Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa
A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB™) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene...
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| creator | Bhoora, Raksha Quan, Melvyn Franssen, Linda Butler, Catherine M. Van der Kolk, Johannes H. Guthrie, Alan J. Zweygarth, Erich Jongejan, Frans Collins, Nicola E. |
| description | A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB™) probe was developed for the detection of
Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the
B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The
B. caballi TaqMan MGB™ qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported
Theileria equi-specific qPCR assay and showed that it was able to detect the 12
T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either
T. equi or
B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating
T. equi and
B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected
T. equi parasite DNA in 73% of the samples, but none of the samples were positive for
B. caballi, although 19
T. equi-positive samples also hybridized to the
Babesia genus-specific probe. This could indicate a mixed
T. equi and
B. caballi infection in these samples, with either the
B. caballi parasitaemia at a level below the detection limit of the
B. caballi RLB probe, or the occurrence of a novel
Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The
B. caballi TaqMan MGB™ qPCR assay was able to detect
B. caballi parasite DNA in 78% of the samples. The
T. equi-specific qPCR assay could positively detect
T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of
T. equi and
B. caballi infections in field samples. |
| doi_str_mv | 10.1016/j.vetpar.2009.11.011 |
| format | Article |
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Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the
B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The
B. caballi TaqMan MGB™ qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported
Theileria equi-specific qPCR assay and showed that it was able to detect the 12
T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either
T. equi or
B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating
T. equi and
B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected
T. equi parasite DNA in 73% of the samples, but none of the samples were positive for
B. caballi, although 19
T. equi-positive samples also hybridized to the
Babesia genus-specific probe. This could indicate a mixed
T. equi and
B. caballi infection in these samples, with either the
B. caballi parasitaemia at a level below the detection limit of the
B. caballi RLB probe, or the occurrence of a novel
Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The
B. caballi TaqMan MGB™ qPCR assay was able to detect
B. caballi parasite DNA in 78% of the samples. The
T. equi-specific qPCR assay could positively detect
T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of
T. equi and
B. caballi infections in field samples.</description><identifier>ISSN: 0304-4017</identifier><identifier>EISSN: 1873-2550</identifier><identifier>DOI: 10.1016/j.vetpar.2009.11.011</identifier><identifier>PMID: 20031328</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Animals ; Babesia ; Babesia - genetics ; Babesia caballi ; babesiosis ; Babesiosis - diagnosis ; Babesiosis - veterinary ; disease detection ; DNA, Protozoan - analysis ; fluorescent antibody technique ; Genotype ; horse diseases ; Horse Diseases - diagnosis ; Horse Diseases - parasitology ; Horses ; indirect fluorescent antibody test ; Male ; Polymerase Chain Reaction ; Real-time PCR ; Reproducibility of Results ; ribosomal RNA ; RNA, Ribosomal, 18S - genetics ; Sensitivity and Specificity ; South Africa ; Theileria ; Theileria - genetics ; Theileria equi ; Theileriasis - diagnosis ; theileriosis</subject><ispartof>Veterinary parasitology, 2010-03, Vol.168 (3), p.201-211</ispartof><rights>2009 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c417t-96cf360bbf983ee9a4ea35d99d0e7ca6a732d37fb73ecb7fde33ed73028a8f203</citedby><cites>FETCH-LOGICAL-c417t-96cf360bbf983ee9a4ea35d99d0e7ca6a732d37fb73ecb7fde33ed73028a8f203</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.vetpar.2009.11.011$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20031328$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bhoora, Raksha</creatorcontrib><creatorcontrib>Quan, Melvyn</creatorcontrib><creatorcontrib>Franssen, Linda</creatorcontrib><creatorcontrib>Butler, Catherine M.</creatorcontrib><creatorcontrib>Van der Kolk, Johannes H.</creatorcontrib><creatorcontrib>Guthrie, Alan J.</creatorcontrib><creatorcontrib>Zweygarth, Erich</creatorcontrib><creatorcontrib>Jongejan, Frans</creatorcontrib><creatorcontrib>Collins, Nicola E.</creatorcontrib><title>Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa</title><title>Veterinary parasitology</title><addtitle>Vet Parasitol</addtitle><description>A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB™) probe was developed for the detection of
Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the
B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The
B. caballi TaqMan MGB™ qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported
Theileria equi-specific qPCR assay and showed that it was able to detect the 12
T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either
T. equi or
B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating
T. equi and
B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected
T. equi parasite DNA in 73% of the samples, but none of the samples were positive for
B. caballi, although 19
T. equi-positive samples also hybridized to the
Babesia genus-specific probe. This could indicate a mixed
T. equi and
B. caballi infection in these samples, with either the
B. caballi parasitaemia at a level below the detection limit of the
B. caballi RLB probe, or the occurrence of a novel
Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The
B. caballi TaqMan MGB™ qPCR assay was able to detect
B. caballi parasite DNA in 78% of the samples. The
T. equi-specific qPCR assay could positively detect
T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of
T. equi and
B. caballi infections in field samples.</description><subject>Animals</subject><subject>Babesia</subject><subject>Babesia - genetics</subject><subject>Babesia caballi</subject><subject>babesiosis</subject><subject>Babesiosis - diagnosis</subject><subject>Babesiosis - veterinary</subject><subject>disease detection</subject><subject>DNA, Protozoan - analysis</subject><subject>fluorescent antibody technique</subject><subject>Genotype</subject><subject>horse diseases</subject><subject>Horse Diseases - diagnosis</subject><subject>Horse Diseases - parasitology</subject><subject>Horses</subject><subject>indirect fluorescent antibody test</subject><subject>Male</subject><subject>Polymerase Chain Reaction</subject><subject>Real-time PCR</subject><subject>Reproducibility of Results</subject><subject>ribosomal RNA</subject><subject>RNA, Ribosomal, 18S - genetics</subject><subject>Sensitivity and Specificity</subject><subject>South Africa</subject><subject>Theileria</subject><subject>Theileria - genetics</subject><subject>Theileria equi</subject><subject>Theileriasis - diagnosis</subject><subject>theileriosis</subject><issn>0304-4017</issn><issn>1873-2550</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAURi0EotPCGyDwjlUGO87EyQapDOVHqgSi7dq6sa8Zj5J4xnYi9WV4VjykZQkrW9b5Ptv3EPKKszVnvH63X8-YDhDWJWPtmvM14_wJWfFGiqLcbNhTsmKCVUXFuDwj5zHuGWMVq-VzcpYjgouyWZFfH3HG3h8GHBOF0VCcoZ8gOT9Sb2lA6IvkBqTftz8oxAj3kVofaNohPU4wJpcyPCM1mFA_xj5Ah9EB1dBB37s_xbc7dD2GfIrHyVE32oWPeUt3PkTMzcEP9MZPaUcvbXAaXpBnFvqILx_WC3L36ep2-6W4_vb56_byutAVl6loa21FzbrOto1AbKFCEBvTtoah1FCDFKUR0nZSoO6kNSgEGilY2UBjSyYuyNul9xD8ccKY1OCixr6HEf0Ulaxqvqmaqvw_KYQoW962mawWUgcfY0CrDsENEO4VZ-qkUO3VolCdFCrOVVaYY68fLpi6Ac3f0KOzDLxZAAtewc_gorq7KRkXjDdciPb0m_cLgXlks8OgonY4ajQu5KEr492_3_Abe8y7Ng</recordid><startdate>20100325</startdate><enddate>20100325</enddate><creator>Bhoora, Raksha</creator><creator>Quan, Melvyn</creator><creator>Franssen, Linda</creator><creator>Butler, Catherine M.</creator><creator>Van der Kolk, Johannes H.</creator><creator>Guthrie, Alan J.</creator><creator>Zweygarth, Erich</creator><creator>Jongejan, Frans</creator><creator>Collins, Nicola E.