Commercial Enzyme-linked Immunosorbent Assay Test Research Articles

Equine Infectious Anemia diagnosis in a 3 months old foal

within the insect, the infectious virus can remain in its mouthparts for several hours after a bite. Several studies have shown that EIAV infection has occurred among horses in North and South America, France, Germany, Italy and Romania. So far no evidence of the presence of EIAV in horses was reported in Serbia. In order to determine EIA prevalence among the 5000 horses of the Vojvodina region of Serbia, 316 horse sera have been collected during 2013 and 2014. Serological analysis was performed using Agar Gel Immuno Diffusion test (AGID) as described by the World Organization of Animal Health (OIE) chapter 2.5.6 and by Enzyme Linked Immuno Sorbent Assay (ELISA). Those sera have been tested with 3 different commercial AGID test (Idexx, ID-Vet and VMRD) and two different commercial ELISA tests (Synbiotics and VMRD). With the three AGID kit 311 (98,4%) among the 316 tested sera were found negative and only five (1,6%) sera were found positive for EIA. With ELISA kits results were slightly different and were dependent of the kit producer. Indeed, Synbiotics ELISA gave the same results as AGID tests but VMRD ELISA found 295 negative samples, 5 positive samples and 16 samples were doubtfull. Our study shows for the first time that EIA is present in Serbia and more specifically in the Vojvodina region. Euthanasia and viral characterization of field isolate collected in Serbian horses will be investigated.

Analysis of the large intestinal and faecal microbiota of horses with grass sickness using denaturing gradient gel electrophoresis

within the insect, the infectious virus can remain in its mouthparts for several hours after a bite. Several studies have shown that EIAV infection has occurred among horses in North and South America, France, Germany, Italy and Romania. So far no evidence of the presence of EIAV in horses was reported in Serbia. In order to determine EIA prevalence among the 5000 horses of the Vojvodina region of Serbia, 316 horse sera have been collected during 2013 and 2014. Serological analysis was performed using Agar Gel Immuno Diffusion test (AGID) as described by the World Organization of Animal Health (OIE) chapter 2.5.6 and by Enzyme Linked Immuno Sorbent Assay (ELISA). Those sera have been tested with 3 different commercial AGID test (Idexx, ID-Vet and VMRD) and two different commercial ELISA tests (Synbiotics and VMRD). With the three AGID kit 311 (98,4%) among the 316 tested sera were found negative and only five (1,6%) sera were found positive for EIA. With ELISA kits results were slightly different and were dependent of the kit producer. Indeed, Synbiotics ELISA gave the same results as AGID tests but VMRD ELISA found 295 negative samples, 5 positive samples and 16 samples were doubtfull. Our study shows for the first time that EIA is present in Serbia and more specifically in the Vojvodina region. Euthanasia and viral characterization of field isolate collected in Serbian horses will be investigated.

Monitoring influenza A virus infection in pigs by using a competitive enzyme-linked immunosorbent assay to detect virus antibodies in pen-based oral-fluid specimens

Objective: To investigate monitoring an influenza A virus (IAV) infection in a finishing pig herd by testing pen-based oral fluids for antibodies against the virus using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Materials and methods: Oral fluids were collected weekly from pigs 12 to 24 or 22 weeks of age in four pens (approximately 25 pigs per pen) in two consecutive batches. Serum samples were also collected from two randomly selected pigs in each pen at 12, 16, and 20 weeks of age in both batches and at 24 weeks in Batch 1 only. Oral-fluid and serum samples were tested for antibodies against IAV by a commercial competitive ELISA test kit. Oral fluids were also tested for IAV by reverse transcription-polymerase chain reaction (RT-PCR). Results: One week after initial detection of IAV in oral-fluid samples by RT-PCR, antibodies against the virus were detected in oral fluids as well as in serum samples. Oral fluids continued to test positive for antibodies 4 to 7 weeks after initial detection of virus, but with a decreasing trend in the amounts of virus antibodies detected by ELISA. All samples in Batch 1 tested negative after 9 weeks. Implications: The longitudinal profile of antibodies against IAV detected in oral fluids promises to be a useful tool for monitoring IAV infection in a pig population. A commercial competitive ELISA test kit could easily be adapted for oral fluids by modifying dilution of the specimen.

Multiple circulating infections can mimic the early stages of viral hemorrhagic fevers and possible human exposure to filoviruses in Sierra Leone prior to the 2014 outbreak.

Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.

