An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retrospective serological study on hepatitis E infection in pigs from 1985 to 1997 in Spain

The objective of the present work was to ascertain the date in which hepatitis E virus (HEV) was introduced in the Spanish pig population. For this, a serological retrospective study was carried out using archived sera. A total of 2871 serum samples gathered between 1985 and 1997 and collected in 208 farms of Spain were tested for anti-HEV IgG by an in-house ELISA. Of the 2871 sera analyzed by ELISA, 1390 were positive for anti-HEV antibodies (48.4%, 95% CI: 46.9-49.9%) and that corresponded to 204/208 farms (98%, 95% CI: 96.1-99.9%) having at least one positive pig. Our results show that HEV was present and widespread in Spanish swine farms at least since 1985. Any significant changes in prevalence were detected from 1 year to another and therefore, HEV infection in swine should be considered endemic in Spain.     

Immunoglobulins IgG and IgM in synovial fluids of swine with erysipelothrix polyarthritis

Concentrations of IgG and IgM immunoglobulins in synovial fluids and sera from a group of swine with experimentally produced Erysipelothrix rhusiopathiae polyarthritis were measured to determine if there was local synthesis of these immunoglobulins by plasma cells in arthritic synovial tissue. IgG and, to a lesser extent, IgM were significantly higher in arthritic than in nirmal synovial fluids from the same group of swine and this increase could only partly be explained by the increased permeability of the arthritic synovial membrane to plasma proteins. When synovial fluid values of IgG and IgM were calculated on the basis of companion serum concentration it was found that 82% of IgG, and 25% of IgM estimations were significantly elevated above levels in normal joints indicating that IgG was the dominant immunoglobulin synthesized.     

Comparison of the microtitration agglutination test and the enzyme-linked immunosorbent assay for the detection of herds affected with swine dysentery

A method was designed to evaluate and compare the microtitration agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA) to detect antibodies in swine sera to Treponema hyodysenteriae and thereby establish a method for determining the prevalence of swine dysentery (SD) in herds. According to sampling criteria based on the hypergeometric distribution, sera were collected from 3 age groups of swine from farms having a history of SD on the premises (SD+) recently or being free of the disease (SD-). The highest degree of test sensitivity was obtained when sera from market age swine were evaluated with the ELISA. Of 14 SD+ herds from which sera were obtained from market-age swine, 13 were positive with the ELISA (93%); none of the 8 SD- herds was positive. The detection rates of individual swine in the SD+ herds for the 2 tests by age group were as follows: MAT--adult swine 1.4%, market-age swine 6%, and weaned pigs 0.8%; ELISA--adult swine 16%, market swine 31%, and weaned pigs 0.5%.     

Age-related variations in serum concentrations of the third component of complement in swine

Single radial immunodiffusion was used to determine the concentration of the third component of complement (C3) in serum from swine in the following age groups; 36 to 60 hours (neonates), 6 to 7 weeks (weanlings), 4 to 5 months (adolescents) and greater than 1 year (adults). Mean serum C3 concentrations, expressed as the percentage of pooled reference sera from 15 adult swine for the 4 groups were 23, 123, 119, and 98%. With the exception of mean values for weanlings and adolescents, all comparisons of group means were significantly different. Regression models were developed to estimate serum C3 concentration in neonates as a function of litter size, birth weight, and total serum IgG concentration. Increases in birth weight and litter size were accompanied by increased serum C3 concentration, possibly reflecting ontogenic variation of the complement system at the end of gestation. Passive transfer of colostral immunoglobulins, as measured by total serum IgG concentration, was inversely related to serum C3 concentration in neonatal swine, suggesting that colostral absorption of C3 has minimal impact on complement activity in neonatal swine.     

Quantitative and Qualitative Study of Enzyme-linked Immunosorbent Assay to Detect IgG against Japanese Encephalitis Virus in Swine Sera

This study was designed to investigate the application of indirect enzyme-linked immunoassay (ELISA) in detecting IgG against Japanese encephalitis virus in swine sera and the qualitative nature of this test. The attenuated strain SA14-14-2 of Japanese encephalitis virus (JEV) was inoculated into 9-day-old chicken embryos and virus was harvested, purified and suspended in 0.9% saline as JEV antigen. The control antigen was prepared by the same method as for the antigen. In the ELISA, the optimal concentrations of antigen coated and dilution factor were selected using chi2 test. Ninety-two swine sera negative to haemagglutination inhibition (HI) were tested by this assay and the positive threshold was determined. The results of this study indicate that indirect ELISA has high specificity, sensitivity and reproducability. Simultaneous testing of 74 serum samples from nine pig farms was carried out to compare the existing HI test and the indirect ELISA. The coincidence rate of the two assays was 85.1% (63/74) and no significant difference was observed between them (p > 0.05). This ELISA test can detect 46 swine serum samples qualitatively and the titre of eight swine serum samples through endpoint dilution quantitatively within one 96-well plate.     

