UZH-Logo

Maintenance Infos

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


Schares, G; Maksimov, A; Basso, W; Moré, G; Dubey, J P; Rosenthal, B; Majzoub, 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(3-4):208-216.

Abstract

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely
understood. 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 genomicDNAin a buffer containing 100 ng/ul bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification
was possible in samples containing ≥1pg B. besnoiti genomic DNA with a coefficient of variation of ≤2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from
cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used,
90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings,
were significantly correlated. 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. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA
of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was
specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely
understood. 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 genomicDNAin a buffer containing 100 ng/ul bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification
was possible in samples containing ≥1pg B. besnoiti genomic DNA with a coefficient of variation of ≤2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from
cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used,
90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings,
were significantly correlated. 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. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA
of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was
specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.

Citations

20 citations in Web of Science®
21 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

2 downloads since deposited on 14 Jul 2011
0 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Parasitology
04 Faculty of Medicine > Institute of Parasitology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
600 Technology
Language:English
Date:2011
Deposited On:14 Jul 2011 12:21
Last Modified:10 Nov 2016 14:13
Publisher:Elsevier
ISSN:0304-4017
Publisher DOI:https://doi.org/10.1016/j.vetpar.2011.01.038
PubMed ID:21324596
Permanent URL: https://doi.org/10.5167/uzh-48679

Download

[img]
Content: Published Version
Filetype: PDF - Registered users only
Size: 2MB
View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations