Single radial hemolysis test for the detection of rinderpest antibody

Summary The single radial hemolysis [SRH] test was employed for detection of rinderpest antibodies in post-vaccinated serum samples as also in serum samples from animals recovered from rinderpest infection. The results were compared with counterimmunoelectrophoresis [CIE] and serum neutralisation [SN] tests. The CIE test was found to be more sensitive than SRH but because of ease and simplicity SRH can also be used for monitoring antibody development after vaccination.

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Resumen Se utilizó la prueba radial sencilla de hemólisis (RSH) para detectar anticuerpos de rinderpest, en muestras de suero de animales vacunados y recuperados de la enfermedad. Los resultados se compararon con las pruebas de contrainmunoelectroforésis (CIE) y sero neutralización (SN). La prueba CIE fue más sensitiva que la RSH, pero debido a la sencillez de manejo, se recomienda la RSH para medir el nivel de anticuerpos post vacunales.

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Résumé Le test d’hémolyse radiale simple (HRS) a été utilisé pour la détection des anticorps antibovipestiques dans des échantillons de sérums après vaccination et aussi dans des échantillons de sérums d’animaux convalescents. Les résultats ont été comparés avec les tests de contre-immunoélectrophorèse (CIE) et de séroneutralisation (SN). On a trouvé que le test CIE est plus sensible que le HRS mais par suite de son aisance et de sa facilité, le HRS peut aussi être employé pour suivre le développement des anticorps après vaccination.

     

山羊痘血清学诊断技术研究

本实验研制了山羊痘的五种血清学检测方法并进行了比较分析，结果表明：琼脂凝胶扩散试验（AGP）适于基层兽医开展山羊痘病例的诊断，对流免疫电泳技术（CIE）能提高对抗原或抗体的分辨能力，荧光抗体技术（FAT）能对感染细胞进行山羊痘病毒定位检测，反向间接血凝试验（RPHA）主要用于临床痘疹痂皮和感染细胞中山羊痘抗原的定量测定，而正向间接血凝试验（IHA）不失为一种山羊痘抗体的最佳监测方法。     

Diagnosis of Rinderpest in Tanzania by a Rapid Chromatographic Strip-test

A simple chromatographic strip-test based on Clearview technology, is under development as a pen-side test for the detection of rinderpest antigen in eye swabs taken from cattle in the field. An outbreak of rinderpest occurred in the northern zone of Tanzania from late February to June 1997. The affected cattle exhibited very mild clinical signs, which made clinical diagnosis difficult. One hundred and seven eye swabs were collected from cattle suspected of infection with rinderpest. These were tested in the field using a prototype of the pen-side test and 13 (12.15%) of the samples were found to be positive for the presence of rinderpest antigen. These were confirmed by ICE. The positive cases were predominantly found in the Ngorongoro district. This demonstrates the usefulness of such a simple, rapid pen-side diagnostic assay, particularly when clinically `mild' strains of rinderpest are present.     

REVERSE PHASE PASSIVE HAEMAGGLUTINATION AND SINGLE RADIAL IMMUNODIFFUSION TO DETECT EPSILON ANTIGEN OF CLOSTRIDIUM PERFRINGENS TYPE D

Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem.     

Detection of duck plague virus by reverse passive hemagglutination test

A reverse passive hemagglutination (RPHA) test was developed to detect duck plague virus (DPV). The technique used sheep erythrocytes stabilized with formaldehyde and pyruvaldehyde and coated with immunoglobulin G (IgG) containing anti-DPV antibody prepared from antiserum produced in sheep. Optimum coating of stabilized erythrocytes occurred at 25 C and pH 4.0 with a concentration of IgG of 20-40 micrograms/ml and a 90-min incubation period. The coated cells were stable for 40 days when stored at 4 C or for at least 4 months (the longest period tested) when frozen at -70 C or -196 C. The RPHA test was conducted at 25 C and read after 3 hours. The high specificity of the test is indicated by the absence of cross-reactions with heterologous virus strains, with specimens prepared from normal duck livers, and with normal chicken embryo chorioallantoic fluid, as well as by the inhibition of hemagglutination only with DPV antiserum. The RPHA test detected six strains of DPV in all virus-containing specimens as well as the immunofluorescence (IF) test did; however, conventional plaque assays (PA) failed to detect virus in five specimens that contained three non-plaque-forming strains of DPV. The mean quantity of DPV that could be detected in the RPHA test was 25 plaque-forming units or 65 fluorescent units per ml. Although the RPHA test was less sensitive than either the PA or the IF test, there was a positive correlation in the titers of DPV antigens between all three tests. The RPHA test is a rapid, simple procedure that is sufficiently sensitive for diagnostic detection of DPV in acute infections, especially in tissues of ducks dying of the disease.     

