Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.     

Risk factors for avian influenza and Newcastle disease in smallholder farming systems, Madagascar highlands

Newcastle disease (ND) and avian influenza (AI) are issues of interest to avian producers in Madagascar. Newcastle disease virus (NDV) is the major constraint for village aviculture, and avian influenza viruses type A (AIAV) are known to circulate in bird flocks. This study aims at classifying smallholder poultry farms, according to the combination of risk factors potentially associated with NDV and AIAV transmission and to assess the level of infection for each farm class. Two study sites, Lake Alaotra and Grand Antananarivo, were chosen with respect to their differences in terms of agro-ecological features and poultry productions. A typology survey involving 526 farms was performed to identify possible risk factors for (i) within-village, and (ii) between-village virus transmission. A cross-sectional serological study was also carried out in 270 farms to assess sero-prevalences of NDV and AIAV for each farm class and the link between them and risk factor patterns. For within-village transmission, four classes of farms were identified in Grand Antananarivo and five in Lake Alaotra. For between-village virus transmission, four classes of farms were identified for each site. In both sites, NDV sero-prevalence was higher than for AIAV. There was no evidence of the presence of H5 or H7 subtypes of AIAV. Sero-prevalences were significantly higher in Lake Alaotra than in Grand Antananarivo for both viruses (OR=2.4, p=0.02 for NDV, and OR=9.6, p<0.0001 for AIAV). For within-village NDV transmission in Grand Antananarivo, backyard chicken farms (OR=3.6, p<0.001), and chicken farms with biosecurity awareness (OR=3.4, p<0.01) had greater odds of having antibodies against NDV than the others. For between-village virus transmission, farms with multiple external contacts, and farms using many small markets had greater odds of having antibodies against NDV than the others (OR=5.4, p<0.01). For AIAV, there were no differences in sero-prevalences among farm classes. In Lake Alaotra, the observed high density of palmipeds and widespread rice paddies were associated with high sero-prevalences for both viruses, and a homogeneous risk of virus transmission between the different farm classes. In Grand Antananarivo, farm visits by collectors or animal health workers, and farm contacts with several markets were identified as potential risk factors for NDV transmission. Further studies are needed to identify the circulating virus genotypes, model their transmission risk, and provide adapted control measures.     

检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立

以大肠杆菌系统表达的H9N2亚型禽流感病毒（AIV）核蛋白（NP）为抗原，建立了禽流感间接酶联免疫吸附试验抗体检测技术（NP—ELISA）。对263份待检血清（包括临床收集的243份血清和20份H9N2亚型AIV免疫鸡阳性血清）进行检测，NP—ELISA与琼脂免疫扩散试验（AGP）的总符合率为83．3％，与血凝抑制试验（HI）的总符合率为92％。特异性试验表明，NP—ELISA方法可以检测H5、H7和H9亚型AIV特异性抗体，检测为阳性的血清样品能够被阳性鸡胚尿囊液阻断。敏感性试验证实，NP—ELISA最早可以检测鸡感染后7d的血清样品，并于感染后10d确定100％血清阳性，而AGP检测直到首免后21～28d才出现部分血清阳性，HI检测直到10～14d才出现部分血清阳性，并且NP-ELISA要比HI敏感4～40倍。试验证明，NP—ELISA是检测AIV血清型特异性抗体的一种特异、敏感、快速、经济的血清学检测技术。     

A trivalent antigen for the detection of type I avian adenovirus precipitin

A double immunodiffusion antigen prepared from cell-culture-propagated CELO virus was not capable of detecting precipitin directed against all of the type I avian adenovirus (fowl adenoviruses) isolates tested. However, an antigen pool containing CELO-4, B-3, and IBH-2 (Tipton) fowl adenovirus isolates detected precipitin directed against representative isolates of 10 type I serotypes. Additionally, this antigen pool markedly improved detection of adenovirus field exposure.     

Effect of sera from fed and fasted pigs on proliferation and protein turnover in cultured myogenic cells

The effects of fasting on the ability of swine serum to affect proliferation, protein synthesis and protein degradation in L6 myoblast cell culture bioassays were evaluated. Barrows (15 to 20 kg) were fitted with jugular catheters. Blood samples were collected at four evenly spaced intervals between 0800 and 1700 on collection days. Prefast blood samples were obtained on d 1 and 2 of the study, after which pigs were subjected to a 5-d fast. Fasted samples were obtained on the 1st, 3rd and 5th d of the fast (d 3, 5 and 7 of the study). Serum from each collection day was pooled and tested in the proliferation bioassay for each pig. Prefast and fasted serum pools were formed by pooling prefast days (1 and 2) or fasted days (3, 5 and 7), respectively, from all pigs in a study. These pools were tested in the proliferation and protein turnover bioassays as well as in a Somatomedin-C (SmC) radioimmunoassay. Serum from the fasted collection days showed a decrease in mitogenic activity compared with serum from the two prefast days (P less than .001). At high concentrations, sera obtained from fasted pigs inhibited muscle cell proliferation (P less than .001). Additionally, adding fasted serum to control swine serum (CSS) inhibited the mitogenic activity of CSS in a dose-dependent manner (P less than .025). Therefore, fasted sera showed a decreased ability to promote muscle cell proliferation and, in addition, appeared to contain a factor(s) that inhibits muscle cell proliferation. Fasted serum also caused a 21.6% increase in protein degradation compared with prefast serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the results of ELISA in the quantitative determination of antibodies against infectious bursitis in poultry]

