Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia

The sensitivities and specificities of an absorbed enzyme-linked immunosorbent assay (ELISA) and an agar-gel immuno-diffusion (AGID) test for the detection of Johne’s disease in sheep were estimated using data from six known infected and 12 assumed uninfected sheep flocks. Sensitivities were estimated for all histologically positive sheep, as well as by histological lesion score, lesion type (paucibacillary or multibacillary) and sheep body-condition score, with ELISA sensitivities estimated at 95 and 99% specificity. Logistic-regression analysis was used to test for significant effects of lesion score and condition score, with flock included in the model as a random effect.

Estimated specificities were 95% (95% CI: 93.4, 95.6%) and 99% (98.4, 99.4%) for ELISA cut-point ratios of 2.4 and 3.6, respectively, and 100% (99.7, 100.0%) for the AGID. Estimated sensitivities for all infected sheep were 41.5% (35.0, 48.3%), 21.9% (16.6, 27.9%) and 24.6% (19.1, 30.7%) for ELISA cut-point ratios of 2.4 and 3.6 and for AGID, respectively. Sensitivities of all tests and cut-points varied significantly between flocks and between categories of lesion score and condition score. Sensitivity ranged from 25 to 73, 10 to 47 and 9.2 to 63% between flocks, for the ELISA with cut-points of 2.4 and 3.6, and for the AGID, respectively. Sensitivity was highest in thin sheep and in sheep with multibacillary lesions. The effects of lesion type and condition score on test sensitivity were significant in the logistic regressions for the AGID and ELISA at both cut-points and the flock effect was significant for the AGID but not for the ELISA at either cut-point.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Evaluation of the gamma interferon ELISA in sheep subclinically infected with Mycobacterium avium subspecies paratuberculosis using a whole-cell sonicate or a johnin purified-protein derivative.

The aim of the study reported here was to estimate the sensitivity and specificity of the gamma interferon (IFN-gamma) ELISA for paratuberculosis in sheep using receiver-operating characteristic analysis. Bacteriologic culture of tissues was used to define the reference positive population (n = 33). Two reference negative populations were used: culture-negative sheep from infected flocks (n = 77), and sheep from noninfected flocks (n = 358). We also evaluated the accuracy of 2 Mycobacterium avium subspecies paratuberculosis (MAP) antigen preparations, a whole-cell sonicate (MpS) and a johnin purified-protein derivative (PPDj). The source of the reference negative sheep used in the analysis affected overall accuracy of the IFN-gamma ELISA. The area under the curve was 0.683 (95% confidence interval 0.574-0.787), using culture-negative sheep from infected flocks, was 0.831 (0.764-0.889), using sheep from noninfected flocks for the MpS, and was 0.809 (0.726-0.881) and 0.897 (0.862-0.925) for the PPDj, respectively. Using the MpS, the cut point that classified the most sheep correctly was an optical density reading of 0.20, for sensitivity of 40.7% (19.4-57.6) and specificity of 88.7% (77.0-95.7) or 97.6% (93.04-99.5), depending on the reference negative population used. Using the PPDj, the cut point that classified the most sheep correctly was 0.25 for sensitivity of 66.7% (47.2-82.7) and specificity of 93.5% (85.5-97.9) or 98.3% (96.4-99.4), respectively. The PPDj was more accurate at identifying MAP-infected sheep than was the MpS (P = 0.034).     

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.Abbreviations AGID agar gel immunodiffusion - bp base pair - ELISA enzyme-linked immunosorbent assay - FBS fetal bovine serum - IB immunoblot - kD kilodalton - OLV ovine lentivirus - MEM minimal essential medium - p.i. post-infection - r recombinant - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TM transmembrane - WV whole virus     

Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Brucella ovis in infected flocks.

