Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis / Padronização de um ELISA indireto (iELISA) para diagnóstico da leucose enzoótica bovina

Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa natural dos bovinos com distribuição mundial causada pelo "bovine leukemia virus" (BLV). A imunodifusão em gel de ágar (IDGA) foi considerada por muitos anos o teste de eleição, porém ensaios imunoenzimáticos (ELISA) apresentam sensibilidade mais elevada e leitura mais rápida e objetiva. No entanto, a importação de kits de ELISA é um processo dispendioso e demorado, e atualmente não há kits de IDGA comercialmente disponíveis no Brasil. Desta forma, o objetivo deste trabalho foi padronizar um ELISA indireto (iELISA) para diagnóstico da LEB utilizando antígenos produzidos a partir do cultivo do BLV em linhagem celular Tadarida brasiliensis "lung" (Tb1Lu) livre de "bovine viral diarrhea virus" (BVDV), diferentemente do que acontece com as linhagens "fetal lamb kidney" (FLK) atualmente utilizadas na produção desses antígenos para uso em ensaios sorológicos. Após a padronização do iELISA, os resultados foram comparados com aqueles obtidos por IDGA e pelo ELISA comercial "Chekit Leucose-Serum". Comparado ao IDGA, o iELISA apresentou 94,44% de sensibilidade, 75,68% de especificidade, valor preditivo positivo (VPP) de 79,1% e valor preditivo negativo (VPN) de 93,3%, com concordância entre os testes de 84% e o índice Kappa 0,699. Quando comparado ao ELISA "Chekit Leucose-Serum", o iELISA apresentou sensibilidade de 92,6%, especificidade de 87,09%, VPP de 90,27% e VPN de 90%, com concordância de 90,27% e o índice Kappa 0,801. Portanto, devido à alta concordância com os testes tradicionais e ausência da ocorrência de reações inespecíficas com BVDV, o ensaio desenvolvido pode ser utilizado como ferramenta diagnóstica para o controle da LEB no Brasil.(AU)

Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen

Canine adenovirus type 1 (CAV-1) causes infectious hepatitis in members of the family Canidae, including dogs. An indirect enzyme-linked immunosorbent assay (I-ELISA) that detects CAV-1 antibodies is required for large-throughput tests of dog sera. We collected 165 serum samples from dogs of Chungbuk and Gyeongbuk provinces between February 2016 and October 2018. The Korean CAV-1 vaccine strain CAV1V was propagated in Madin-Darby canine kidney (MDCK) cells and purified via Nuvia cPrime anion-exchange chromatography; the virus served as an I-ELISA antigen. Virus-neutralizing anti-CAV-1 titers in dog sera were measured using the virus neutralization (VN) method. The I-ELISA was optimized using purified CAV-1 antigen and serum samples. This kit was used to evaluate dog sera. The VN and I-ELISA data were compared. The sensitivity, specificity, and accuracy of the I-ELISA were 97.0%, 74.2%, and 92.7% compared to the VN assay, respectively. The I-ELISA data significantly correlated with those of VN (r = 0.88). These results suggest that the I-ELISA is useful for serosurveillance of CAV-1 in dog sera.

Comparative Evaluation of Serological Tests for Diagnosis of Brucellosis in Bovines with History of Abortion in Punjab

