Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

Carelli, Grazia; Decaro, Nicola; Lorusso, Alessio; Elia, Gabriella; Lorusso, Eleonora; Mari, Viviana; Ceci, Luigi; Buonavoglia, Canio

doi:10.1016/j.vetmic.2007.03.022

Cited by 131 publications

(139 citation statements)

References 23 publications

(24 reference statements)

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“…However, when the two re-actions were subjected to a comparative field trial using samples from animals from an enzootic stability region, a significant difference was not observed. Previously described real-time PCR assays for the detection of Anaplasma marginale have also shown high levels of sensitivity, as well as those reported by Carelli et al (2007) and Decaro et al (2008) (detection limit = 5 copies of DNA). However, in both cases a hydrolysis probe based system was used (TaqMan system), whereas in the present stud the SYBR Green system was used.…”

Section: Discussionmentioning

confidence: 65%

“…Recently, several types of polymerase chain reactions (PCR) such as nested PCR, multiplex PCR, real-time PCR) have been developed and used for the diagnosis of various pathogens (Courtney et al 2004, Carelli et al 2007). Real--time PCR has a number of advantages, including high sensitivity and specificity, usefulness as a quantitative assay, and is able to deliver faster results than end point PCR (Bell & Ranford-Cartwrightet 2002).…”

Section: Introductionmentioning

confidence: 99%

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Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Bacanelli

Ramos

Araújo³

2014

Pesq. Vet. Bras.

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Pesq. Vet. Bras. 34(1):29-33, janeiro 2014 29 RESUMO.-[Diagnóstico molecular de Anaplasma marginale em bovinos: avaliação quantitativa de uma PCR em tempo real baseada no gene msp5.] 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. 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.

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Section: Discussionmentioning

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Bacanelli

Ramos

Araújo³

2014

Pesq. Vet. Bras.

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“…For PCR assays of A. marginale, one set of specific primers was used to amplify an approximately 95 bp fragment as previously described by Carelli et al (2007). The sequences of the primers are as follows: forward 5'-TTGGCAAGGCAGCAGCTT-3', and reverse 5'-TTCCGCGAGCATGTGCAT-3'.…”

Section: Methodsmentioning

confidence: 99%

Significance of clinical variables and selected biochemical markers in predicting the outcome of bovine anaplasmosis

El-Ashker¹,

Salama²,

El-Sebaei³

et al. 2015

Vet. Med. - Czech

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ABSTRACT:The present study was aimed at evaluating the usefulness of selected inflammatory and oxidative stress markers in predicting the clinical outcome of cattle infected with Anaplasma (A). marginale. The study population consisted of 39 cattle naturally infected with A. marginale. The presumptive diagnosis of this infection was initially achieved on the basis of case history, microscopy and clinical examination findings, and confirmed using A. marginale-specific PCR assays. The diseased cattle were categorised according to the clinical outcome into survivors (n = 26) and non-survivors (n = 13). For comparison, ten clinically healthy cattle were randomly selected and served as controls. Blood was drawn from all examined animals to measure the respective levels of selected cytokines, acute phase proteins, oxidative stress markers, and antioxidant enzyme levels. We found that the clinical examination alone was not conclusive and should be used in conjunction with other diagnostic methods. Nonetheless, the non-surviving animals showed anorexia, frequent coughing, dyspnoea, bloody faeces, recumbency, pale or icteric mucous membranes, and haemoglobinuria. Biochemically, tumour necrosis factor alpha, interleukin (IL)-1β, IL-6, serum amyloid A, fibrinogen, malondialdehye, superoxide dismutase, and catalase levels were significantly higher in diseased cattle compared with controls, and were higher in non-survivors than survivors (P < 0.05). In contrast, reduced glutathione (G-SH) was significantly lower in non-surviving cattle than survivors and controls. Interestingly, a significant correlation was found between parasitaemia of the diseased cattle and most of the measured biochemical variables, with IL-1β and G-SH showing the highest correlation. Our findings clearly demonstrate that A. marginale infection is associated with marked inflammatory and oxidative stress responses, which are higher in non-surviving cattle compared with survivors. The overall degree of cytokine and anti-oxidative disruption may have an important prognostic value for the disease outcome.

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“…Anaplasmosis is globaly the most prevalent tick-borne disease of ruminants and has a worldwide distribution with areas in endemicity on six continents. High prevalence rates are reported in tropical and subtropical regions of the world [2][3][4][5][6] . Prevalence of disease depends on distribution and density of reservoir host and tick vectors [2,5,6] .…”

Section: Introductionmentioning

confidence: 99%

“…High prevalence rates are reported in tropical and subtropical regions of the world [2][3][4][5][6] . Prevalence of disease depends on distribution and density of reservoir host and tick vectors [2,5,6] . Anaplasma species is transmitted biologically by infected ticks, mechanically by biting flies (Tabanids) or blood contaminated fomites.…”

Section: Introductionmentioning

confidence: 99%

Klinik Olarak Anaplasmosis Şüphesi Olan Ruminantlarda cELISA’nın Tanısal Değerinin Belirlenmesi

Selçuk¹,

Alver²,

Çatik³

et al. 2015

Kafkas Univ Vet Fak Derg

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The aim of this study was to determine diagnostic value of cELISA in anaplasmosis in clinically suspected animals and to compare the cELISA results with the clinical examination results. For this purpose a total of 720 ruminants (457 cattle, 146 sheep, 117 goat) were examined in terms of clinical signs. Eighty-eight ruminants consisting of 61 cattle, 11 sheep and 16 goat which had the symptoms of anemia, fever, icterus, weakness, depression and lack of appetite were selected for the study. Blood samples were collected from the jugular vein of all clinically suspected animals and serum samples were separated. A commercially available competitive enzyme-linked immunosorbent assay (C-ELISA) kit was used for determine antibodies to Anaplasma species. cELISA based diagnosis revealed that 47 of 88 serum samples (53.4%) were positive for anaplasmosis. In serological examination Anaplasma specific antibodies were determined in 45.9% of cattle, 63.6% of sheep and 56.2% of goats. Seropositivity rate was statistically differ among the age groups of cattle and the highest seropositvity rate was found in <12 month age (P<0.005). However no difference was found in the seropositivity rate of Anaplasma in sheep and goat in relation to age group. From the data obtained in this study it can be concluded that clinical findings are not sufficient criteria for the diagnosis of anaplasmosis and must be supported by serological examination.

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Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

Cited by 131 publications

References 23 publications

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Significance of clinical variables and selected biochemical markers in predicting the outcome of bovine anaplasmosis

Klinik Olarak Anaplasmosis Şüphesi Olan Ruminantlarda cELISA’nın Tanısal Değerinin Belirlenmesi

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