Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test

Abstract

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.

Molecular of Anaplasma marginale Theiler (Rickettsiales: Anaplasmataceae) in horseflies (Diptera: Tabanidae) in Uruguay

“…The A. marginale DNA positive control was kindly supplied by F. Riet-Correa. The optimal PCR parameters were initial denaturation for 4 min at 94 °C, followed by 35 cycles of 94 °C for 1 min, 60 °C for 30 s, and 72 °C for 45 s, with a final extension of 4 min at 72 °C 30 .…”

Technologies for Detection of Babesia microti: Advances and Challenges

“…Multiplex PCR assays using standard PCR protocols and fragment size differentiation have been employed for decades [71], but now the RT-PCR platform can easily be adapted to distinguish separate species with the use of fluorescent probes specific for distinct target genes. Historically, these assays have been of particular use in veterinary fields, as livestock and domesticated animals tend to be exposed to a far greater breadth of tick-borne pathogens that need to be distinguished [71,72]. Another application of multiplex PCR assays is for the surveillance of the tick population in a given region to establish the probable rate of exposure to given pathogens [73,74].…”

Molecular Survey of Babesia and Anaplasma Infection in Cattle in Bolivia

“…In Latin American countries, the most severe and prevalent TBDs are babesiosis and anaplasmosis [4][5][6][7][8]. Bovine babesiosis is a globally distributed tick-borne hemoprotozoan Vet.…”

“…In Latin American countries, the most severe and prevalent TBDs are babesiosis and anaplasmosis [ 4 , 5 , 6 , 7 , 8 ]. Bovine babesiosis is a globally distributed tick-borne hemoprotozoan disease caused by pathogenic species, such as Babesia bovis , Babesia bigemina , and Babesia divergens .…”