Highly sensitive antigen detection procedures for the diagnosis of infectious bovine rhinotracheitis: amplified ELISA and reverse passive haemagglutination

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of bovid herpesvirus 1 antigen was increased by up to 50-fold using the biotin-avidin interaction to amplify the reaction, when compared with a simple sandwich ELISA. An alternative immunoassay, reverse passive haemagglutination (RPHA), had a similar sensitivity to the amplified ELISA, and was technically simpler to perform. Both the amplified ELISA and the RPHA could detect viral antigen in the nasal secretions of calves undergoing experimental primary infection with the virus from Day 3 to Day 7 after inoculation. Neither assay was as sensitive as virus isolation in cell culture and they failed to detect antigen in virus-positive samples from the calves from 8 days after inoculation, and from vaccinated calves undergoing challenge infection.     

Detection of antibodies to Mycobacterium johnei by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis (CIE) was applied in the detection of antibodies to Mycobacterium johnei in 110 sheep, 11 goat and 31 cattle sera and compared to immunodiffusion (ID) test. One per cent Noble agar, 7 ml per slide of 5 cm x 10 cm; barbitone-tris buffer, mu = 0.03, pH 8.6; a constant current of 5 mA per slide and M johnei protoplasmic antigen at 4 mg per ml were found to impart high sensitivity to CIE and give rapid results. CIE detected 97 sheep, 11 goat and 31 cattle positive sera, a total of 139, as compared to 44, 11, 28 and 83 respectively, detected by ID. Strongly positive sera could be demonstrated within 30 minutes by CIE and the test was run for only 90 minutes while earliest reactions were not observed before 18 hours and some reactions took 144 hours to develop in ID test.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

A comparison of three rapid diagnostic methods for the detection of rotavirus infection in calves

Three techniques for the detection of rotavirus in faecal samples from calves with neonatal gastroenteritis were compared. A preliminary study indicated that reverse passive haemagglutination (RPHA) was at least as sensitive as the enzyme-linked immunosorbent assay (ELISA). These two immunoassays were compared with the detection of viral RNA by polyacrylamide gel electrophoresis (PAGE) on 209 field samples. Of the 77 samples in which at least one test gave a positive result, 69 were positive by both RPHA and PAGE, but only 49 were also positive by ELISA, indicating a lower sensitivity for the latter test. The overall agreement between RPHA and PAGE was 96%. The reasons for the discrepancies between the tests are discussed.     

Pneumococcal antigen detection in cerebrospinal fluid: a comparative study on counter immunoelectrophoresis,latex agglutination and coagglutination

The sensitivity, specificity, accuracy and predictive values of counter immunoelectrophoresis (CIE), latex agglutination (LA) and coagglutination (CoAg) tests were compared for detection of pneumococcal antigen in cerebrospinal fluid (CSF) of patients suspected of meningitis. A total of 95 CSF samples comprising 15 culture proven, 47 clinically suspected but culture negative cases of meningitis and 33 controls were screened by above tests. Among three tests, LA was found to have high sensitivity and moderately high negative predictive value than CIE and CoAg tests. However, CIE had slightly better specificity than LA and CoAg tests. Accuracywise CIE and LA tests were comparable than CoAg test. CIE and LA tests had high positive predictive value than CoAg test.     

Comparison of rinderpest and peste des petits ruminants viruses using anti-nucleoprotein monoclonal antibodies

Monoclonal antibodies (MAbs) were obtained using a purified preparation of the RBOK strain of a rinderpest vaccine virus. The cytoplasmic immunofluorescent staining test showed that these clones had specificity for the nucleoprotein (N) of the virus. Six clones which immunoprecipitated the N protein corroborated these results. Thirteen anti-N MAbs were used to compare geographically widespread rinderpest viruses (RPV) and peste des petits ruminants viruses (PPRV) to two other morbilliviruses, measles (MV) and canine distemper (CDV). The N protein antigen profiles of the 23 isolates determined by immunofluorescent staining and enzyme linked immunosorbent assay (ELISA) on infected cells enabled us to classify the strains into groups. A differential identification of the morbilliviruses can be made using one MAb or associations of the MAbs. The potential to distinguish between RPV and PPRV and between virulent and avirulent strains of rinderpest is of primary interest.     