Specific antibodies were investigated in serums of chicks vaccinated with live vaccine and revaccinated with inactivated vaccine against the infectious bursal disease virus, using three methods. An ELISA technique was used to determine antibody titres at a fourfold serum dilution; the reaction was evaluated visually. The results were compared with the titres of neutralizing antibodies and percentage of samples with precipitin activity (Fig. 1). The average values of neutralizing antibody titres and ELISA titres were found to have an analogical pattern; a decrease in maternal antibodies on the first days of chick life and an increase in the antibodies after vaccination, accompanied by an increase in precipitin activity, were typical in these tests. Using the different ELISA technique, 138 samples of fowl serum were examined (Fig. 2). The high correlation, r = 0.85, was found between the ELISA titres determined by visual reading of the reaction within the fourfold serum dilutions and the absorbance values determined at single serum dilution. Applying a spectrophotometer programme, a scale of antibody quantification was made up pursuant to the intensity of immunoenzymatic reaction. For the purposes of method reproducibility, the evaluation was made within the average values of absorbance of positive serum on the one hand and of negative serum on the other. The span of these values was divided into ten equal parts, designated by degrees 0 to 9. Corresponding degrees of positivity were assigned to the examined samples in dependence on the determined value of absorbance (Fig. 3). The correlation r = 0.61 was found between the ELISA values and the titres of neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

应用改良阻断ELISA检测禽网状内皮组织增殖病血清抗体

应用禽网状内皮组织增殖病病毒纯化抗原和抗ＲＥＶ单克隆抗体建立了改良阻断ＥＬＩＳＡ用于鸡血清中ＲＥＶ抗体检测，并对北京地区鸡群中随机采样的３６份血清样本进行了检测，阳性率为５．６％。与间接ＥＬＳＩＡ的检测结果进行了统计学比较，两种方法的阳性率无显著差异。结果表明本试验所建立的改良阻断ＥＬＩＳＡ可以用于鸡群ＲＥＶ感染的血清学调查。     

Enzyme immunoassay studies on the serological response of turkeys to hemorrhagic enteritis virus

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies in turkey serum to hemorrhagic enteritis virus. The ELISA antigen was extracted from turkey spleens and partially purified with fluorocarbon. Antibodies were demonstrated in serum samples of breeding and meat flocks that had been naturally exposed to infection. These samples were also examined in parallel by agar-gel precipitin (AGP); most of the sera were AGP-positive. ELISA, however, was more sensitive in detecting antibodies in day-old sera that were AGP-negative. The passively acquired antibodies were no longer detected by 4 weeks of age. A brisk but short-lived secondary response was detected by ELISA in the sera of turkeys immunized with beta-propiolactone-inactivated extract of infected spleens.     

"Haysickness" in Icelandic horses: precipitin tests and other studies

Blood samples were taken from 18 healthy horses (Group A), 15 horses clinically diagnosed to have "haysickness" ("farmer's lung") (Group B), 10 closely related horses (Group C) and 14 inbred horses (Group D). Precipitins in sera were measured by double gel diffusion test against Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus, Alternaria, Penicillium and Rhizopus species. In Group A, all the horses were precipitin negative except one with a faint reaction to Rhizopus species. In Group B all had precipitin against M faeni. One horse also had precipitins against Rhizopus species and another against A fumigatus. In Group C, seven of the 10 horses had precipitins against M faeni. Of these, five had a history of respiratory signs, but two horses with a faint reaction had no such history. In Group D, four out of 14 horses had positive precipitin tests against M faeni. Of these four horses, three also had a faint reaction to A fumigatus and one a faint reaction to Alternaria species. All were asymptomatic. These results indicate that "farmer's lung" in man and "haysickness" in horses are of the same origin. However, further studies are necessary to substantiate the diagnostic or prognostic value of these precipitin tests in equine practice. The question of whether hereditary factors play a role in causing this disease also warrants further studies.     

