title: Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers.
abstract: Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior cortex in 3 out of 5 fetuses from the TAI+NC-1 group. We concluded that heifers infected after TAI had a higher incidence of the parasite at the fetus CNS. Identification of N. caninum by TaqMan RT-PCR would assist in the investigation of infection and in the evaluation of vaccines or therapeutic drugs to control neosporosis in cattle.
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Gabriel Ribas
authors.initials: GR
authors.lastname: Pereira
authors.affiliation: Laboratório de Doenças Parasitárias - LaDoPar, Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. Electronic address: fefevogel@gmail.com.
authors.forename: Fernanda Silveira Flores
authors.initials: FS
authors.lastname: Vogel
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Rodrigo Camponogara
authors.initials: RC
authors.lastname: Bohrer
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Janduí Escarião
authors.initials: JE
authors.lastname: da Nóbrega
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Gustavo Freitas
authors.initials: GF
authors.lastname: Ilha
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Paulo Roberto Antunes
authors.initials: PR
authors.lastname: da Rosa
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Werner Giehl
authors.initials: WG
authors.lastname: Glanzner
authors.affiliation: Laboratório de Doenças Parasitárias - LaDoPar, Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil.
authors.forename: Giovana
authors.initials: G
authors.lastname: Camillo
authors.affiliation: Laboratório de Doenças Parasitárias - LaDoPar, Departamento de Medicina Veterinária Preventiva (DMVP), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil.
authors.forename: Patricia
authors.initials: P
authors.lastname: Braunig
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: João Francisco Coelho
authors.initials: JF
authors.lastname: de Oliveira
authors.affiliation: Laboratório de Biotecnologia e Reprodução Animal - BioRep, Departamento de Clínica de Grandes Animais (DCGA), Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil.
authors.forename: Paulo Bayard Dias
authors.initials: PB
authors.lastname: Gonçalves
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology* / Q000473:pathology; D003048:Coccidiosis / Q000473:pathology / Q000662:veterinary*; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D005333:Fetus / Q000469:parasitology; D007315:Insemination, Artificial / Q000662:veterinary; D018688:Neospora / Q000235:genetics / Q000502:physiology*; D011247:Pregnancy; D018873:Pregnancy Rate; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*
chemical_list: D016054:DNA, Protozoan
keywords: Abortion; Cattle; Neospora caninum; TaqMan real-time PCR