Brucellosis is highly infectious zoonotic disease that causes huge economic losses to livestock farmers by affecting the reproductive potential of animals causing last trimester abortions and infertility. In the present study evaluation of different serological tests to diagnose the seroprevalence of brucellosis in bovines with history of abortion using various serological tests [Rose Bengal Plate Test (RBPT), modified rose bengal plate test (mRBPT), microtitre plate agglutination test (MAT) and indirect enzyme linked immunosorbent assay (i-ELISA)] was carried out. A total of 134 blood samples of cattle and buffalo with history of abortion were collected from organized and unorganized farms. Seroprevalence by mRBPT, RBPT, MAT and i-ELISA was 75.37%, 67.91%, 72.38% and 72.38%, respectively. In organized farms, prevalence of 78.12%, 81.25%, 78.12% and 81.25% while in unorganized farms prevalence of 64.70%, 73.52%, 70.58% and 69.60% was reported by RBPT, mRBPT, MAT and i-ELISA, respectively. The sensitivity and specificity of serological tests by keeping i-ELISA as gold standard were also calculated and the results revealed that sensitivities of RBPT, mRBPT and MAT were 91.75%, 97.94% and 96.91%, respectively, whereas specificities were 94.59%, 83.78% and 91.89%, respectively.

Ferramentas diagnósticas de Lentivirose de Pequenos Ruminantes: padronização da técnica de ELISA indireto / Diagnostic tools of small ruminant lentiviruses: standardization of indirect ELISA

As Lentiviroses de Pequenos Ruminantes (LVPR) incluem a Maedi-Visna (MV) em ovinos e a Artrite Encefalite Caprina (CAE). Essas enfermidades estão difundidas no mundo e são responsáveis por grandes perdas na produtividade destes animais. Os LVPR são vírus RNA da subfamília Lentivirinae que causam uma infecção persistente, sendo a detecção precoce uma das formas mais eficientes para limitar sua disseminação no rebanho. Visando contribuir com essas questões, este experimento foi realizado na Universidade Federal do Piauí (UFPI) em parceria com a Embrapa Caprinos e Ovinos, com o objetivo de padronizar a técnica de ensaio imunoenzimático indireto e compará-lo com a imunodifusão em gel de agarose no diagnóstico da CAE. Foram utilizadas 696 amostras de soros de caprinos machos e fêmeas oriundas do banco de soros da Unidade de Pesquisa de LVPR do Centro de Ciências Agrárias da UFPI. As amostras foram coletadas no período de janeiro de 2007 a março de 2010. Na padronização, verificou-se que 0,25 µg de proteína/poço, diluição de 1:200 do soro e concentração de 1:3.000 do conjugado anticorpo anti-IgG cabra apresentaram os melhores resultados. O ponto de corte obtido foi de 0,36. Na comparação, o Imunodifusão em Gel de Ágar (IDGA) detectou 128 (18,4%) amostras positivas, e o ELISA indireto (ELISA-i), 259 (37,2%). A sensibilidade e a especificidade do teste ELISA-i com relação ao IDGA foi de 94,5% e 75,7%, respectivamente. Verificou-se maior índice de positividade em caprinos acima de seis meses (p < 0,05), e nos machos obteve-se prevalência de 56,7% em comparação às fêmeas, 35,4%, (p < 0,01).(AU)

The Small Ruminant Lentiviruses (SRLVs) include Maedi-Visna (MV) of sheep and Caprine Arthritis-Encephalitis (CAE). These diseases are widespread and responsible for major production losses regarding sheep and goats. The SRLV is a RNA virus of the subfamily Lentivirus genus that causes persistent infections in goats. Early detection is one of the best ways to limit its spread in the herd. To contribute to these issues, this experiment was conducted at Universidade Federal do Piauí in partnership with Embrapa Goats and Sheep, with the objective of standardizing the technique of indirect ELISA (i-ELISA) and to compare it with Immunodiffusion in Agarose Gel to diagnose Caprine Lentiviruses (LC). Six hundred ninety six serum samples were used from the University Veterinary Hospital, Universidade Federal do Piauí, from January 2007 to March 2010. Standardization showed that 0.25 µg protein/well, a 1:200 dilution of the serum and concentration of 1:3,000 of the conjugated anti-goat IgG presented the best results. It was observed that the Agar Gel Immunodiffusion (AGID) detected 128 (18.4%) positive samples, and ELISA, 259 (37.2%). The sensitivity and specificity of i-ELISA regarding AGID were 94.5% and 75.7%, respectively. A higher prevalence was observed among animals older than six months (p < 0.05). The prevalence among males was of 56.7%, and among females, 35.4% (p < 0.01).(AU)