</creator><general>Elsevier B.V</general><general>Amsterdam; New York: Elsevier</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>M7N</scope></search><sort><creationdate>20100325</creationdate><title>Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa</title><author>Bhoora, Raksha ; Quan, Melvyn ; Franssen, Linda ; Butler, Catherine M. ; Van der Kolk, Johannes H. ; Guthrie, Alan J. ; Zweygarth, Erich ; Jongejan, Frans ; Collins, Nicola E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c417t-96cf360bbf983ee9a4ea35d99d0e7ca6a732d37fb73ecb7fde33ed73028a8f203</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Animals</topic><topic>Babesia</topic><topic>Babesia - genetics</topic><topic>Babesia caballi</topic><topic>babesiosis</topic><topic>Babesiosis - diagnosis</topic><topic>Babesiosis - veterinary</topic><topic>disease detection</topic><topic>DNA, Protozoan - analysis</topic><topic>fluorescent antibody technique</topic><topic>Genotype</topic><topic>horse diseases</topic><topic>Horse Diseases - diagnosis</topic><topic>Horse Diseases - parasitology</topic><topic>Horses</topic><topic>indirect fluorescent antibody test</topic><topic>Male</topic><topic>Polymerase Chain Reaction</topic><topic>Real-time PCR</topic><topic>Reproducibility of Results</topic><topic>ribosomal RNA</topic><topic>RNA, Ribosomal, 18S - genetics</topic><topic>Sensitivity and Specificity</topic><topic>South Africa</topic><topic>Theileria</topic><topic>Theileria - genetics</topic><topic>Theileria equi</topic><topic>Theileriasis - diagnosis</topic><topic>theileriosis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bhoora, Raksha</creatorcontrib><creatorcontrib>Quan, Melvyn</creatorcontrib><creatorcontrib>Franssen, Linda</creatorcontrib><creatorcontrib>Butler, Catherine M.</creatorcontrib><creatorcontrib>Van der Kolk, Johannes H.</creatorcontrib><creatorcontrib>Guthrie, Alan J.</creatorcontrib><creatorcontrib>Zweygarth, Erich</creatorcontrib><creatorcontrib>Jongejan, Frans</creatorcontrib><creatorcontrib>Collins, Nicola E.</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><jtitle>Veterinary parasitology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bhoora, Raksha</au><au>Quan, Melvyn</au><au>Franssen, Linda</au><au>Butler, Catherine M.</au><au>Van der Kolk, Johannes H.</au><au>Guthrie, Alan J.</au><au>Zweygarth, Erich</au><au>Jongejan, Frans</au><au>Collins, Nicola E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa</atitle><jtitle>Veterinary parasitology</jtitle><addtitle>Vet Parasitol</addtitle><date>2010-03-25</date><risdate>2010</risdate><volume>168</volume><issue>3</issue><spage>201</spage><epage>211</epage><pages>201-211</pages><issn>0304-4017</issn><eissn>1873-2550</eissn><abstract>A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB™) probe was developed for the detection of
Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the
B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The
B. caballi TaqMan MGB™ qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported
Theileria equi-specific qPCR assay and showed that it was able to detect the 12
T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either
T. equi or
B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating
T. equi and
B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected
T. equi parasite DNA in 73% of the samples, but none of the samples were positive for
B. caballi, although 19
T. equi-positive samples also hybridized to the
Babesia genus-specific probe. This could indicate a mixed
T. equi and
B. caballi infection in these samples, with either the
B. caballi parasitaemia at a level below the detection limit of the
B. caballi RLB probe, or the occurrence of a novel
Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The
B. caballi TaqMan MGB™ qPCR assay was able to detect
B. caballi parasite DNA in 78% of the samples. The
T. equi-specific qPCR assay could positively detect
T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of
T. equi and
B. caballi infections in field samples.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>20031328</pmid><doi>10.1016/j.vetpar.2009.11.011</doi><tpages>11</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0304-4017 |
| ispartof | Veterinary parasitology, 2010-03, Vol.168 (3), p.201-211 |
| issn | 0304-4017 1873-2550 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Animals Babesia Babesia - genetics Babesia caballi babesiosis Babesiosis - diagnosis Babesiosis - veterinary disease detection DNA, Protozoan - analysis fluorescent antibody technique Genotype horse diseases Horse Diseases - diagnosis Horse Diseases - parasitology Horses indirect fluorescent antibody test Male Polymerase Chain Reaction Real-time PCR Reproducibility of Results ribosomal RNA RNA, Ribosomal, 18S - genetics Sensitivity and Specificity South Africa Theileria Theileria - genetics Theileria equi Theileriasis - diagnosis theileriosis |
| title | Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa |
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