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Title: Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle

Author
item SCHARES, G - Federal Research Institute
item MAKSIMOV, A - Federal Research Institute
item BASSO, W - Federal Research Institute
item MORE, G - National University Of Laplata
item Dubey, Jitender
item Rosenthal, Benjamin
item MSJXOUB, M - Ludwig-Maximilians University
item ROSTAHER, A - Ludwig-Maximilians University
item SELMAIR, J - Collaborator
item LANGENMAYER, M - Ludwig-Maximilians University
item SCHARR, J - Ludwig-Maximilians University
item CONRATHS, F - Federal Research Institute
item GOLLNICK, N - Ludwig-Maximilians University

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2011
Publication Date: 3/1/2011
Citation: Schares, G., Maksimov, A., Basso, W., More, G., Dubey, J.P., Rosenthal, B.M., Msjxoub, M., Rostaher, A., Selmair, J., Langenmayer, M.C., Scharr, J.C., Conraths, F.J., Gollnick, N.S. 2011. Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle. Veterinary Parasitology. 178:208-216.

Interpretive Summary: Assays were developed to identify and quantify bovine infections with Besnoitia besnoiti, a parasite responsible for significant cattle production losses in Africa, Asia, and now emerging in Europe. The assays employ quantitative real-time PCR, are able to detect samples containing more than 1 picogram of parasite DNA, do not cross-react with several other bovine parasites, but can be used to detect closely related parasites known to occur in ungulate hosts.

Technical Abstract: Bovine besnoitiosis, an economically important disease in cattle in many countries of Africa and Asia, has re-emerged in Europe. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5’-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/µl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing more or equal than 1 pg B. besnoiti genomic DNA with a coefficient of variation of less or equal to 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae were taken from animals with clinical signs of chronic bovine besnoitiosis. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp. Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei.