Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20-22 and 10kDa.     

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.     

The effect of dose, route, number of injections and the use of adjuvant on the clearance of and immune response to, haemocyanin following injection into brown trout (Salmo trutta)

The primary and secondary immune responses were investigated in trout injected with haemocyanin (HCN) intramuscularly (IM) or intraperitoneally (IP), with or without adjuvant. HCN was detected in the blood 30 min after injection and clearance times varied after one injection from 8 to 56 days. Fish given two and three injections cleared the antigen faster. Precipitins against HCN were first detected 21 to 30 days after injection and were still present on Day 56. However, antibodies detectable by indirect haemagglutination (IHA) and complement fixation (CFA) were initially demonstrated 7 to 21 days after a single injection and highest titres were reached between 33 and 56 days according to the experimental protocol. In fish given two injections, maximum titres were reached between Days 42 and 56 (IM), and Days 50 and 56 (IP). With three injections, maximum CFA and IHA values occurred on Days 62 and 66 respectively in the case of IP, and on Days 103 and 106 respectively with IM. Overall, higher titres were found with IHA than by CFA.     

3种ELISA试剂盒检测不同亚型外源性鸡白血病病毒的比较

为建立一种稳定、快速从感染鸡体内检测或分离外源性鸡白血病病毒(ALV)的简易方法,作者使用A亚型(ALV-A)、C亚型(ALV-C)以及2株J亚型(ALV-J-PY和ALV-J-WS)鸡白血病病毒(ALV)按高、中、低(即100、10、1μL)3种接种量人工接种DF1细胞,在接种后不同时间用A、B、C 3种ELISA试剂盒检测ALV抗原(P27)。结果表明,对ALV-A和ALV-J-PY,高剂量接种时,用A试剂盒在接种后第3天即可检测出;低剂量接种时,第7天可检出。使用B试剂盒,2株病毒的检出时间延长(高剂量组分别为第7天和第5天;低剂量组分别为第15天和第13天);使用C试剂盒,所需检出时间最长(接种后第13天)。对ALV-C和ALV-J-WS毒株,A试剂盒对高剂量组,分别能够在接种后第5天和第9天检出,其它中、低接种量组15 d内均没有检出;而B、C2个试剂盒对3个接种剂量组均没有检测出。从以上结果看出,3种ELISA试剂盒对3种亚型ALV毒株的检出时间存在明显不同,A试剂盒灵敏度最高,能最早作出检测;B试剂盒灵敏度次之。同时,无论使用哪种试剂盒ALVs的检出时间与病毒接种量间存在很大的相关性。本研究结果为外源性ALV的检测及病毒分离研究提供一定的科学依据。     

Efficiency of a recombinant MSA-2c-based ELISA to establish the persistence of antibodies in cattle vaccinated with Babesia bovis

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.     

Comparison of an indirect immunofluorescence assay, western blot analysis, and a commercially available ELISA for detection of Ehrlichia canis antibodies in canine sera

OBJECTIVE: To examine the correlation between results for an indirect immunofluorescence assay (IFA) that uses Ehrlichia canis antigen as a substrate (ie, E canis-IFA), 2 western blot (WB) analyses, and a commercially available ELISA in the detection of E canis antibody in dog sera. SAMPLE POPULATION: 54 canine serum samples that were reactive on E canis-IFA and 16 canine serum samples that were E canis-IFA nonreactive. PROCEDURE: Serum samples were evaluated by use of 2 WB analyses and a commercially available ELISA. Correlation between results of the 3 testing modalities (ie, IFA, WB analyses, and the ELISA) was examined by use of nonreactive (E canis-IFA reciprocal titer, < 20), low-titer (reciprocal titer, 80 to 160), medium-titer (reciprocal titer, 320 to 2,560), and high-titer (reciprocal titer, 5,120 to > 20,480) serum samples. RESULTS: For all serum samples in the nonreactive (n = 16), medium-titer (17), and high-titer (18) groups, correlation of results among IFA, WB analyses, and the commercially available ELISA was excellent. A poor correlation was found between IFA results and those of WB analyses and the ELISA for serum samples in the low-titer group (19), with only 4 of the 19 serum samples having positive results on both WB analyses and the commercially available ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: The discrepancy between E canis-IFA, WB analyses, and the commercially available ELISA results for the low-titer serum samples may be related to a high IFA sensitivity or, more likely, a lack of specificity associated with cross-reactivity among Ehrlichia spp.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Heterologous antibodies to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain

An IgM capture ELISA using heterologous antibodies was developed to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain. Detection of parasite in tissues from inoculated dogs was evaluated by mouse bioassay and immunohistochemical techniques. Serum samples were obtained at regular intervals up to 62 days post-inoculation (p.i.), when the animals were necropsied and their tissues examined. Antibody levels were measured by IgM capture ELISA (McELISA), indirect hemagglutination (IHA), indirect fluorescent antibody test (IgG-IFAT) and indirect immunoenzymatic assay (IgG-ELISA). All dogs seroconverted but only one exhibited severe clinical signs of infection. IgM antibodies were detected by McELISA from the seventh day on, with decreasing IgM levels around the 27th day. Similar results were obtained from IHA, although McELISA showed earlier and longer detection of IgM antibodies. IgG antibodies were detected from the seventh day on, and throughout the period of observation. Immunohistochemical findings and mouse bioassay revealed the presence of free tachyzoites in tissues of the clinically affected dog only. These results suggest that T. gondii acute infection in dogs shows a remarkably transient IgM synthesis, and this feature may constitute an important marker of active infection. Furthermore, McELISA was shown to be a potential tool to diagnose canine toxoplasmosis.     

伪狂犬病病毒囊膜蛋白ISCOMs免疫原性测定

应用伪狂犬病病毒囊膜蛋白与Quil A结合制备囊膜蛋白免疫刺激复合物(PRV-ISCOMs)，并通过ELISA、血清中和试验和淋巴细胞转化试验(Brdu-ELISA法)测定其诱导小鼠体液和细胞免疫应答水平。结果如下：1)小鼠分别接种3、7、10ug的PRV-ISCOMs后，于接种后PI7d血清中可测出ELISA IgG而抗体，随后抗体水平逐渐升高。间隔21d加强免疫后，FLISA IgG抗体水平进一步提高，且中和抗体效价均在1：22以上，而未加佐剂的囊膜蛋白对照组均无中和抗体；2)淋巴细胞转化试验初步结果表明PRV-ISCOMs能诱导小鼠的细胞免疫应答。3)免疫保护力测定显示免疫鼠能抵抗强毒攻击。上述结果表明PRV-ISCOMs具有良好的免疫原性，能诱导小鼠的体液免疫和细胞免疫应答。     

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.     

The humoral immune response of the spiny-tailed agamid lizard (Agama caudospinosum) to injection with Leishmania agamae promastigotes

Spiny-tailed agamid lizards (Agama caudospinosum) were given a single intraperitoneal injection of Leishmania agamae promastigotes. Direct agglutinins (DA), indirect haemagglutinins (IHA) and complement-fixing antibodies (CFA) produced against the parasites were non-precipitating, relatively thermostable and dithiothreitol sensitive. Antibodies were also detected by the immobilisation test (IMM) and by enzyme-linked immunosorbent assay (ELISA). The most sensitive method was the ELISA one. Antibodies were detected 7 days post-injection and maximum IMM (2(-6)), DA (2(-8)), CFA (2(-10)), IHA (2(-12)) and ELISA (2(-16)) titres were obtained from 35 to 49 days. In all cases, the levels of antibody following antigenic stimulation were significantly different from the controls (P less than 0.001). Serum lysozyme levels increased three-fold (P less than 0.001) with the highest value of 46 micrograms/ml occurring after 42 days.     

Immune responses of specific pathogen free foals to EHV-1 infection.

Four foals were raised under specific pathogen free (SPF) conditions. At 3 to 4 months of age, SPF foals and 1 other non-SPF foal were intranasally inoculated with equine herpes virus type 1 (EHV-1). Clinical signs included depression, fever, inappetence and intermittent coughing. Clinical recovery was complete by seven days but high titres of virus were detected in nasal mucus for at least 10 days after inoculation. Clinical illness was less severe in the non-SPF foal. Interferon was detected in the nasal mucus of all foals from 2 days post infection (dpi), persisting until 8 or 10 dpi. ELISA antibody was detected in serum from 6 dpi. Titres continued to rise throughout the period of observation, and were slightly stimulated by re-inoculation. EHV antibody, identified as belonging to the IgM class by the double sandwich ELISA, was detected from 6 dpi. Peak IgM titres were observed between day 10 and 18, declining to base levels by day 42. Virus neutralizing antibody was detectable in serum from day 14 and rises in titre were parallel to that of total ELISA antibody. Cellular immunity in EHV-1 infected SPF horses was examined by the antibody dependent cytotoxicity (ADCC) test and the specific lymphocyte transformation test. The ability of foal neutrophils to effect ADCC decreased significantly between 3 to 10 days after inoculation. Peripheral blood mononuclear cells (PBMC) displayed reactivity towards EHV-1 antigens from about day 14, with maximum stimulation indices being obtained between 28 and 42 dpi.     