Cross-Reactivity of Antibodies with Phenolic Compounds in Pistachios during Quantification of Ochratoxin A by Commercial Enzyme-Linked Immunosorbent Assay Kits

Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Analysis of the levels of tumour necrosis factor (TNF), autoantibodies to TNF, and soluble TNF receptors in patients with rheumatoid arthritis

Objectives: To evaluate the potential contribution made by autoantibodies against tumour necrosis factor (TNF) to the pathogenesis of rheumatoid arthritis (RA).Method: We used affinity chromatography methods and a magnetic separation procedure to purify human autoantibodies specific to TNF. The autoantibodies were used as calibration material to determine the absolute content of autoantibodies to TNF using an enzyme-linked immunosorbent assay (ELISA). TNF content and the levels of soluble TNF receptors types I and II (sTNF-RI and sTNF-RII) were determined using commercial ELISA test kits.Results: We demonstrated significant increases in the levels of TNF, sTNF-RI, and sTNF-RII in the sera of patients with acute RA and in patients with RA who had responded positively to therapy compared with healthy controls. Levels of autoantibodies of the immunoglobulin (Ig)G2, IgG3, and IgG4 subclasses were significantly higher in sera from patients with acute RA than in sera from healthy controls. Level of autoantibodies of the IgG2 subclass were significantly higher in sera from patients with acute RA than in RA patients who had responded positively to therapy.Conclusions: Acute RA is associated with changes in levels of TNF and soluble receptors for TNF and also in levels of autoantibodies to TNF. Given the magnitude of the changes in levels of different subclasses of autoantibodies to TNF, we propose that these autoantibodies might contribute to the pathogenesis of RA.

Detectability of Lupine Seeds by ELISA and PCR May Be Strongly Influenced by Potential Differences between Cultivars

Accurate methods for allergen detection are needed for the verification of allergen labeling and the avoidance of hidden allergens. But systematic data on the influence of different cultivars of allergenic crop species on their detectability in enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are lacking. As one example, seeds of 14 different cultivars of lupine (Lupinus albus, Lupinus angustifolius, Lupinus luteus) were investigated for total protein according to a Kjeldahl method, and for their relative quantitative detectability in three commercial lupine-specific ELISA tests and four lupine-specific PCR methods. Total Kjeldahl nitrogen allowed an accurate quantification of total protein. Relative differences in quantitative response between cultivars of 390-5050% and 480-13,600% were observed between ELISA kits and PCR methods, respectively. Hence, quantitative results of selected ELISA and PCR methods may be strongly influenced by the examined lupine cultivar.

A Multi-Analyte Assay for the Non-Invasive Detection of Bladder Cancer

Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.

Detection of Mycoplasma gallisepticum in experimentally infected broiler chickens using Culture, SPA, ELISA, and PCR methods

Avian mycoplasmosis is caused by several pathogenic mycoplasmas of which Mycoplasma gallisepticum and Mycoplasma synoviae are the most important. This experiment was designed to study the diagnosis of M. gallisepticum infection in broiler chicken receiving M. gallisepticum. One hundred and twenty one-day-old chickens were raised in separate isolated rooms and equally allocated into two groups and renamed groups A and B were 121-day-old chickens. At 3 weeks old, group A was considered as the control group while group B was challenged with M. gallisepticum containing 1 × 106 CFU/bird via an eye drop. In this study, culture, serum plate agglutination (SPA) test, the commercially available enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) tests were compared for the detection of M. gallisepticum infection during the 1–7 weeks of age. Results revealed that M. gallisepticum was found in group B using culture and PCR during the 4–7-week-old period. The SPA results showed positive reactors in group B during the 5–7-week-old period. The positive reactors were detected by commercial ELISA test kit from 6 to 7 weeks of age. In this study, sensitivity of the PCR method was higher as compared to culture and serological tests. Its high sensitivity and fast turnaround time is making it the most frequently used test to monitor M. gallisepticum infection beside other tests.

Quantitative Detection of Human Tumor Necrosis Factor α by a Resonance Raman Enzyme-Linked Immunosorbent Assay

Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3',5,5'-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.

Progress towards measles and rubella elimination in Tuscany, Italy: the role of population seroepidemiological profile

As a part of the National Plan for Measles and Congenital Rubella Elimination, a catch-up campaign targeting children aged 7-14 years with Measles-Mumps-Rubella (MMR) vaccine was conducted during 2004-05 in Tuscany, Central Italy. To assess the profile of measles and rubella susceptibility, immunoglobulin G antibodies against measles (945 subjects) and rubella (1110 subjects) were detected using two commercial enzyme-linked immunosorbent assay tests in sera from a population aged 1-49 years. Overall immunity towards measles increased in the last years, but the WHO-Euro recommended threshold of susceptibility for elimination was attained only in 2- to 4-year-olds. All fertile women up to 29 years had rates of susceptibility clearly higher than those required by WHO-Euro (5%) to eliminate congenital rubella. Despite the reduction of susceptibility among subjects targeted by the catch-up campaign, continuous efforts to increase immunization coverage are needed to eliminate measles and rubella circulation. Our results are predictive of a high chance of measles and rubella outbreaks because of the accumulation of susceptible individuals. Additional catch-up vaccination strategies targeting the adult population (particularly fertile women) are strongly needed to eliminate the risk of measles and congenital rubella syndrome for future generations.