The aim of this work was to compare the performance of 6 serological tests using outer or internal antigens from Brucella for the diagnosis of Brucella ovis infection in sheep in an endemic area. Outer membrane antigens included a hot saline extract (HS) and the rough lipopolysaccharide (R-LPS) from B. ovis. Internal antigens were LPS-free total cytosolic proteins (CP) and an 18-kDa cytosolic protein (p18) from Brucella spp. Sera from 200 sheep from naturally infected flocks were assayed by agar gel immunodiffusion test (AGID) and by complement fixation test (CFT), both using HS, and by 4 ELISA using HS, R-LPS, CP, and p18, respectively. The percentage of positive results was 45.5% for ELISA with HS, 42.0% for ELISA with p18, 39.5% for CFT, 33.5% for ELISA with R-LPS, 29.0% for ELISA with CP, and 18.0% for AGID. Taking CFT as the reference test for calculating relative test parameters, the ELISA with HS had the best sensitivity (96.2%), while AGID and the ELISA with R-LPS had the best specificity (96.6%). The ELISA with CP was not more sensitive than the ELISA with p18 (67.1% vs. 79.7%) in spite of the higher number of antigens in CP. The lower relative sensitivity of tests using internal antigens might reflect a lack of antibodies to cytosolic proteins in some infected animals or a shorter persistence of these antibodies relative to antibodies to outer membrane components after recovery from infection.     

Skin test and gamma interferon enzyme-linked immunosorbent assay results in sheep exposed to dead Mycobacterium avium subspecies paratuberculosis organisms.

Cell-mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the Johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While these CMI tests tend to be relatively specific in noninfected flocks, in MAP-infected flocks, these tests often identify animals that cannot be confirmed infected by any other reference test, including necropsy and culture. The aim of this study was to determine if antigen exposure by inhalation or oral ingestion of killed MAP organisms would cause a detectable CMI response in sheep. Forty-eight lambs 4 months of age were randomly divided into a control group, an orally exposed group (dosed with 1 x 10(10) autoclaved MAP organisms 3 times), and an inhalation-exposed group (dosed once with 1 x 10(5) dead organisms). Lambs were skin tested and/or bled pre-exposure and 1, 2, 3, 4, and 12 months postexposure. No significant difference was seen with either the oral- or inhalation-exposed groups of lambs versus controls with either the IFN-gamma ELISA or the skin test at any time pre- or postexposure. These results suggest that infection/invasion of MAP organisms must occur in order to have a positive skin test or IFN-gamma ELISA beyond the false-positive rate. Simple exposure is not enough to elicit a detectable CMI response.     

Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without Mycobacterium paratuberculosis infection

The present study was designed to evaluate a commercial ELISA kit (Institut Pourquier) for the diagnosis of ovine and caprine paratuberculosis under Australian conditions and to compare its accuracy with the existing AGID test. The sensitivity of the ELISA in sheep and goats was 34.9% and 56.4%, with a specificity of 98.8% and 100.0%, respectively. Sensitivity of AGID was 13.8% for sheep and 39.5% for goats, with specificity of 100.0% for both species. The sensitivity of the ELISA in sheep depended on the category of histological lesions. AGID and ELISA were conditionally independent, and appeared to detect overlapping but distinct subgroups of infected animals. The ELISA was significantly more sensitive than the AGID. The ELISA was simple to perform, robust and repeatable. Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches

The diagnostic accuracies of the modified agglutination test (MAT) and indirect ELISA test for the detection of serum antibodies against Toxoplasma gondii in sheep were evaluated through Bayesian approaches on two populations of sheep created from three different groups of animals (T. gondii-aborted ewes, colostrums-deprived newborn lambs, and ewe-lambs and adult ewes with unknown T. gondii infection status). Tests showed a high degree of agreement (kappa statistic = 0.93; 95% confidence interval = 0.87, 0.98) and a significant specificity (Sp) correlation (gamma(Sp) = 0.26; 95% credibility interval = 0.017, 0.61). When prior information was used for all unknown parameters the posterior medians for the sensitivity (Se) and Sp of the MAT and ELISA were, respectively, 92.6% (95% credibility interval = 85.2, 96.9), 95.5% (89.9, 98.7), 90.5% (83.4, 95.6), and 97.8% (94.2, 99.5). These estimates remained similar when uninformative priors were included. The Se estimates of the MAT and ELISA were higher than those obtained on pigs in other study using the same approach (Se = 80.6% and Sp = 89.5% for the MAT, and Se = 71.5% and Sp = 85.5% for the ELISA [Georgiadis, M.P., Wesley, O.J., Gardner, I.A., Singh, R., 2003. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl. Stat. 52, 63-78]. This finding supported the believe that test performances may vary when applied on different animal species. Thus, if these tests are planned to be used on animal species other than sheep or pigs, their diagnostic accuracy should be re-assessed to prevent biased inferences from their results.     