Enzyme-linked immunoassay in the diagnosis of leptospirosis in domestic animals using peroxidase-conjugated protein-A

The ELISA test for detection of antibodies to Leptospirosis in domestic animals was performed using Staphylococcal protein-A coupled to peroxidase in place of antisera to IgG. Genus- and type-specific antigens were extracted with SDS technique from four pathogenic serotypes and two non-pathogenic ones, and they were identified with the aid of ELISA using specific rabbit antisera. Micro-agglutination (MA) and ELISA were compared using a total of 48 positive swine sera and a 100% agreement was obtained, since with sera from 16 dogs clinically suspected of Leptospirosis the ELISA resulted highly more sensitive and precocious than MA in detecting specific antibodies.     

Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures

The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.     

猪戊型肝炎病毒Ⅳ型衣壳蛋白单克隆抗体的制备与鉴定

为获得猪戊型肝炎病毒(Hepatitis E virus,HEV)Ⅳ型衣壳蛋白单克隆抗体,将猪HEV衣壳蛋白的C端267(408—675)个氨基酸基因序列克隆入原核表达载体pET-28a(+),构建重组质粒pET-28a-ORF2-C,转化E.coli Rosetta(BL21)感受态细胞进行诱导表达,SDS-PAGE和Western blot鉴定,纯化后免疫小鼠。取免疫小鼠的脾脏与鼠骨髓瘤细胞SP2/0融合制备单克隆抗体。通过间接ELISA和竞争ELISA方法筛选并鉴定单抗。结果表明蛋白得到正确、高效表达,获得3株识别不同的抗原表位区的单克隆抗体,分别命名为Mab-1E4(IgG1)、Mab-2C7(IgG1)和Mab-2G9(IgG2b),其中1E4和2G9能阻断临床阳性猪血清,提示该2株单克隆抗体识别的抗原表位是猪HEVⅣ型衣壳蛋白上重要的抗原表位区,而单抗Mab-2C7不能阻断。本研究为猪HEVⅣ型的诊断及研究提供重要工具。     

A serologic study on Toxoplasma gondii infection in slaughtered sheep and goats in Qazvin Province,Iran

Background

Toxoplasma gondii is a common protozoan parasite among all mammals, in particular small ruminants, worldwide. Traditional husbandry can be a major risk factor for infection of sheep and goats with this parasite.

Objectives

The present study aimed to determine the current status of the prevalence for T. gondii in livestock of Qazvin Province.

Methods

In this cross-sectional study, the sera of 455 sheep and 375 goats were examined to detect anti-Toxoplasma IgG antibodies by using in-house indirect ELISA.

Results

Overall, 33.62% (153/455) of sheep and 36.41% (130/375) of goats were positive for anti-Toxoplasma IgG antibodies with no statistically significant difference. The prevalence rate of T. gondii among the sheep of Qazvin County was significantly higher than in Abyek and Abhar counties (p < 0.001).

Conclusions

The results of the present study indicate that the prevalence of T. gondii in sheep and goats of the study area is high. Therefore, the meat of the animals reared in this area can be a potential source of human infections by this parasite.

     

Increased urban seroprevalence of Toxoplasma gondii infecting swine in Panama

Toxoplasmosis gondii causes one of the most common zoonoses worldwide. The rate in Panama is one of the highest in the world. Infections are primarily asymptomatic in immune competent individuals; however, in immunocompromised patient and congenital cases can be lethal. Exposure to the pathogen is hypothesized to occur when handling or consuming infected food such as swine meat. In this study, we analyzed 290 swine sera collected from six provinces in Panama by Indirect Immunofluorescence antibody test (IFAT) against T. gondii. Toxoplasma-specific IgG were found in 32.1% of the samples. The highest seroprevalence was found in the province of Panama.     

广东部分地区中小型猪场猪伪狂犬病野毒感染的血清学调查

采用阻断ELISA法,对广东省9个地区经猪伪狂犬基因缺失苗免疫的中小型猪场2005年-2006上半年送检的375份血清进行猪伪狂犬病野毒感染的血清学检测。结果表明,有9个地区猪场血清呈阳性,其中阳性血清174份,平均阳性率为46.4%,最高阳性率达65.0%,提示该地区中小型猪场有猪伪狂犬病野毒感染。     

Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies

Three monoclonal antibodies, specific for porcine IgG, IgM and IgA, were used to develop isotype-specific immunoperoxidase monolayer assays for the detection of antibodies against African swine fever virus. A mixture of anti-IgM and anti-IgG monoclonal antibodies was used in an assay designed for screening sera. This test was compared with a commercially available ELISA by using experimental sera and field sera obtained after an outbreak of African swine fever on two farms in the Netherlands in 1986. Although the ELISA was less sensitive than the immunoperoxidase monolayer assay on sera taken early after infection, the tests were equally useful for screening purposes. The isotype-specific assays gave epizootiological information about the stage of infection on the two farms.     