Detection of Rinderpest Virus Using N-Protein Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.     

The Use of Antigen-capture Enzyme-linked Immunosorbent Assay (ELISA) for the Diagnosis of Rinderpest and Peste des Petits Ruminants in Ethiopia

Rinderpest had been reported in most parts of Ethiopia when the Pan African Rinderpest Campaign (PARC) was launched. As a result of intensive disease investigation and strategic vaccination, most parts of the country are now considered provisionally free, and widespread vaccination has been replaced by clinical and serological surveillance. Details of any episodes of disease are recorded and followed up after laboratory confirmation of suspected cass using antigen-capture ELISA. This paper is based on observations on the performance of the antigen detection ELISA compared to the agar gel immunodiffusion (AGID) test, which also differentiates rinderpest from peste des petits ruminants (PPR). The stability of the specific viral antigen was monitored for 4 days, and rinderpest and PPR antigens were still detected, depending on the type of specimen. Antigen capture ELISA is more rapid, sensitive and virus specific than the AGID. Even if the cold chain of the specimen is compromised for a day or two during sample collection and submission, the specimen may still be suitable for testing by ELISA.     

Re-infection of wildlife populations with rinderpest virus on the periphery of the Somali ecosystem in East Africa

We report surveillance for rinderpest virus in wildlife populations in three major ecosystems of East Africa: Great Rift Valley, Somali and Tsavo from 1994 to 2003. Three hundred and eighty wild animals were sampled for detection of rinderpest virus, antigen or genome and 1133 sampled for antibody in sera from Kenya, Uganda, Ethiopia and Tanzania from 20 species. This was done modifying for wildlife the internationally recommended standards for rinderpest investigation and diagnosis in livestock. The animals were selected according to susceptibility and preference given to gregarious species, and populations were selected according to abundance, availability and association with livestock. Rinderpest virus, antigen and/or genome were detected in Kenya; within Tsavo, Nairobi and Meru National Parks. Serological results from 864 animals (of which 65% were buffalo) from the region were selected as unequivocal; showing the temporal and spatial aspects of past epidemics. Recent infection has been only in or peripheral to the Somali ecosystem (in Kenya). Our evidence supports the hypothesis that wildlife is not important in the long-term maintenance of rinderpest and that wildlife are infected sporadically most likely from a cattle source, although this needs to be proven in the Somali ecosystem. Wildlife will continue to be a key to monitoring the remaining virus circulation in Africa.     

An immunoenzyme test used for the detection of antibodies to rinderpest: the advantage of using a purified virus cultivated in a cell line

Two types of in vitro assays (enzyme immunoassay and sero-neutralization test) were compared for their ability to detect antibodies to rinderpest virus in field sera from West African bovines. Purified virus grown on Vero cells (Ag/Vero) or bovine kidney cells (Ag/BK), were tested as antigen in enzyme-linked immunoassays (EIA). The results of the comparative evaluation of the two antigens by EIA, prove that Ag/Vero did enhance the sensitivity and the specificity of the test in comparison to Ag/BK and offers a 94% correspondence with seroneutralization.     

Microelisa test for detecting antibodies to rinderpest virus antigens

Summary A microplate enzyme-linked immunosorbent assay (ELISA) was developed which detected antibodies to a soluble antigen prepared from sonicated rinderpest virus-infected cells. The ELISA detected titres of antibody to the virus in the sera of cattle 3 weeks after immunisation with tissue culture rinderpest virus vaccine which were similar to those detected by the virus neutralisation test. The ELISA test shows potential as a rapid and economic technique for screening large numbers of sera for antibody to rinderpest virus.