Evaluation of three serological tests for diagnosis of Maedi-Visna virus infection using latent class analysis

Maedi-Visna virus (MVV) infection in sheep is present in several European countries, including Norway. The current Norwegian surveillance and control programme for MVV infection uses three serological tests: an agar gel immunodiffusion test (AGID) and two commercially available indirect ELISAs (Institut Pourquier, P-ELISA and HYPHEN BioMed, H-ELISA). From 18 flocks with suspected or confirmed MVV infection, sera from naturally infected sheep were obtained, and sensitivity (Se) and specificity (Sp) of the three tests were estimated in absence of a perfect reference test using latent class models in a Bayesian analysis. The AGID had higher Sp (95% posterior credibility interval (PCI) [98.4; 99.9]) than either ELISA (95% PCI: P-ELISA, [95.1; 99.0]; H-ELISA, [91.4; 96.6]), but much lower Se (95% PCI: AGID, [41.4; 59.8]; P-ELISA, [92.7; 100.0]; H-ELISA, [90.9; 99.4]). Currently the P-ELISA is used for screening and positive samples are subsequently confirmed by a setup using all three tests in a serial reading. The Se and Sp of the serial interpretations with and without the H-ELISA were estimated. The results suggested that the H-ELISA could be dropped as a confirmatory test as the Se of the three test serial reading was reduced significantly without adding a significant improvement of the Sp compared to the serial reading of the P-ELISA and AGID alone. However, the perceived cost of false positives versus false negatives will influence this decision. Estimates of the predictive values for the tests and combinations suggested that the P-ELISA is a good choice of screening, but confirmatory tests are needed to achieve acceptable levels of positive predictive values.     

Bovine leukemia virus infection in Taiwan: evaluation of the enzyme-linked immunosorbent assay and agar gel immunodiffusion test.

I evaluated an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test simultaneously for the detection of bovine leukemia virus (BLV) antibodies. Total 1,293 serum samples were tested for ELISA and AGID test and the results were compared. The results of ELISA and AGID agreed by 1,156 out of 1,293 (89.4%). All of AGID-positive 356 sera were positive by ELISA. However, of 451 ELISA-positive sera, 95 sera were either negative or equivocal by AGID test. Eleven animals which showed ELISA-positive but AGID-negative or equivocal became AGID-positive in a year. It may be inferred that ELISA detects infected cattle earlier and with greater sensitivity than AGID.     

Evaluation of a serological test (indirect ELISA) for the diagnosis of sarcoptic mange in red foxes (Vulpes vulpes)

Sarcoptic mange occurs in many parts of the world and is common in populations of domestic and wild canids, including red foxes (Vulpes vulpes). In recent years, an indirect antibody enzyme-linked immunosorbent assay (ELISA), with higher sensitivity and specificity than traditional diagnostic methods, has been successfully applied in the diagnosis of sarcoptic mange in dogs. The same ELISA has also demonstrated specific antibodies to Sarcoptes scabiei in experimentally infected red foxes. The aim of this study was to evaluate the indirect ELISA when used to detect antibodies to S. scabiei in field sera from Swedish red foxes. One cohort of both infected and non-infected red foxes (cohort 1; n = 88), and one cohort of apparently non-infected foxes (cohort 2; n = 67) were examined for skin lesions and presence of S. scabiei by thorough visual examination at autopsy and skin scrapings. Samples of blood-tinted body liquid from the abdomen or thorax cavity were collected and analysed by the indirect ELISA. The relative sensitivity and specificity of the ELISA at different cut-offs (OD values) were estimated by comparing the test results to the infection status as determined by examination and skin scrapings. The highest combination of relative sensitivity and specificity, calculated based on cohort 1, was 95.4 and 100.0%, respectively. These estimates were constant for cut-offs 0.150-0.225, which included the cut-off based on the mean plus three standard deviations of test results from cohort 2 (0.165). It is concluded that this test can be useful in diagnosis and epidemiological studies of S. scabiei infection in red foxes.     