用ELISA检测接种猪瘟疫苗猪的血清抗体

用ＥＬＩＳＡ检测了接种猪瘟疫苗猪的血清抗体。共计４８６份猪血样,检测到血清抗体阳性者４４１份,阳性率为９０．７４％。同时对其中的１１１份血清进行了自然强毒血清抗体的检测,检测到强毒抗体阳性４份,阳性率为３．６０％。结果表明青海省猪瘟疫苗注射密度较高,但也反映出在青海省猪群中可能存在猪瘟自然强毒感染。     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

Seroprevalence of porcine respiratory coronavirus in selected Korean pigs

A total of 446 serum samples from 88 herds in Korea were examined for antibody to porcine respiratory coronavirus (PRCV) using blocking enzyme-linked immunosorbent assay (ELISA). All serum samples were collected from 24- to 26-week-old finishing pigs between December 1998 and June 1999. By ELISA, 237 out of 446 sera tested (53.1%) and 54 out of 88 sampled herds (61.3%) were positive against PRCV. Of 446 sera from 88 herd tested, 185 (41.5%) serum samples from 22 (25%) herds were seronegative against PRCV and transmissible gastroenteritis virus infection. Our data suggested that seropositive herds for PRCV are distributed diffusely throughout South Korea.     

青海省英得尔种羊场种羊流产血清学调查

通过间接血凝试验(IHA)和虎红平板凝集试验(RBPT),对来自青海省英得尔种羊场的353份羊血清进行布鲁氏菌、弓形虫和衣原体三种主要疫病的血清抗体检测。结果表明,18.9%(33/174)的山羊血清和3.9%(7/179)的绵羊血清其衣原体抗体血清凝集效价≥1:16(++),被判为阳性;抗布鲁氏菌和弓形虫的血清抗体均未检测到。     

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country.The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.     

Isolation and purification of a low molecular weight protein from bovine urine

A low molecular weight protein was separated from urine samples obtained from a heifer with spontaneous renal disease and from cows with CaNa2EDTA-induced renal dysfunction. The molecular weight and electrophoretic mobility of the separated protein were examined. The low molecular weight protein collected by gel filtration chromatography was further separated into two fractions by ion exchange chromatography using DEAE-cellulose. One of the two fractions, the lowest molecular weight protein showed a single band in SDS-PAGE, and its molecular weight was approximately 12,000. An antiserum against this protein formed a single precipitin line with the urine from cows with experimentally induced renal dysfunction and a heifer with spontaneous renal disease by the double immunodiffusion technique. However, the antiserum did not form any precipitin line with the concentrated urine of healthy cow and human beta 2-microglobulin. In cellulose acetate membrane electrophoresis, this protein migrated in the same position as that of serum gamma-globulin from healthy cow.     

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.     

禽脑脊髓炎的调查及病毒分离

对陕西省宝鸡、渭南等地的24个鸡群进行了禽脑脊髓炎（AE）的调查,所调查鸡群都有产蛋下降史或产蛋正在下降,下降幅度平均为17.2%。用琼扩试验（AGP）检测所采集的256份血清,AE阳性率平均高达85.6%。有10个鸡群阳性率达100%。跟踪调查2个种鸡群,发现孵出的雏鸡从3日龄开始发病,而且有AE的症状,采发病雏鸡的脑组织,接种6龄鸡胚分离病毒,连传3代,琼扩检测分离毒,呈AE抗原阳性,人工感染1日龄雏鸡复制出与原发病鸡群相同的疾例,表明分离毒是AE病毒。     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

OBJECTIVE: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 Ontario herds. PROCEDURE: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis. RESULTS: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.     

鼬獾群体中狂犬病中和抗体调查与分析

采用荧光抗体病毒中和试验（FAVN）即固定病毒一稀释血清的中和试验方法,对从我国浙江、江西、安徽和广东地区采集的外观健康鼬獾的215份血清进行了狂犬病中和抗体的检测。结果显示,鼬獾体内存在比例较高（38％）的狂犬病中和抗体,但是自不同地区或群体采集的血清样品,狂犬病中和抗体阳性率不同,有些群体样品中完全没有抗体,有些群体样品中狂犬病中和抗体阳性比例100％,但是中和抗体的水平普遍不高,说明鼬獾种群感染狂犬病几率较高,并可能存在狂犬病的一过性或隐性感染。     

Identification of common antigens in ribosome-rich extracts from Fusobacterium necrophorum

Ribosome-rich extracts (RRE) were prepared by differential and ultracentrifugation from 25 bovine and 6 ovine isolates of Fusobacterium necrophorum (FN) including both biotypes A and B. A pooled rabbit antiserum was prepared against whole-cell and sonicated whole-cell bacterins of F necrophorum isolate FN 3080, and a 2nd pooled rabbit antiserum was prepared against a RRE of FN 3080. The RRE of the 25 bovine isolates were tested against the FN 3080 whole-cell antiserum, using Ouchterlony double-immunodiffusion procedures. One to 3 precipitin lines were observed with the 25 isolates. The individual bovine isolates were found to have lines of identity with 5 to 21 of the other 24 isolates. The 25 bovine isolates and the 6 ovine isolates were then compared, using the FN 3080 RRE antiserum. One to 3 precipitin lines were observed for the 31 isolates with the RRE antiserum, and lines of identity were observed between all 31 of the isolates. These results indicated that common antigens are present in the RRE from a wide variety of F necrophorum isolates including both A and B biotypes.