Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene

Background & objectives: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. Methods: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. Results: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. Interpretation & conclusion: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition

Detecção sorológica e molecular de Anaplasma marginale em búfalos na Ilha de Marajó, Pará / Serological and molecular detection of Anaplasma marginale in water buffaloes on Marajó Island, State of Pará, Brazil

O objetivo do estudo foi testar a prevalência sorológica e molecular de Anaplasma marginale em búfalos do municipio de Soure, Ilha de Marajó, estado do Pará, Brasil. Para a pesquisa sorologica foram selecionados randomicamente 800 animais e para a pesquisa molecular 50 destes animais foram aleatoriamente escolhidos. Para quantificar a prevalência sorológica utilizou-se o ensaio de imunoadsorção enzimático indireto (iELISA) com antígeno total contendo proteínas de superfície externa e para quantificar a prevalência molecular utilizou-se a reação em cadeia da polimerase (PCR), envolvendo a amplificação de fragmento gênico da proteína de superfície maior 5 (MSP5). A prevalência de animais positivos no ELISA para A. marginale foi de 25% (200/800). Na PCR foi detectada a presença de A. marginale em 2% (1/50) dos animais. Embora apenas um animal tenha sido positivo na PCR, observou-se que o mesmo foi negativo no ELISA. A presença do agente, mesmo em baixa prevalência, mostra que os bubalinos podem funcionar como um importante reservatório desse patógeno para os rebanhos bovinos da região norte do Brasil.

The aim of the study was to test the molecular and serological prevalence of Anaplasma marginale in water buffaloes of the Marajó Island, State of Pará, Brazil. For serologic research were randomly selected 800 buffaloes and for molecular research 50 of these animals were randomly chosen. To quantify the serological prevalence we used the indirect enzyme linked immunosorbent assay (iELISA) with total antigen containing proteins outer surface. To quantify the prevalence molecular was used the polymerase chain reaction (PCR) involving gene amplification fragment larger surface protein 5 (MSP5). The prevalence of positive animals in iELISA was 25% (200/800). In the PCR we detected the presence of A. marginale in 2% (1/50) of animals. Although only one animal was positive in PCR, we found that it was negative in ELISA. The presence of the agent, even in low prevalence, shows that buffaloes can act as an important reservoir for transmission of the pathogen to cattle in northern Brazil.

Diagnóstico molecular de Anaplasma marginale em bovinos: avaliação quantitativa de uma PCR em tempo real baseada no gene msp5 / Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

A riquétsia Anaplasma marginale é considerada o principal agente da anaplasmose bovina. Devido a não especificidade dos sinais clínicos, a confirmação da infecção nos animais depende de testes laboratoriais. Recentemente, métodos de diagnóstico molecular têm sido aplicados para detecção de A. marginale em bovinos. No entanto, a grande quantidade de testes com diferentes sensibilidade e custos tem dificultado a escolha do ensaio mais adequado. No presente estudo, uma PCR em tempo real baseada no gene msp5 foi avaliada quantitativamente e comparada a uma reação de PCR convencional. As reações foram submetidas à avaliação de sensibilidade e especificidade com DNA plasmidial e amostras provenientes de bovinos experimentalmente infectados por A. marginale. Uma avaliação comparativa a campo foi realizada entre os testes utilizando amostras provenientes de bovinos criados em uma região de estabilidade enzoótica para A. marginale. Embora os testes não tenham apresentado diferença estatisticamente significativa, a PCR em tempo real apresentou valor de sensibilidade maior do que a PCR convencional. A PCR em tempo real foi capaz de detectar uma cópia de msp5 em 100ng de DNA plasmidial, e mais de 80% de resultados positivos entre bovinos experimentalmente infectados apenas sete dias após infecção. Além disso, baseado em análise in silico, a PCR em tempo real avaliada aqui pode ser útil para detecção de Anaplasma ovis.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Immune response of calves inoculated with proteins of Anaplasma marginale bound to an immunostimulant complex / Resposta imune de bezerros inoculados com proteínas de Anaplasma marginale ligadas a um complexo imunoestimulante