Seroepidemiology of bovine herpesvirus 1 (BHV1) infection among Estonian dairy herds and risk factors for the spread within herds

The objectives of this study were to reassess the herd level and within-herd prevalence of bovine herpesvirus 1 (BHV1) infection in Estonian dairy cattle, estimate the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for bulk tank milk (BTM) testing and determine the risk factors related to high prevalence of the infection in herds. To estimate the herd prevalence, BTM samples from each of the 1,205 herds that sell milk to dairy companies were analysed for BHV1 antibodies. One hundred and three herds with known BHV1 infection status were selected to estimate within-herd prevalence and to calculate the sensitivity and specificity of BTM ELISA. In these herds serum samples were collected from cows and youngstock, together with BTM samples. A commercial blocking ELISA test was used to analyse samples for antibodies against BHV1. A questionnaire was completed to collect herd data. The sensitivity and specificity of the BTM ELISA were 76.5% and 97.2%, respectively, and the true herd prevalence of BHV1 was calculated to be 22.0%. The herd prevalence increased significantly with herd size, being 3.4% in the smallest category (less than 20 cows) and 85.7% in herds of size over 400. The mean within-herd prevalence was 37.8% (range 1–100, median 31.5). The mean within-herd prevalence increased with herd size. Data from 59 infected herds was used to determine the risk factors associated with high within-herd prevalence (>50%) of BHV1, using logistic regression analysis. As, in some infected herds, the youngstock were uninfected, risk factors for the presence of BHV1 among youngstock from 6 months until calving were analysed. The results indicate the importance of iatrogenic spread of the virus, since the overall within-herd prevalence was higher in those herds in which a veterinarian was an employee of the farm and an inseminator worked only for the particular farm. The presence of bovine viral diarrhoea virus (BVDV) in a herd was associated with a higher prevalence of BHV1.

Effect of Heat Treatment on the Quantitative Detection of Egg Protein Residues by Commercial Enzyme-Linked Immunosorbent Assay Test Kits

This study examined the changes in the solubility of egg proteins as affected by different heat treatments and compared the performances of three commercial test kits for the quantitation of protein residues in heat-treated samples. National Institute of Standards and Technology (NIST) whole egg standard reference material #8415 and Henningsen spray-dried whole egg powder were subjected to heating in the presence of water at 60 and 100 degrees C, autoclaving for 5 or 10 min, or dry heating at 60-400 degrees C for 10 min. The amount of protein in the heated samples was assayed using the bicinchoninic acid total protein assay as well as egg-specific commercial enzyme-linked immunosorbent assay (ELISA) kits. Elevated heat resulted in a lower level of proteins extracted. Neogen's Veratox kit, which is reactive to multiple proteins in egg, greatly underestimated the amount of residual proteins in the boiled or autoclaved samples. Tepnel BioSystems' Biokits assay, which employs antibodies specific to a heat-stable marker protein (ovomucoid), registered a higher level of protein in these samples. Both test kits substantially underestimated the amount of residual proteins in samples dry-heated at temperatures >176 degrees C. The Morinaga test, using an improved extraction buffer, registered the highest level of protein in the heat-treated NIST samples but not the Henningsen samples. The underestimation by the commercial test kits was attributed to changes in the immunoreactivity of residual proteins after heat treatments and not the differences in the amount of protein extracted. These results suggest that thermal processing may affect the quantitative analysis of allergens and needs to be taken into account in the validation of commercial ELISA test kits.

Application of an enzyme-linked immunosorbent assay for the detection of clenbuterol residues in swine urine and feeds

The present study outlines applications of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol residues. Antisera were raised from rabbits immunized with diazotized clenbuterol-bovine serum albumin (BSA) conjugate. The assay was specific to clenbuterol with a half-maximum inhibition concentration (IC50) of 1.8 ng/mL and 2.5 ng/mL in blank swine urine and phosphate buffer solution, respectively. The assay had high cross-reactivity (86%) with mabuterol, but low with other adrenergic agonists and antagonists. The average recovery of clenbuterol, as measured with the ELISA, ranged from 90% to 112% in swine urine samples and from 86% to 95% in feeds, respectively. This new assay was compared with commercial ELISA test kits. An excellent correlation (r 2 = 0.98) between the two methods and satisfactory recoveries suggest that the new assay can be suitable for the determination of clenbuterol residues in real samples. The assay was used to analyze clenbuterol residues in 103 swine urine samples and 68 feed samples collected from northern China. Approximately 50% of the urine samples and 25% of the feed samples analyzed were found positive (concentration of clenbuterol ≥ 1 ppb). The results indicate that clenbuterol was misused in some of the areas surveyed.

