Detection of antibodies to the Ehrlichia ruminantium MAP1-B antigen in goat sera from three communal land areas of Zimbabwe by an indirect enzyme-linked immunosorbent assay

A total of 1,286 caprine serum samples collected from three communal land areas in Zimbabwe from March 1999 to February 2000 were tested for Ehrlichia ruminantium antibodies using the indirect MAP1-B enzyme-linked immunosorbent assay. Of the 480 samples tested from Mudzi, a non-heartwater area, 425 (89.4 %) were positive. In the heartwater endemic areas, of the 441 samples 352 (79.4 %) from Gwanda and 300 of the 365 samples (83.2 %) from Bikita tested positive. The seroprevalence in the Bikita and Gwanda (approaching 90 %) is consistent with reports in related serological surveys that puts the seroprevalence of E. ruminantium in goats from endemic areas of Zimbabwe at 90 %. However, the high seroprevalence in the non-heartwater area of Mudzi is unexpected and can be a result of the presence of a serologically cross-reacting organism, which has to be isolated and characterized. The results need to be confirmed by alternative tests, based on molecular diagnostic tools. There were no significant differences in seroprevalence between the three sampling areas as there were between the three sampling periods. The highest corresponded with the period January to February (peak tick activity) and the lowest with the period July to September (minimal tick activity).     

Efficacy of a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G₁ that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay.

At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation

     

Comparison of indirect fluorescent antibody test and enzyme linked immunosorbent assay in the detection of exposure of cattle to Theileria parva in Kenya.

Appraisal of the indirect fluorescent antibody test (IFAT) and antigen enzyme linked immunosorbent assay (ELISA) serological tests as carried out to detect cattle exposed to Theileria parva at the National Veterinary Research Centre, Muguga (NVRC), Kenya is reported. Using sera from T. parva naive cattle and cattle experimentally exposed to T. parva, the two tests were appraised in terms of their sensitivity and specificity. IFAT and ELISA had the same sensitivity of 90% while ELISA had a higher specificity (90%) than IFAT (80%). A comparison was also made of the capability of the two tests to detect exposure of dairy cattle to T. parva prior to immunization against East Coast fever (ECF). The positive outcome from the IFAT was significantly higher (chi 2 = 30.36; P < 0.001) than that from the ELISA. The agreement between the two tests was low (Kappa = 0.21). The two tests indicated a higher risk of ECF in the study area than was expected. Indications are that the ELISA has been effectively adopted at NVRC.     

Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bovis

An indirect enzyme linked immunosorbent assay (ELISA) using an unpurified antigen was assessed for its accuracy in detecting Mycobacterium bovis infected cattle. The ELISA test recorded sensitivities of 88.7% and 63%, respectively, for infected cattle tuberculin tested positive and for infected cattle never tuberculin tested. Specificity was determined at 52.6% for cattle from confirmed free herds which had never been tuberculin tested. Significant differences in the mean ELISA values were recorded between the 3 groups. No evidence was found for long term effects of tuberculin testing upon the titre of antibodies detected by the ELISA in unaffected cattle. The indirect ELISA using the unpurified antigen of this assay was considered to be unsuitable as an alternative to tuberculin testing for the detection of M. bovis infected animals.     

The detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum by an enzyme-linked immunosorbent assay

A sensitive and reliable enzyme-linked immunosorbent assay for the detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum nymphae is described. For the screening of antibodies, C. ruminantium from A. hebraeum nymphae, partially purified by wheat-germ lectin affinity chromatography, was used as antigen. To screen nymph populations, sera from either Ball 3 strain-infected sheep or Kumm-strain infected mice were used. By using appropriate controls the assays were rendered specific with respect to C. ruminantium.     

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.     

Comparison of blood polymerase chain reaction and enzyme-linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep.