Despite our current knowledge of the immunology, pathology, and genetics of Anaplasma marginale, prevention in cattle is currently based on old standbys, including live attenuated vaccines, antibiotic treatment, and maintaining enzootic stability in cattle herds. In the present study, we evaluated the use of an immunostimulant complex (ISCOMATRIX) adjuvant, associated with a pool of recombinant major surface proteins (rMSP1a, rMSP1b, rMSP4 and rMSP5) to improve the humoral immune response triggered in calves mainly by IgG2. Ten calves were divided in three groups: 4 calves were inoculated with the ISCOMATRIX/rMSPs (G1); 2 calves were inoculated with ISCOMATRIX adjuvant (G2); and 4 calves received saline (G3). Three inoculations were administered at 21-day intervals. In G1, the calves showed significant increases in total IgG, IgG1 and IgG2 levels 21 days after the second inoculation, compared to the control group (p < 0.05), and G1 calves remained above the cut-off value 28 days after the third inoculation (p < 0.05). The post-immunized sera from calves in G1 reacted specifically for each of the rMSPs used. In conclusion, the ISCOMATRIX/rMSPs induced antigen-specific seroconversion in calves. Therefore, additional testing to explore the protection induced by rMSPs, both alone and in conjunction with proteins previously identified as subdominant epitopes, is warranted.

Apesar dos avanços da imunologia, patologia e genética de Anaplasma marginale, a prevenção em bovinos ainda é baseada nas vacinas vivas atenuadas, na terapia com antibiótico e estabilidade enzoótica dos rebanhos bovinos. No presente estudo, avaliou-se o uso de um complexo imunoestimulante (ISCOMATRIX), associado às proteínas recombinantes de superfície (rMSP1a, rMSP1b, rMSP4 e rMSP5) para melhorar a resposta imune humoral desencadeada em bezerros, principalmente por IgG2. Dez animais foram divididos em três grupos: 4 bezerros foram inoculados com o ISCOMATRIX/rMSPs (G1), 2 bezerros foram inoculados com ISCOMATRIX adjuvante (G2) e 4 bezerros receberam salina (G3). Três doses vacinais foram administradas em intervalos de 21 dias. No G1, os bezerros apresentaram aumentos significativos nos níveis de IgG total, IgG1 e IgG2 21 dias após a segunda inoculação, em comparação com o grupo de controle (p <0,05). Nos bezerros do G1 esses níveis de anticorpos permaneceram acima do ponto de corte 28 dias após a terceira inoculação (p < 0,05). Os soros pós-imunização de bezerros do G1 reagiram especificamente com cada uma das rMSPs utilizadas. Em conclusão, o ISCOMATRIX/rMSPs induziu soroconversão antígeno-específica em bezerros. Portanto, se justifica a realização de ensaios adicionais para explorar a proteção induzida pela rMSPs, tanto sozinhas como em conjunto com novas proteínas identificadas com epítopos subdominantes.

Serological evidence of Anaplasma spp. in small ruminants from Venezuela using recombinant MSP5 in immunoenzymatic assay / Evidencia serológica de Anaplasma spp. en pequeños rumiantes de Venezuela utilizando MSP5 recombinante en ensayos inmunoenzimáticos

Anaplasma marginale causes a disease in cattle characterized by fever, anemia and decrease in milk and meat production. Small ruminants do not show signs of disease when infected, but it has been suggested they could act as reservoirs. Goat and sheep breeding is socially and economically important in arid and semi-arid areas in Venezuela, and these species often share space and food with cattle. The aim of this work was to detect antibodies against Anaplasma spp. in Venezuelan goat and sheep flocks. To accomplish this goal, an indirect ELISA using recombinant MSP5 as antigen of A. marginale was performed. Sera obtained from experimental infection in goat and a hyperimmune sheep serum were used as positive controls. Blood sera were obtained from 45 sheep and 48 goats located in Guárico State, an endemic area to bovine anaplasmosis. After standardization of assay for each species, 80.46% of the sheep and 59.25% of the goat sera showed to have antibodies against MSP5. No signs of clinical disease were detected in sampled animals. These results suggest that small ruminants could harbour A. marginale and consequently may be reservoirs for neighbouring cattle if appropriate vectors are present. The development of clinical diseases caused by A. marginale under stress situations and the existence of other Anaplasma species (e.g. A. ovis) in small ruminants should also be investigated.