Comparison of Brucellacapt and iELISA in serological diagnosis of brucellosis / 中国人兽共患病学报

Brucellacapt is a new serological test based on immunocapture-agglutination technique that has been used in many countries ,but there was no related article reported in China .The value of the Brucellacapt method in diagnosing human brucellosis was evaluated in this study .Among 120 suspected patients ,75 patients and 45 negatives were diagnosed by SAT and RBPT method combination with their clinical symptoms .Sera from all 120 people were tested by the method of Brucella-capt and iELISA ,and the results were ,consequently ,analyzed and compared .It showed that sensitivity ,specificity ,consis-tency rate ,Kappa value ,and area under ROC curve were found to be 82 .7% ,88 .9% ,85 .0% ,0 .69 ,and 0 .86 ,respectively for Brucellacapt ,whereas they were found to be 90 .7% ,64 .4% ,80 .8% ,0 .57 ,and 0 .78 for iELISA .In conclusion ,specific-ity ,consistency rate ,Kappa value ,and area under ROC curve were higher in Brucellacapt method than that in iELISA .How-ever ,the sensitivity of iELISA is higher .

Prevalência de anticorpos anti-Toxoplasma gondii em búfalos (Bubalus bubalis) no Estado do Pará / Serological prevalence of Toxoplasma gondii in water buffaloes (Bubalus bubalis) in Marajó Island, State of Pará, Brazil

O objetivo do estudo foi conhecer a prevalência sorológica de Toxoplasma gondii em búfalos (Bubalus bubalis) do Estado do Pará, Brasil. Foram selecionados randomicamente 319 bubalinos distribuídos em sete municípios da Ilha do Marajó. Para efeito comparativo também foram avaliados 128 bubalinos pertencentes a cinco municípios do Estado do Pará. A prevalência sorológica de Toxoplasma gondii foi avaliada pelo Ensaio de Imunoadsorção Enzimático Indireto (iELISA). As amostras diagnósticadas como positivas no iELISA foram submetidas a Reação de Imunofluorescência Indireta (RIFI). Foram avaliados os fatores de risco: localidade, raça, gestação, co-infecção por Brucella abortus e co-infecção por Mycobacterium bovis. As frequências de animais positivos no iELISA para T. gondii foram comparadas pelo teste de Qui-quadrado (χ2) com 95% de confiabilidade. As variáveis com p<0,2 foram submetidos à análise de regressão logística, sendo o modelo construído baseado no teste da "odds ratios". A prevalência de T. gondii observada no iELISA foi de 41,6% (186/447). Na RIFI, 86,5% (161/186) das amostram positivas no iELISA tiveram sua positividade para T. gondii confirmada. A prevalência média nos municípios da Ilha do Marajo e do Continente foi de 32% (103/319) e 55% (70/128), respectivamente. Os municípios que apresentaram as maiores prevalências foram Soure (53%) e Salvaterra (49%) na Ilha do Marajó e Castanhal (55%) e Tailândia (50%) no Continente. Os fatores de risco raça e co-infecção por Brucella abortus ou Mycobacterium bovis não influenciaram na prevalência de T. gondii. Além disso, animais gestantes foram 57% mais positivos para T. gondii do que animais não gestantes. A circulação de anticorpos é um indicativo da presença do agente da toxoplasmose em búfalos no Estado do Pará. Esses achados representam um risco não apenas para os animais de produção, mas à saúde pública, como uma fonte de infecção.