A study was carried out to compare the performance of enzyme-linked immunosorbent assay (ELISA) and blood polymerase chain reaction (PCR) for diagnosis of paratuberculosis in cattle and sheep. For cattle, a set of 278 samples from 1 paratuberculosis-affected Friesian farm was used; it included 80 ELISA-positive samples and 198 ELISA-negative samples from an age-matched group. Ninety-four samples were from heifers and 184 were from 2-5-year-old cows. The overall analysis showed a clear association (Fisher exact test [FET] P = 0.0049) but a weak negative agreement (45.3%, kappa = -0.1665 +/- 0.0994) between the 2 tests. It reflected a moderate agreement among heifers (87.7%, kappa = 0.4471 +/- 0.2435) and a moderate disagreement among cows (62.7%, kappa = -0.3670 +/- 0.1057). For sheep, 496 blood samples from 53 Latxa dairy flocks were used; 180 of the blood samples were from dam/offspring pairs. The overall association between the 2 tests on ovine samples was strong (FET, P = 0.0005), whereas the agreement was low (kappa = 0.1622 +/- 0.1188). There was slightly better agreement for ewes (kappa = 0.2135 +/- 0.1992) than for lambs (kappa = 0.1193 +/- 0.1301). There was also a highly unlikely proportion of dam/offspring positive results (FET, P < 0.0001, kappa = 0.6269 +/- 0.1854). Four of 6 lambs that were necropsied 1 year after testing had paratuberculosis microscopic lesions in the ileocecal valve (3 lambs) or a PCR-positive result (4 lambs). These results suggest that blood PCR testing might be a potentially useful new approach in paratuberculosis diagnosis, especially in young animals.     

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog

An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia burgdorferi in dog sera. The concordance of the two tests was 93.5% for sera from dogs from Maryland (n = 93), 98.0% for sera from dogs from North Carolina (n = 446), and 97.2% for the combined sample groups (n = 539). Twenty-five of the 27 samples with discordant or low positive results were tested, and showed immunoblot reactions to 1 to 10 different bands. Reaction patterns and intensity of the bands were quite variable, and did not explain a reason for the discordance.     

鸡肌肉组织中氯霉素残留ELISA检测方法的研究

本文利用活化酯法合成了氯霉素抗原 ,作为免疫原免疫新西兰大耳白兔得到氯霉素的多克隆抗体 ,建立了氯霉素间接竞争酶联免疫检测方法 ,进行了药物交叉反应性试验 ,并对鸡肌肉进行添加回收试验。结果显示抗体效价可达 1∶ 6 4 0 0 0 0 ,IC50 为 1.3ng/ ml,最低检测限达到 0 .0 5 ng/ ml,线性检测范围为 0 .1～ 36 .4 5 ng/ ml。在 0 .5、1、2 .5、5 ng/ g浓度水平添加到鸡肌肉组织中 ,测得回收率为 5 5 .4 %～ 119.0 % ,变异系数为 4 .3%～ 10 .4 %。该 EL ISA方法快速、灵敏、方便 ,满足了氯霉素残留检测的要求。     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

用改良的RT-PCR技术快速检测传染性法氏囊病病毒

本研究将最新的病毒核酸纯化技术和单管ＲＴ－ＰＣＲ技术相结合,应用于ＩＢＤＶ的诊断研究,对４０余个病毒样品进行扩增反应,均取得令人满意的效果。该技术灵敏快速,从核酸纯化到电脉检测ＰＣＲ产物只需５～６ｈ,反应灵敏度比传统方法至少提高１００倍。所设计的引物对毒株的适用范围较广,对１２个ＩＢＤＶ毒株和１２个病变法氏囊样品均得到分子量一致并与设计相符的扩增产物     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

Test protocol of an enzyme‐linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus

Summary

In this communication the test procedure is described for an indirect enzyme‐linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus (BLV). Test sera are incubated in polystyrene microtiter plates sensitized with a partly‐purified preparation of BLV. Bovine antibodies are detected with anti‐species immunoglobulin conjugated to the enzyme horseradish peroxidase, followed by the addition of the enzyme substrate.     

Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection.

The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig.     

Comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis

OBJECTIVE: To determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis and examine the correlation between results for an ELISA, indirect immunofluorescent antibody (IFA) test, and polymerase chain reaction (PCR) assay. SAMPLE POPULATION: Blood samples obtained from 90 adult dogs admitted to an animal shelter in eastern Tennessee. PROCEDURE: Serum samples were analyzed for antibodies against E. canis by use of a commercially available ELISA kit, 2 IFA tests, and a PCR assay; testing was performed at the University of Tennessee (TN) and North Carolina State University (NCSU). The PCR amplification was performed by use of DNA extracted from EDTA-anticoagulated blood and primers designed to amplify DNA of Ehrlichia spp. RESULTS: Antibodies against E. canis were detected in only 1 dog by use of the ELISA. By IFA testing at TN, 10 of 90 (11%) dogs were seroreactive against E. canis antigens, all of which had medium to high titers to E. canis. Only 5 of the 10 TN seroreactors were also reactive against E. canis antigens in IFA tests conducted at NCSU, and all 5 had low to medium titers. The DNA of Ehrlichia spp was not amplified in any blood samples by use of PCR assays conducted at the TN or NCSU. CONCLUSIONS AND CLINICAL RELEVANCE: The discordant ELISA, IFA, and PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E. canis, such as E. ewingii.     

Mycobacterium paratuberculosis detection in bovine feces is improved by coupling agar culture enrichment to an IS900-specific polymerase chain reaction assay.

The feasibility of coupling an agar culture enrichment step with gene amplification (ACE-PCR) as a means to improve turnaround time and detect Mycobacterium paratuberculosis (Mpt) in the presence of contaminants was investigated. Fecal samples from 463 Pennsylvania dairy cows were cultured in duplicate sets. One replicate from each set was processed and interpreted according to standard culture (SC) protocol, whereas cultures from the second replicate were harvested at 6 weeks postinoculation; DNA extracts from the harvested material were evaluated by a polymerase chain reaction (PCR) test for the Mpt-specific IS900 gene. One hundred seventy-six of 463 culture sets were positive by either method. One hundred sixty-five of these (94%) were ACE-PCR positive, and 151 (86%) were positive by SC. Eleven SC-positive samples were ACE-PCR negative, and 9 ACE-PCR-positive samples were negative by SC; these discrepancies could be a consequence of a low organism burden (< or = 5 organisms/g) or slow growth rate of Mpt in cultures of these samples. One hundred thirty-nine of 463 culture sets (30%) were reported as inconclusive because of culture contamination according to SC protocol; 16 of these (11.5%) were ACE-PCR positive. Seventy-four ACE-PCR-positive sets (42% of all positives) were negative or inconclusive by SC at 6 weeks postinoculation. Agar culture enrichment prior to IS900 PCR testing significantly improves Mpt culture turnaround time and sensitivity.     

Molecular assay for the detection of Cochlosoma anatis in house flies and turkey specimens by polymerase chain reaction

A 1520 bp region of Cochlosoma anatis mtDNA 16S gene was subjected to DNA sequencing and a 466 bp portion was compared with other protozoan 16S sequences to develop PCR primers specific for C. anatis. This PCR diagnostic method allowed identification of C. anatis from house flies, Musca domestica L., turkey gut, and fecal samples within 6 h after field-collected samples reached the laboratory. House flies detected carrying C. anatis using the diagnostic 374 bp amplicons represented the first record of this protozoan in house flies.     

Evaluation of a quantitative enzyme-linked immunosorbent assay for feline leukemia virus p27 antigen and comparison to proviral DNA loads by real-time polymerase chain reaction

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. While higher viral RNA and proviral DNA loads have been correlated with progressive infections and disease, a similar correlation has been suggested for p27 antigen concentrations. This analytical study compared the results of a quantitative ELISA for p27 antigen with quantitative real-time PCR results for FeLV proviral DNA in patient samples. A significant positive correlation between copies of proviral DNA and the concentration of p27 antigen was identified (r = 0.761, P < 0.0001). Samples with high proviral DNA loads, at least 1 × 10⁶ copies/mL of whole blood, typically had p27 antigen concentrations greater than 30 ng/mL in plasma. Samples with proviral DNA loads below this level all had concentrations of p27 antigen in plasma that were less than 10 ng/mL. Given this correlation, it is hypothesized that the concentration of p27 antigen at a given point in time may help to indicate the likelihood of a progressive or regressive infection similar to what has been demonstrated for proviral DNA loads.