 0.01) increased plasma prolactin, cortisol (P < 0.01), and milk fat cholesterol (P < 0.05) without affecting total cholesterol, HDL, LDL, glucose, and NEFA concentrations. The buffaloes of treatment group produced more milk (@ 0.526 kg/animal/day) suggesting thereby that A. racemosus is galactopoietic. It was concluded that feeding of A. racemosus increases plasma prolactin and cortisol and decreased plasma total cholesterol and LDL concentration.