A probe-based real-time PCR assay for the detection of Neospora caninum in clinical samples from cattle

An outbreak of Neospora caninum abortion in a dairy herd from the State of Georgia, United States

“…method for the identification of N. caninum in aborted fetal tissues, particularly for a probe-based Taqman real-time qPCR assay that offers an accurate and rapid detection of the parasite from contaminated tissues. The qPCR results were confirmed by two different assays (Barry et al, 2019;Brower et al, 2008) (Nusinovici, Seegersa, Joly, Beaudeaua, & Fourichon, 2012). In addition, positive serology to one of the aborted cows to BHV-4, also raises a question about the role of this herpesvirus in the present outbreak investigation, because this virus has been isolated from several epidemic events of metritis and abortions.…”

“…Detection of N. caninum specific DNA by PCR is a highly sensitive method for the identification of N. caninum in aborted fetal tissues, particularly for a probe‐based Taqman real‐time qPCR assay that offers an accurate and rapid detection of the parasite from contaminated tissues. The qPCR results were confirmed by two different assays (Barry et al., 2019; Brower et al., 2008) and the positive results were in 100% agreement. Both assays consistently yielded Ct values between 30 and 34 in 40 cycle PCR runs for all tissue DNA extracts further confirming the presence of N. caninum DNA.…”

“…In addition, abortion panel testing that comprised of bacterial culture, virus isolation and fluorescent antibody tests were performed on fetal membranes from aborted cows and organs from aborted fetuses. The fresh tissues were also evaluated by PCR tests (probe‐based Taqman real‐time qPCR assays; Barry et al., 2019; Brower et al., 2008) for the presence of DNA of abortifacient infectious agents: Leptospira pathogenic serovars, Tritrichomonas foetus , Campylobacter fetus , Anaplasma marginale , BVDV, Bovine Herpes virus‐1&4, Salmonella spp., Chlamydophila spp , Listeria monocytogenes, Coxiella burnetii and N. caninum . All microbiological testing and histopathology based testing (Table 2) were performed in accordance with the approved standard operating procedures of the quality system at the TVDIL with appropriate controls for each assay.…”

“…Three fetuses and one placental tissue (abortions from 26 to 28 May 2019) were submitted to the TVDIL for necropsy (fetuses 1, 2 and 3) along with samples of blood from the dams. During necropsy, samples were collected for histopathology, bacterial cultures, serology and virology and PCR testing (probe‐based Taqman real‐time qPCR assays; Barry et al., 2019; Brower et al., 2008) (Table 2). In addition, ocular fluid from fetuses 1 and 2 were collected for nitrate test.…”

Toxoplasma gondii and Neospora caninum Infections in Stray Cats and Dogs in the Qinghai–Tibetan Plateau Area, China

“…Although several studies have examined the seroprevalence of T. gondii in animals in the QTPA [ 17 , 18 , 19 , 20 , 21 ], to date, the information about the epidemiology of toxoplasmosis and neosporosis in stray cats and dogs in the QTPA is limited, and little is known about the diversity of the diseases. The surface antigen 1 (SAG1) and dense granule protein 7 (GRA7) of T. gondii and N. caninum have been identified and tested as important candidates for the serological diagnosis of toxoplasmosis and neosporosis [ 22 , 23 , 24 , 25 , 26 ], while the Tg B1 and Nc Nc5 genes have been used to molecularly amplify the T. gondii and N. caninum in quantitative PCR (qPCR) from fecal and tissue DNA [ 27 , 28 , 29 , 30 , 31 ]. Therefore, the aim of this study was to perform indirect ELISA tests based on recombinant Tg SAG1, Tg GRA1, Nc SAG1 and Nc GRA7 proteins and qPCR amplification based on the Tg B1 and Nc Nc5 genes to establish a detailed record of the seroprevalence of T. gondii and N. caninum -specific IgG and IgM antibodies in serum samples and the molecular epidemiology in feces from stray cats and dogs in the QTPA.…”