Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein

BACKGROUND: Growth hormone (GH) has been shown to promote wound healing and to improve protein metabolism in burned patients. Through immunomodulation, GH has also protected rats infected with Salmonella typhimurium and mice infected with Escherichia coli. In spite of advances in the management of patient care for those with thermal injuries, high mortality rates of burned patients as a result of infections are of special concern. An improvement in the resistance of burned patients to certain infections will make the beneficial role of GH very clear. In this study, therefore, the immunomodulating effects of recombinant human GH (rhGH) in thermally injured mice exposed to opportunistic herpesvirus infections were investigated. METHODS: (1) Burned mice, exposed to herpes simplex virus type 1 (HSV-1), were treated subcutaneously with rhGH (4 mg/kg) and observed for 21 days to determine the protective antiviral effect of rhGH. (2) Because of reports describing a lack of interferon-gamma (IFN-gamma) responsiveness in burned mice, the IFN-gamma-producing ability of the splenic mononuclear cells (SMNC) from burned mice treated with rhGH was examined. (3) Because the generation of burn-associated suppressor macrophages that can inhibit the IFN-gamma production by SMNC has been previously described, the suppressor cell activities of macrophages from burned mice treated with rhGH were examined. RESULTS: After exposure to lethal amounts of HSV-1, mice treated with rhGH displayed a reduced mortality rate compared with control mice treated with saline. SMNC from burned mice treated with rhGH produced IFN-gamma, whereas this cytokine was not produced by SMNC from burned mice treated with saline. Also, an inhibition of the generation of burn-associated suppressor macrophages was displayed in burned mice treated with rhGH. CONCLUSION: Exogenous administration of rhGH caused an improvement in the resistance of burned mice to HSV-1 infection. In burned mice treated with rhGH, the impaired IFN-gamma responsiveness was restored and the generation of burn-associated suppressor macrophages was inhibited. IFN-gamma, a typical antiviral cytokine induced by rhGH through the regulation of the suppressor macrophage generation, may therefore play a role in the protection of burned mice infected with a lethal amount of HSV-1.     

Survey for antibodies to bovine adenoviruses in six- to nine-month-old feedyard cattle

OBJECTIVE: To determine the prevalence of antibody to bovine adenovirus (BAdV) serotypes 1-8 and 10 in calves at a farm and after 5 weeks in a feedyard. ANIMALS: 2- to 5-month-old calves of mixed English breeding (n = 100) from 4 farms. PROCEDURE: Serum BAdV antibody was measured by use of a microtitration test. RESULTS: Serum antibodies were found to the 9 BAdV serotypes studied. Seroconversion to each virus had occurred in some calves by the time the second serum sample had been obtained, indicating that the BAdV were present and inducing active infection in these calves. CONCLUSIONS: Antibody to BAdV serotypes 1-8 and 10 are present in cattle populations of the United States, indicating existence of these serotypes, although only BAdV serotypes 1-4, 7, and 10 have been isolated.     

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a fourfold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

Monoclonal antibody for the demonstration by ELISA of antibodies to protein p24 of enzootic bovine leukosis virus in individual and pooled blood serum and milk samples

A sensitive and specific ELISA for the demonstration of antibodies to the protein p24 of enzootic bovine leukosis virus (EBVL) using a 'capture' monoclonal antibody to this protein (MAb p24) was developed. The method is sensitive enough to detect the international reference serum E4/10 in pooled blood serum samples collected from up to 50 cows, or, if a 10-fold concentrate of milk whey is tested, in samples of bulk milk collected from up to 400 cows. The application of MAb p24 has considerably increased not only the sensitivity, but also the specificity of ELISA. Moreover it is possible to differentiate reliably between positive and 'false positive' reagents by testing a suspicious sample in a pair of wells of which one is coated with MAb p24 alone and the other with the complex MAb p24 + EBLV antigen and the subsequent calculation of 'specific absorbance'. This method, showing the highest sensitivity of detection of antibodies to EBLV p24 described so far, can become an effective tool on the sanitation of infected herds as well as in checks of the EBL-free status. A diagnostic kit suitable for commercial manufacture has been devised.     

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.     