The aim was to study the seroprevalence of Toxoplasma gondii in water buffaloes (Bubalus bubalis) from State of Pará, Brazil. Three hundred and nineteen buffaloes were randomly selected into seven municipalities of Marajó Island. For comparative purposes, 128 buffaloes of five municipalities in the state of Pará were also evaluated. The seroprevalence of T. gondii was evaluated by Indirect Enzyme Linked Immunosorbent Assay (iELISA). The samples diagnosed as positive in iELISA were subjected to Immunofluorescence Antibody Test (IFAT). We evaluated risk factors: location, breed, pregnancy and co-infection with Brucella abortus or Mycobacterium bovis. The frequency of animals positive for T. gondii in iELISA were compared by chi-square (χ2) with 95% confidence. Variables with p <0.2 were subjected to logistic regression analysis; the model was built based on the "odds ratios" test. The prevalence of T. gondii in iELISA was 41,6% (186/447). In IFAT, 86,5% (161/186) had their positivity for T. gondii confirmed. The average prevalence in the municipalities of the Marajó Island and of the mainland was 32% (103/319) and 55% (70/128), respectively. The municipalities with the highest prevalence were Soure (53%) and Salvaterra (49%) in Marajó Island, and Castanhal (55%) and Thailândia (50%) in the Continent. The breed and co-infection with Brucella abortus or Mycobacterium bovis presented no influence on the prevalence of T. gondii. Additionally, pregnant animals were 57% more positive for T. gondii than nonpregnant animals. The presence of antibodies is an indicative of T. gondii in buffaloes in the state of Pará, and these findings represent a risk not only for farm animals, but to public health as a source of infection.

Comparação dos testes sorológicos de Imunofluorescência Indireta, Conglutinação Rápida, ELISA indireto e ELISA por competição para a detecção de anticorpos contra o Anaplasma marginale em soros de bovinos de diferentes áreas enzoóticas / Comparison of the serologic tests of Indirect Immunofluorescence,Rapid Conglutination, Indirect ELISA and Competition ELISA fordetection of antibodies against Anaplasma marginale in cattle sera fromdifferent enzootic areas

Os Testes Sorológicos de Conglutinação Rápida (TCR) Imunofluorescência Indireta (IFI) e Imunoenzimáticos Indireto (iELISA) utilizando ELISA por competição (cELISA), como padrão ouro, foram avaliados comparativamente para a detecção de anticorpos contra o Anaplasma marginale. Foram utilizadas 453 amostras de soros sangüíneos de bovinos vacinados e não-vacinados e de áreas de estabilidade e instabilidade enzoótica. O iELISA, IFI e TCR apresentaram respectivamente, índice kappa=0,77 (substancial), 0,57 e 0,49 (moderado), sensibilidade de 90,6, 90,2 e 73,7 e especificidade de 86,6, 62,8, e 79,3. O iELISA apresentou o melhor desempenho e maior especificidade, podendo ser indicado na avaliação do perfil sorológico de rebanhos, na detecção de animais persistentemente infectados e de animais submetidos a programas de vacinação. As técnicas de IFI e TCR, mesmo apresentando desempenho inferior, podem ser recomendadas para a realização de inquéritos epidemiológicos e para o monitoramento de animais em trânsito entre as diferentes regiões geográficas

The serological techniques Rapid Conglutination Test (RCT), Indirect ELISA (iELISA) and IndirectImmunofluorescent Assay (IFA), using the competition ELISA (cELISA) as gold test, were comparativelyevaluated to detect antibodies against Anaplasma marginale. A total of 453 sera from vaccinated andnon vaccinated cattle and, collected from enzootic stability and instability areas were tested. iELISA, IFAand TCR presented kappa index = 0.77 (substantial); 0.57 and 0.49 (moderate), sensibility of 90.6%;90.2% and 73.7% and specificity of 86.6%; 62.8%, and 79.3%, respectively. Therefore, iELISA presentedbetter specificity than IFA and RCT, and can be indicated for more detailed serological investigations,detection of persistently infected animals in cattle herds and for monitorating of vaccination programs.IFA and TCR can be used in prevalence studies and to monitor cattle movement between differentgeographical regions

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.