Anaplasma marginale ocasiona una enfermedad en los bovinos caracterizada por fiebre, anemia y disminución de la producción de leche y carne. Los pequeños rumiantes generalmente no muestran signos clínicos, por lo que pudieran actuar como reservorio. En Venezuela, los ovinos y caprinos tienen gran importancia económica y socialmente en zonas áridas y semi- áridas e incluso, en muchas ocasiones comparten su espacio y alimento con los bovinos. El objetivo de este trabajo fue detectar anticuerpos contra Anaplasma spp. en rebaños de ovinos y caprinos. Para ello, se estandarizó un ELISA indirecto con la MSP5 recombinante de A. marginale, empleando sueros provenientes de infecciones experimentales en caprinos y un suero hiperinmune ovino como controles positivos. Posteriormente, fueron obtenidos sueros sanguíneos de 45 ovinos y 48 caprinos localizados en una zona endémica a anaplasmosis bovina del estado Guárico. De estos, 80,46% de los ovinos y 59,25% de los caprinos presentaron anticuerpos que reconocieron la MSP5, sin embargo, ninguno de estos animales positivos presentaron signos clínicos de la enfermedad. Estos resultados sugieren que los pequeños rumiantes son portadores de A. marginale y por ende, pueden estar actuando como reservorio de la enfermedad para los bovinos en el caso que se encuentren los vectores apropiados. Por lo tanto, se debe profundizar en los estudios sobre el desarrollo de sintomatología clínica en condiciones de estrés y la existencia de otras especies de Anaplasma (como A. ovis) en los ovinos y caprinos de Venezuela.

Soroprevalência de Anaplasma marginale em bovinos da região Centro-Sul do estado do Paraná, Brasil, por um teste imunoenzimático competitivo utilizando proteína recombinante MSP5-PR1 / Seroprevalence of Anaplasma marginale in cattle from Center-South Region of Paraná State, Brazil by a competitive ELISA test with recombinant MSP5-PR1 protein

Foi determinada a prevalência de Anaplasma marginale em 223 soros de bovinos com idade maior ou igual a dois anos, das regiões de Ponta Grossa, Guarapuava e Laranjeiras do Sul, região Centro-Sul do estado do Paraná. O teste imunoenzimático de competição PR1 (cELISA-PR1) foi utilizado para determinar a presença ou ausência de anticorpos anti-A. marginale. Dos 223 soros analisados, 130 (58,74 por cento) foram positivos pelo cELISA-PR1, sugerindo ser essa região de instabilidade de enzoótica, com uma porcentagem significativa de animais susceptíveis à infecção por A. marginale, com risco potencial para desenvolver a anaplasmose. As características de tipo de exploração da propriedade, sistema de criação, manejo e forma de comercialização dos animais foram avaliadas. A análise estatística não demonstrou haver diferença significativa entre as variáveis estudadas e a positividade dos animais.

Anaplasma marginale prevalence was determined in 223 sera samples in 2-year old or older cattle, from the Center- Southern Region of the Paraná State, including Ponta Grossa, Guarapuava and Laranjeiras do Sul municipalities. A survey of antibodies IgG class against Anaplasma marginale was performed through a competitive immune absorbent assay (cELISA-PR1). From the 223 sera examined, 130 (58.74 percent) reacted to cELISA-PR1 test, suggesting an region of enzootic instability, with a significant percentage of animals susceptible to infection by A. marginale and potentially in risk to develop anaplasmosis. The kind of exploration in the property, the breeding and handling system, the presence of other animals (ovine/caprine, horses, wild animals), and means of commercialization of animals were analyzed. The statistical analysis showed that there were no significant differences among the analyzed variables.

Detecção de anticorpos para Anaplasma sp. em pequenos ruminantes no semi-árido do estado de Pernambuco, Brasil / Detection of antibodies against Anaplasma sp. in small ruminants from the semi-arid region against Pernambuco State, Brazil

Neste trabalho é descrita a detecção de anticorpos para Anaplasma sp. em caprinos e ovinos da região do semi-árido do Estado de Pernambuco, Brasil, utilizando-se um ensaio de imunoadsorção enzimática baseado em MSP5 recombinante de Anaplasma marginale. Foram analisados soros de 243 caprinos e 68 ovinos provenientes do município de Ibimirim, e observadas freqüências de anticorpos de 11,93 por cento (29/243) e 16,17 por cento (11/68) para caprinos e ovinos, respectivamente. A importância epidemiológica dos achados foi discutida.