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La Prueba Micro-Elisa Para Detectar Anticuerpos Producidos Por Antigenos Del Virus De Rinderpest

Resumen Se utilizó la prueba micro-ELISA para detectar anticuerpos producidos por un antígeno soluble preparado con células sonicadasinfectadas con el virus de rinderpest. La prueba ELISA detectó anticuerpos en el suero de bovinos, 3 semanas después de que éstos fueron inmunizados con la vacuna de rinderpest, preparada ésta en cultivos celulares. Los anticuerpos detectados fueron similares a los estudiados mediante la prueba de neutralización viral. La prueba ELISA se perfila como una técnica rápida y económica para trabajar un número apreciable de muestras de suero con el fin de detectar anticuerpos del virus de rinderpest.

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Un Micro-Test Elisa Pour Deceler Les Anticorps Specifiques Du Vir Us Bovipestique

Résumé Un test immuno-enzymatique (ELISA) a été mis au point déceler les anticorps correspondant à un antigène préparé par traitement aux ultra-sons de cellules infectées. Les titres sériques obtenus par cette méthode dans les sérums de bovins immunisés trois semaines auparavant avec du vaccin de culture cellulaire se sont révélés comparables à ceux obtenus par la méthode classique de séroneutralisation. Le test ELISA apparait comme un moyen rapide et économique pour rechercher les anticorps spécifiques du virus bovipestique dans des sérums en grand nombre.

     

Detection of goat pox antigen and antibody by the counter immunoelectrophoresis test

Summary The counterimmunoelectrophoresis (CIE) test was standardised for the detection of goat pox antigen and antibody using inactivated antigens. The chloroform inactivated and live antigens were equally sensitive for detection of goat pox precipitins. The precipitinogens of goat pox virus (GPV) were found to be soluble in nature. The CIE test was quick as well as more sensitive than the agar gel precipitation test for detection of GPV antibody/antigen. The CIE employing inactivated antigen has been used for the first time in the detection of GPV antibodies/antigens.

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Deteccion Del Antigeno Y Anticuerpos De Viruela Caprina Mediate La Prueba De Contrainmunoelectroforesis

Resumen Se estandarizó la prueba de contrainmunoelectroforesis, para la detección del antígeno y anticuerpos del virus de la viruela caprina, usando antígenos inactivados. El antígeno inactivado con cloroformo y el antígeno vivo, fueron igualmente sensitivos para la detección de precipitinas de viruela caprina. Los precipitógenos del virus de la viruela caprina se encontraron que eran solubles. La prueba de contrainmunoelectroforesis fue más rápida y más sensitiva que la precipitación agar gelatina para la detección de anticuerpos/antígenos del virus de la viruela caprina. La prueba de contrainmunoelectroforesis con antígeno inactivado ha sido utilizada por vez primera en la detección de anticuerpos/antígenos del virus de la viruela caprina.

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Detection De l'Antigene Et De l'Anticorps Variole Caprine Par Un Test De Contrimmuno-Electrophorese

Résumé Le test de contrimmuno-électrophorèse (CIE) a été standardisé pour la détection de l'antigène et de l'anticorps variole caprine avec des antigènes inactivés. Les antigènes vivants et inactivés par le chloroforme sont de sensibilité équivalente pour la détection des précipitines variole caprine. On a montré que ces précipitogènes du virus variole caprine (VVC) étaient de nature soluble. Le CIE est rapide et plus sensible que le test de précipitation en gélose pour la détection des antigènes et anticorps VVC. C'est la première fois que le CIE mettant en oeuvre un antigène inactivé a été utilisé.

     

Detection of rinderpest virus antigens in vitro and in vivo by direct immunofluorescence

Cell-culture attenuated and virulent strains of rinderpest virus (RV) were inoculated on to bovine kidney cell cultures. A direct immunofluorescent antibody test detected RV antigens in cell cultures within one to three days after inoculation whereas RV cytopathic effects usually took three to nine days to develop. Cells containing RV antigens were also detected in impression smears and frozen sections of tissues collected from RV infected animals at post mortem examination, and in smears of lymph node biopsies taken from cattle with clinical rinderpest. These techniques may offer additional methods for rapid diagnosis of rinderpest.     

The risk of rinderpest re-introduction in post-eradication era

In 2011, ten years after the last reported outbreak, the eradication of rinderpest was declared. However, as rinderpest virus stocks still exist, there remains a risk of rinderpest re-introduction.