Serological survey to determine the prevalence of bovine leukaemia virus antibodies in dairy cattle on selected farms in the Gauteng and Mpumalanga provinces

Cattle from a farm where enzootic bovine leukosis had been diagnosed were tested to determine the prevalence of bovine leukaemia virus antibodies. Farmers who had bought cattle from this farm were identified and their herds also tested. Of 381 adult dairy cattle tested, 14 animals reacted positively (3.67%). Cattle (n = 81) from 3 selected herds, not associated with the affected farm were also bled and 7 animals reacted positively (8.64%).     

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.     

Experimental infection with Babesia divergens in cattle persistently infected with bovine virus diarrhoea virus

Nine Norwegian Red cattle, aged 7-14 months, persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with a Swedish strain of Babesia divergens. Six persistently infected cattle of the same age and breed were kept as controls. Blood and serum samples were collected regularly during the observation period. Rectal temperatures were recorded every morning for 25 days post infection, and the animals were examined clinically on a daily basis. Sera were examined for antibodies to B. divergens by indirect immunofluorescence antibody test (IFAT). Eight of the infected animals developed fever of 2-5 days duration. Babesia divergens organisms appeared in the erythrocytes of all infected animals on the day after inoculation. The parasitaemia lasted for 4-11 days. One animal had a transient haemoglobinuria. Compared with the control group, there was a 20% decrease in the haematocrit. There was a transient lymphopenia and thrombocytopenia during the period of fever. There were no differences in mean numbers of neutrophils between the two persistently infected groups. Compared with cattle free of BVDV, the persistently infected cattle had consistently lower total leucocyte count that was mainly due to decreased mean numbers of neutrophils and monocytes. All infected animals develop antibodies > or = 1:1280 between day 7 and 10 post infection. The magnitude of the antibody response was considerably lower than that of BVDV-free animals inoculated with the same strain and dosage of B.divergens.     

Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity was revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining revealed that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin.     

Epidemiology of bovine virus diarrhoea in cattle on communal alpine pastures in Switzerland

The goal of this study was to determine the influence of communal pasturing on the spread of bovine virus diarrhoea (BVD). The investigation involved 990 Swiss Braunvieh cattle from 149 different owners on seven communal pastures in the Swiss Alps. Prior to pasturing, blood samples were collected from all animals for examination for BVD antigen and antibodies. Serological examinations were also performed during and after pasturing to determine possible increases in seroprevalence and to determine whether seroprevalence was different on pastures with and without persistently infected cattle. At the start of pasturing, nine (0.9%) animals were persistently viraemic. On three alpine pastures, no persistently viraemic animals were detected. The prevalence of persistently infected cattle on the remaining four pastures varied from 0.3 to 3.9%. Of the 990 animals tested at the start of pasturing, 632 (63.3%) were seropositive. Seroprevalence differed among pastures and varied from 21.8 to 85.9%. During the summer, seroprevalence increased on all pastures surveyed, and at the end of the pasture season, 778 (80.1%) of the 971 cattle that were examined twice were seropositive. The incidence of seroconversion was significantly higher on pastures with persistently infected cattle compared with those without; it ranged from 32.7 to 100.0% in the former and from 6.0 to 22.2 in the latter. The results of this study suggest that communal alpine pasturing does play a role in the spread of BVD. The extent of this role depends on the presence of persistently infected animals.     

Detection of antibodies to melanocytes in vitiligo by western immunoblotting

To more fully define the nature of the antibody response to melanocytes which is associated with vitiligo, a Western immunoblot assay was used to test the sera of 28 patients with vitiligo (21 with active non-segmental, and 7 with stable segmental diseases) and 26 normal individuals for antibodies to antigens in detergent extracts of melanocyte membrane fractions. Antibodies to melanocytes were found in 26 (93%) of the patients with vitiligo, and in 16 (62%) of the control individuals. Patients with vitiligo and control individuals both had antibodies to an 80 approximately 83 kD antigen. The patient with vitiligo, in addition, had antibody responses to antigens with MWs of 45, 65, and 110 kD. Antibodies to these antigens were present in 46, 25, and 31% of vitiligo patients, but in only 19%. 0%, amd 0%, respectively, of the normal individuals. The heterogeneity of the antibody responses to melanocytes in vitiligo was further confirmed by the presence of antibodies to at least 3 distinct antigens in one-third of vitiligo patients but in none of the normal individuals. There was no difference in antibody response between patients with generalized and segmental vitiligo, suggesting that the pathogenesis of diseases was similar in both cases.     