Evaluation of a New Antibody-Based Enzyme-Linked Immunosorbent Assay for the Detection of Bovine Leukemia Virus Infection in Dairy Cattle

The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collected from 524 adult Holstein cows originating from 6 dairy herds in Central Argentina. The M108 and S108 were compared with agar gel immunodiffusion (AGID), polymerase chain reaction and a commercial ELISA. Because there is currently no reference test capable of serving as a gold standard, the test sensitivity (SE) and specificity (SP) were evaluated by the use of a latent class model. Statistical inference was performed by classical maximum likelihood and by Bayesian techniques. The maximum-likelihood analysis was performed assuming conditional independence of tests, whereas the Bayesian approach allowed for conditional dependence. No clear conclusion could be drawn about conditional dependence of tests. Results with maximum likelihood (under conditional independence) and posterior Bayes (under conditional dependence) were practically the same. Conservative estimates of SE and SP (with 95% confidence intervals) for M108 were 98.6 (96.7; 99.6) and 96.7 (92.9; 98.8) and for S108 99.5 (98.2; 99.9) and 95.4 (90.9; 98.1), respectively. The ELISA 108 using either milk or serum to detect BLV-infected animals had comparable SE and SP with the official AGID and a commercial ELISA test, which are currently the most widely accepted tests for the serological diagnosis of BLV infection. Therefore, ELISA 108 can be used as an alternative test in monitoring and control programs.

A Multilaboratory Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Test for the Detection of Antibodies against Mycobacterium Avium Subsp. Paratuberculosis in Cattle

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

The seroprevalence of Johne's disease in Georgia beef and dairy cull cattle.

Beef and dairy cattle serum samples, collected during 2000 at sale barns throughout Georgia, were obtained from the Georgia State Brucellosis Laboratory and were used to conduct a retrospective epidemiological study. Statistical samplings of 5,307 sera, from over 200,000 sera, were tested for antibodies to Mycobacterium avium ssp. paratuberculosis, (Johne's disease) using a commercial enzyme-linked immunosorbent assay test kit. An overall period seroprevalence in all classes of cattle tested was 4.73%. The period seroprevalence in dairy cattle was 9.58%, in beef cattle it was 3.95%, and in cattle of unknown breed it was 4.72%. It was concluded that the seroprevalence of Johne's disease in cull beef and dairy cattle in Georgia is economically significant.

An estimated prevalence of Johne's disease in a subpopulation of Alabama beef cattle.

The objective of this study was to estimate the overall prevalence of animals that were infected with Mycobacterium avium ssp. paratuberculosis in a subpopulation of Alabama beef cattle. This was determined using a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of M. avium ssp. paratuberculosis-specific antibodies in serum. Serum was collected from 79 herds that were participating in the Alabama Brucellosis Certification program. A total of 2,073 beef cattle were randomly tested by selecting 30 animals per herd in herds greater than 30 and selecting all animals in herds 30 and less for testing. It has been estimated that the commercial ELISA test used has a 60% sensitivity and a 97% specificity. Of the 79 herds tested, 29 herds were seronegative, 24 herds had 1-2 positive animals, and 26 herds had 3 or more seropositive animals. The average number of infected animals per positive herd was 3.3. In addition, a calculated minimum of 53.5% of the herds were identified as Johne's positive herds with a 95% confidence level. Of the total number of animals tested, 8.0% (166/2,073) of them were positive by the ELISA. After adjustments for test sensitivity and specificity and the proportion of animals sampled per herd, the true prevalence was calculated to be 8.75%. These data suggest that approximately 50% of the herds are infected with M. avium ssp. Paratuberculosis, and the overall prevalence of infection in Alabama beef cattle is approximately 8%, which correlates with other previously published regional estimates.

Evaluation of sample preparation for control of chloramphenicol residues in porcine tissues by enzyme-linked immunosorbent assay and liquid chromatography

The effects of sample preparation on the determination of chloramphenicol (CAP) residues by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography (LC) were investigated. First, the biological matrices were treated with extraction solutions, which were mixtures of acetate buffer and acetonitrile. The isolation of CAP from previously spiked samples was performed by homogenization or ultrasonication. For clean-up, the suitability of solid phase extraction columns (in reverse phase (RP) or mixed mode) was compared. The level of CAP in tested samples was determined by a commercial ELISA test (R-Biopharm) and LC with UV detection at 270 nm, after separation on a RP column. It was found that the best isolation from interfering, endogenous compounds was obtained after homogenization of sample with a mixture of acetate buffer (pH 5.0) and acetonitrile (2:8, v/v). The clean-up of the sample extracts in a mixed mode rather than reverse phase was more efficient. The recovery of CAP from spiked pig muscle was 82%, and from kidney, 85%. The same analytical protocol is suitable for CAP screening in animal tissues by ELISA as well as by LC.