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.     

ELISA和IHA检测弓形虫抗体的比较试验研究

目的:分析ELISA方法和IHA方法检测弓形虫病的可靠性。方法:本文应用酶联免疫吸附试验(ELISA)和间接血凝试验(IHA)两种方法,平行检测来自龙岩市某个猪场的32份猪血清中的抗弓形虫特异性抗体。结果:ELISA和IHA对猪弓形虫抗体阳性检出率分别为62.5%(20/32)、53.13%(17/32),这两种方法的阳性检出符合率较高,为71.88%(23/32)。其中,ELISA方法的敏感性(93.33%)、检测效率(81.25%)以及Youden指数(63.92%)都在IHA方法之上,但ELISA方法的特异性(12/17,70.59%)略低于I-HA方法(13/17,76.47%)。结论:ELISA和IHA两种方法均可用于猪弓形虫病的诊断和血清学调查,但ELISA方法更适用于猪弓形虫病的血清学调查。     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

某猪场猪瘟免疫程序的调整及免疫效果评价

为了更好地预防和控制猪瘟,找出适合四川某猪场的猪瘟免疫程序,笔者根据实际情况对免疫程序进行了调整。然后用ELISA和IHA法对免疫程序调整前后的不同日龄猪血清进行了抗体检测,结果显示:调整前用ELISA法检测到3日龄、25日龄、50日龄、90日龄和120日龄猪只的猪瘟抗体阳性率分别为80%、54.54%、77.78%、80%和95%,用IHA法测得的阳性率分别为100%、63.64%、77.78%、90%和95%;调整后用ELISA法测得的猪瘟抗体阳性率分别为100%、80%、77.27%、77.78%和95%,用正向IHA法测得的抗体阳性率分别为97.73%、94.44%、96.15%、100%和100%。调整前用ELISA和IHA测得的平均阳性率为77.46%、85.28%,调整后则变为86.01%、97.66%,虽然均符合农业部颁布的猪群猪瘟抗体阳性率应不低于70%的标准,但免疫程序调整后的抗体阳性率明显高于调整前,可见调整后的免疫程序更适合于该猪场。     

应用斑点酶联免疫吸附试验快速检测猪弓形虫抗体

将斑点酶联免疫吸附试验(Dot—ELISA)用于检测猪弓形虫抗体,并与常规ELISA和IHA法进行了比较。结果,对102份滴度下降的猪阳性血清检测,弓形虫抗体阳性检出率,Dot—ELISA为66.67％(68/102),常规ELISA为48.04％(49/102),IHA为27.45％(28/102);对675头商品猪血清检测,弓形虫抗体阳性检出率,Dot—ELISA为48.15％(325/675),常规ELISA为41.93％(283/675),IHA为33.80％(228/675);与3种寄生虫(猪囊虫、猪旋毛虫、住肉孢子虫)阳性血清无交叉反应;对123份弓形虫抗体阳性和158份阴性猪血清进行3次重复性试验,结果完全一致。结果证明,该法敏感性高,特异性强,操作简便快速(于接到病料后2h报告结果),便于在基层推广使用。     

The effects of bovine viral diarrhoea-mucosal disease (BVD) virus on the ovine foetus

Bovine viral diarrhoea-mucosal disease (BVD) virus has been incriminated as a cause of abortion, hairy birth coat and unthriftiness in sheep. Intravenous inoculation of 40 ewes 34 to 45 days pregnant with the V/TOB strain of virus produced death in two of four foetuses 9 days after inoculation and in all but one of 31 foetuses between 11 and 56 days. The highest levels of virus in placentomes and foetal tissues occurred between 9 and 15 days after inoculation and in foetal fluids between 11 and 18 days. Virus was not detected in any foetus later than 21 days after inoculation. Groups of 10 ewes infected between 59 and 62 days (Group B) and 70 and 76 days (Group C) of gestation had 73% and 62%, respectively, of abortions or perinatal foetal deaths. Birth weights of lambs born to infected ewes in groups B and C were significantly lower than those born to uninfected control ewes. Virus was recovered consistently from the cotyledons of the foetal membranes of live lambs, and irregularly from the tissues of full term foetuses that were dead at birth but on no occasion from mummified foetuses. There were no specific gross or microscopic lesions in tissues selected from aborted foetuses and the results highlight the difficulties associated with the diagnosis of BVD abortions and perinatal death of foetuses under field conditions.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.