Evaluation of a hand-held electrical conductivity meter for detection of subclinical mastitis in cattle

OBJECTIVE: To assess, under field conditions, whether a hand-held electrical conductivity (EC) meter could be used to detect subclinical mastitis caused by pathogens most commonly associated with mastitis in dairy cows. ANIMALS: 425 lactating cows on 15 dairies in Costa Rica. PROCEDURE: Immediately prior to milking, milk samples from each quarter were tested, using a hand-held EC meter. A milk sample from the quarter with the highest score was submitted for bacteriologic culture. Results of bacteriologic culture were compared with highest absolute EC score for each cow and with differential EC score (ie, difference between the highest and lowest absolute EC scores for the 4 quarters of each cow). RESULTS: Absolute EC score for cows with subclinical mastitis was significantly higher than that for cows without subclinical mastitis, and absolute EC score was significantly associated with detection of subclinical mastitis. If absolute EC score > or = 7 was considered indicative of subclinical mastitis, sensitivity was 0.43, specificity was 0.83, predictive value of a positive result was 0.39, and predictive value of a negative result was 0.85. Differential EC score for cows with mastitis was significantly higher than that for cows without subclinical mastitis. If differential EC score > or = 2 was considered indicative of subclinical mastitis, sensitivity was 0.53, specificity was 0.77, predictive value of a positive result was 0.37, and predictive value of a negative result was 0.87. CLINICAL IMPLICATIONS: A hand-held EC meter may be used to screen cows for subclinical mastitis.     

Evaluation of the therapeutic and protective efficacy of doramectin against psoroptic scabies in cattle

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.     

Standardization of an indirect ultramicro ELISA for the determination of total antibodies against the measles virus

An indirect ultramicro ELISA system (UME) for the determination of antibodies to measles virus by the cuban technology of Ultramicro Analytical Systems (SUMA) was developed. A number of 448 serum samples from the National System of Seroepidemiological Surveillance of Measles were simultaneously processed by the Ultramicro ELISA system and by the haemagglutination inhibition technique. Results were compared in both assays. Afterwards; 120 serum pairs obtained from the same source were studies and processed in a similar way. Results evidenced the usefulness of such immunoenzymatic system for the serological diagnosis and the positive validation of replacing conventional techniques by this novel technology.     

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.     

Evaluation of serological tests for the detection of pseudorabies gE antibodies during early infection

Two enzyme-linked immunosorbent assays (ELISAs) and a particle concentration fluorescence immunoassay (PCFIA) were compared for their ability to detect antibodies against pseudorabies virus (Aujeszky's disease virus) glycoprotein E (gE) in the early stages of infection in pigs previously vaccinated with gE-deleted pseudorabies vaccines. Seventy pigs were included in the study. Five groups of 6 pigs each were vaccinated with one of 5 different pseudorabies virus (PRV) gE-deleted vaccines, and subsequently infected intranasally with 10(5.6) TCID50 of the Iowa 4892 pneumotropic strain of PRV. This entire procedure was repeated using 10(4.6) TCID50 of the Rice strain of PRV. Five unvaccinated control pigs were also challenged with each virus strain. Three control pigs died before seroconverting, leaving 67 pigs for comparison. Blood samples were drawn from experimentally inoculated pigs on the day of vaccination, the day of challenge, and on 4-10, 14, and 21 days postchallenge (DPC). Serology test sensitivity estimates and comparisons among tests were made for each sampling day. Results of this study demonstrated differences among the tests in the time from inoculation to initial antibody detection, and the time to detect 50% and 75% of the infected pigs. The average time until first detection of pigs as seropositive for gE antibodies by PCFIA was 7.5 DPC. The blocking ELISA detected pigs as seropositive an average of 8.8 DPC, and the indirect ELISA first detected gE antibodies by 9.3 DPC. Fifty percent of the pigs were detected as seropositive by days 7, 8, and 9 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. Similarly, 75% of the pigs were detected as seropositive by days 8, 9, and 10 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. All pigs were detected as seropositive by 14 DPC for all 3 tests.     

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.