This paper reports the detection of antibodies against Anaplasma sp. in goats and sheep from the semi-arid region from Pernambuco State, Brazil, using an enzyme-linked immunosorbent assay with recombinant MSP5 of Anaplasma marginale. Sera from 243 goats and 68 sheep from Ibimirim municipality were analyzed and frequencies of antibodies of 11.93 percent (29/243) and 16.17 percent (11/68) were found for goats and sheep, respectively. The epidemiological relevance of the findings was discussed.

Evaluación y mejoramiento del ensayo inmunoenzimático (ELISA) para el diagnóstico de la anaplasmosis bovina, utilizando las MSP5 recombinante como antígeno / Evaluation and improvement of an immunoenzimatic assay (ELISA) for diagnosis of bovine anaplasmosis, using recombinant MSP5 as antigen

La anaplasmosis, es una enfermedad producida por la bacteria Anaplasma marginale que está ampliamente distribuida en Venezuela, originando efectos negativos en la salud y productividad de los rebaños bovinos. Hasta el presente se han caracterizado 6 proteínas mayoritarias de superficie (MSP) de esta bacteria, de las cuales la MSP5, ha sido señalada como un excelente polipéptido para el diagnóstico de la enfermedad, debido a que esta proteína es altamente conservada e inmunogénica. Esto ha motivado su clonamiento e inserción en un plásmido de E. coli, para usarla purificada como antígeno en ensayos inmunoenzimáticos. Sin embargo, estudios posteriores, indican que proteínas de E. coli recombinante que eluyen conjuntamente con la MSP5 durante el proceso de purificación, interfieren en el ELISA originando falsos positivos. En el presente trabajo se estandarizó un ELISA indirecto, utilizando la MSP5 como antígeno y se logró disminuir las uniones inespecíficas a las proteínas contaminantes de E. coli, por adición de un suero de conejo anti E. coli que bloquea los epítopes de estas proteínas. A través de un cuadro de contingencia de doble entrada, se determinaron los parámetros de validación del ELISA al compararla con la técnica de naranja de acridina-bromuro de etídio, obteniéndose como resultado que la técnica de ELISA mejorada es 96,1 por ciento sensible, 9 por ciento específica y presenta un valor predictivo del 88,6 por ciento. Además, se estudió una población bovina de 48 mautes de la Estación Experimental La Iguana (estado Guárico), utilizando ambas técnicas, obteniendo una seroprevalencia de 93,7 por ciento por ELISA y una prevalencia de 54,1 por ciento por naranja de acridina-bromuro de etidio. Estos resultados muestran que el bloqueo de los epítopes de las proteínas de E. coli contaminates, utilizando para ello un suero de conejo anti-E. coli, permite disminuir los falsos negativos cuando se utilizan proteínas recombinantes.

Anaplasmosis, is a disease produced by Anaplasma marginale widely distributed in Venezuela, causing negative effects on the health and productivity of bovine herds. Until now, 6 constitutive Anaplasma marginale Mayor Surface Proteins (MSPs) have been characterized, including MSP5 which appears to be an excellent polypeptide for the diagnosis of this disease since it is highly conserved and immunogenic. This has motivated its cloning and insertion into a plasmid in E. coli and the use of the purified antigen in immunoenzymatic assays. However, subsequent indicated that E. coli recombinant proteins, that copurify with MSP5, interfere with the ELISA giving rise to false positives. In the present study, it was accomplished the standardization of an indirect ELISA, using MSP5 as the antigen and it was also diminishing the non-specific unions to the contaminating proteins of E. coli by adding anti-E. coli rabbit serum that blocks the epitopes of these proteins. With the use of a double entry contingency table, the parameters of validation of the ELISA were determined, comparing it to the acridine orange-ethidium bromide technique. The result indicate that the improve MSP5 indirect ELISA has a 96.1% sensitivity, 9% specificity and a predictive value of 88.6%. It was also studied a bovine population of 48 cattle from the Experimental Station “La Iguana” (Guárico State), using both techniques, obtaining a seroprevalence of 93.7% with ELISA and a prevalence of 54.1% with orange acridine-ethidium bromide. These results show that the blockage of the contaminating E. coli protein epitopes using an anti-E. coli rabbit serum permits the diminishment of false negatives when using recombinant proteins.