Investigation of some factors which influence the absorption of IgG by the neonatal piglet

The absorption by the neonatal gut of purified swine IgG isolated from maternal serum was studied in 91 motherless piglets raised without swine colostrum, to investigate some of the factors which influence absorption. Factors studied included the amount of IgG administered, the effect of bovine colostrum fed concurrently, the influence of starvation and the effect of lactose.Administration of iodinated IgG within 3 hours after birth resulted in the appearance of IgG in the piglet circulation in unaltered form. Immediate feeding of bovine colostrum followed by administration of iodinated IgG at 72 hours resulted in the appearance of only IgG digestion fragments in the circulation.Administration of 15 g of lactose during the first 24 hours reduced absorption of IgG by 26% compared to controls while treatment with 54 g of lactose reduced absorption by 94%. On the contrary, when bovine colostrum was administered immediately after purified IgG, the amount of swine IgG absorbed was 50–70% greater than in controls. Mature milk failed to have the same influence. Finally, in experiments in which the amount of purified swine IgG administered varied from 1.0 to 8.0 g, absorption was directly proportional to the amount administered. When these data were compared with data on naturally reared piglets, the same proportionality was seen and indicated a consistent 1:2.5 to 1:3 ratio of ingested IgG (g) to the maximal serum level of immunglobulin (mg/ml) resulting from absorption. IgA and IgM follow a similar pattern.The data are consistent with a regulated absorption mechanism, that appears to be lost very quickly after the initial ingestion of food. Heterologous protein and lactose alone can switch off this absorption mechanism, thus arguing against “gut closure” by a mechanism of receptor saturation by absorbed IgG. Bovine colostrum, when compared to mature milk, is able to augment the absorption of swine IgG by an unknown mechanism.     

Detection of antibodies against the virus causing viral gastroenteritis in swine using the modified autoradiography test

A modified method for autoradiographic determination of antibodies to the virus of transmissible gastroenteritis of swine was developed. It is based on the actual reaction between antigen bound in cells of the infected cell cultures and antibodies in tested sera, which is visualized, by help of rabbit antibodies marked 125J (125J RaSw IgG antibody) to porcine IgG, on a sensitive radiograph and evaluated on the basis of darkening at the point of positive immunological reaction. Specificity of the test and mutual comparability and reproducibility of the results were confirmed by examining the known positive and negative sera and by comparison with the results of the virus-neutralisation test. Out of the 36 examined porcine blood sera the antibodies were proved autoradiographically only in the samples positive also by virus-neutralisation. In experimentally infected pigs, the same dynamics of antibody production was recorded by the two tests. They were, however, demonstrated autoradiographically already by the two tests. They were, however, demonstrated autoradiographically already the eighth day after infection, while by virus neutralisation test as late as 14th day. Their level increased gradually until 35th day after infection.     

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.     

Japanese encephalitis virus immunoglobulin M antibodies in porcine sera

A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.     

Prevalence of antibodies to Neospora caninum in Korean native beef cattle

A total of 438 sera from Korean native beef cattle in 9 provinces were tested for Neospora caninum antibodies using an immunofluorescent antibody test (IFAT). Eighteen (4.1%) cattle were positive by IFAT. The titers ranged from 1:200 (10 animals), 1:400 (5 animals), 1:800 (2 animals) to 1:1,600 (1 animal). Although the seroprevalence was slightly higher in Chungnam (8.9%), this was not significantly different from those noted in Kyunggi, Kangwon, Kyungbuk, Kyungnam, and Cheju provinces. Sera obtained from beef cattle in the provinces of Chungbuk, Jeonbuk and Jeonnam were all negative. Neospora positive sera were also tested for anti-Toxoplasma gondii antibodies using a commercial latex agglutination test (LAT). Antibody to T. gondii was detected in only 1 (5.6%) of 18 N. caninum positive sera. These results indicate that N. caninum and T. gondii infection are present at a low level in the Korean native beef cattle.     

The prevalence of toxoplasma antibodies in swine sera in Finland.

A total of 1847 swine sera obtained from the 10 largest abattoirs slaughtering swine in Finland were examined by ELISA for toxoplasma antibodies. The sample represented 0.64% of the total number of swine slaughtered in these abattoirs over a period of 2 months. The prevalence of toxoplasma antibodies in swine sera was 2.5%.