Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Bilgiç, Hüseyin Bilgin; Karagenç, Tülin; Simuunza, Martin; Shiels, Brian; Tait, Andy; Eren, Hasan; Weir, William

doi:10.1016/j.exppara.2012.11.005

Cited by 127 publications

(79 citation statements)

References 48 publications

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“…Such long standing carriers act as the major contributors of infection through the ticks. Transport of carrier cattle to non-endemic areas can lead to disease outbreak (Bilgic et al 2013). Hence, detection of piroplasms in carrier animals becomes more challenging.…”

Section: Introductionmentioning

confidence: 99%

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction

Kundave

Patel

et al. 2013

J Parasit Dis

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Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease of great economic importance in tropical and subtropical regions of the world. The present study was undertaken to detect theilerosis in cattle and buffaloes by polymerase chain reaction (PCR). The diagnosis of theileriosis is usually carried out by blood smear staining technique, which is not sufficiently sensitive to detect the piroplasms in the carrier animals. In this study, a total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Screening of blood smears by Giemsa staining detected 15 samples (12.93 %) positive for Theileria piroplasms out of 116 samples. However, the PCR based screening using the specific primers from the major merozoite-piroplasm surface antigen sequence of T. annulata (Tams1) gene detected 74 samples (63.79 %) positive for T. annulata which included 59 samples found negative by Giemsa staining. Our study suggests that the PCR based screening is more sensitive and accurate method for diagnosis of tropical theileriosis in cattle and buffaloes.

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Section: Introductionmentioning

confidence: 99%

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction

Kundave

Patel

et al. 2013

J Parasit Dis

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“…This indicates that even lower amounts than 0.1 fg of B. bigemina DNA can be detected with the method here described. The CYTb gene has also been successfully used for the molecular detection of the related piroplasmid Theileria annulata (Bilgiç et al, 2010(Bilgiç et al, , 2013.…”

Section: Resultsmentioning

confidence: 99%

Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field

Romero-Salas

Mira²,

Mosqueda

et al. 2016

Veterinary Parasitology

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“…Serological tests have been developed to detect circulatory antibodies against the parasites, but these generally have poor sensitivity and specificity due to involving cross-reactions or nonspecific immune responses (Passos et al , 1998), and only detect previous exposure as opposed to current infection. Conventional PCR methods are useful in the detection of particular haemoprotozoan species for which the reagents and conditions have been developed, but can have limitations in the detection of other species and lack scalability (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). In contrast, the deep amplicon sequencing method is potentially providing more reliable and accurate in the automated high throughput analysis of all haemoprotozoan species.…”

Section: Discussionmentioning

confidence: 99%

“…Determination of sequence variations in the hyper-variable 18S rDNA cistron can discriminate between haemoprotozoan parasites (Gubbels et al , 1999) and overcome limitations of traditional gross parasitological methods for the diagnosis of haemoprotozoa at species level (Agudelo et al , 2013; Haanshuus et al , 2013; Lee et al , 2015; Lefterova et al , 2015; Mens et al , 2006; Rougemont et al , 2004; Steenkeste et al , 2009). Various PCR methods (reverse line blot (RLB)-PCR, quantitative (q)PCR, multiple PCR) have been described to amplify the 18S region for sequence determination, but these are low throughput, hence relatively expensive, and can be error-prone (Bilgic et al , 2013; Chaisi et al , 2013; Gubbels et al , 1999; Kundave et al , 2018). These methods depend on the use of species-specific primers and probes, hence can only identify the tested species.…”

Section: Introductionmentioning

confidence: 99%

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

Chaudhry

Rashid

et al. 2019

Preprint

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Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a "haemoprotobiome". First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The "haemoprotobiome" concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

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Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Abstract: Graphical abstractHighlights► Novel multiplex PCR for Theileria annulata, Babesia bovis and Anaplasma marginale. ► Specific and sensitive tool which can be applied to epidemiological studies. ► Simple and efficient assay which has been validated using field samples.

Cited by 127 publications

References 48 publications

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction

Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

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