Comparison of four diagnostic tests for the identification of serum antibodies in small ruminants infected with Mycoplasma agalactiae

AIM: To determine the diagnostic capability of a newly developed Western blot (WB) assay for the detection of serum antibodies against Mycoplasma agalactiae compared with conventional serological tests, and to identify the best test for routine diagnostic use. METHODS: The serological test methods used were: two commercial indirect enzyme-linked immunosorbent assays (ELISA), viz ELISA-1, using a bacterial antigen preparation, and ELISA-2, using a recombinant protein (lipoprotein p48) antigen; the complement fixation test (CFT); and a newly developed WB assay, the latter both using a bacterial antigen preparation. Thirty sera from goats infected with M. agalactiae and 97 sera from non-infected sheep were tested using all four methods. RESULTS: Staining patterns in the WB were quite variable. An immuno-dominant band of 41 kDa was detected in 63% of sera from infected animals. The same band also appeared, although mostly very weakly, in 10% of sera from non-infected animals. When suspicious or very weak reactors were omitted, the diagnostic sensitivity (DSE) and diagnostic specificity (DSP), respectively, for the four assays were: WB=56.7%, 97.9%; ELISA-1=76.7%, 99.0%; ELISA-2=56.7%, 100%; and CFT=40.0%, 94.8%. CONCLUSIONS: ELISA-1 performed best in this comparison. While the WB can be used, it did not have a technical advantage over the ELISA. The CFT should be discouraged as the primary screening method for contagious agalactia and should be replaced by ELISA-1. Results from this study confirm that serological test methods for contagious agalactia are useful for the detection of infected flocks but will not detect every individual infected animal.     

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.     

Serological differentiation of Brucella-vaccinated and -infected domesticated animals by the agar gel immunodiffusion test using Brucella polysaccharide in mongolia

To investigate Brucella infection in cattle, sheep, goat, reindeer and yak in Mongolia, serological reactions of Brucella-infected and -vaccinated domestic animals were compared by the agar gel immunodiffusion (AGID) test with a polysaccharide (poly-B) of the B. Abortus strain S-19. The sensitivity and specificity were compared with conventional serological tests that are commonly used in Mongolia, such as the rose Bengal test, the tube agglutination test and the compliment fixation test. A total of 73.3, 100, 100, 95.8 and 61.9% of the sera from suspected cattle, yak, goat, sheep and reindeer, respectively, that were positive in the compliment fixation test, were also positive in the AGID test. Sera from vaccinated cattle, sheep and goat were positive over 90% by conventional tests 3 months after vaccination, but were negative by the AGID. These results suggest that the AGID test may be useful to differentiate infected and vaccinated animals in the field.     

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.     

Elevated concentrations in serum immunoglobulins due to infection by ovine progressive pneumonia virus.

Sixty-seven serum samples were obtained from 2 sheep flocks. Agar gel immunodiffusion (AGID) was used to separate progressive pneumonia virus (PPV)-infected sheep from noninfected sheep by the presence of precipitating antibodies. Immunoglobulin (Ig), total protein, and albumin concentrations were then measured from all 67 sera to determine whether differences existed between PPV-infected and non-infected sheep. A significant difference (P less than 0.0005) was found in both total protein and Ig concentration between PPV-infected and noninfected sheep. This corresponding difference was absent in albumin measurements. The significant differences (P less than 0.0005) in Ig and total protein concentrations were then used to evaluate a field test for diagnosing progressive pneumonia. The possibility of using either total protein or Ig concentrations as a field test was found to be highly unlikely due to variation in individual values.     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.