title: Adaptation of a commercial ELISA to determine the IgG avidity in sheep experimentally and naturally infected with Neospora caninum.
abstract: Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.
authors.affiliation: Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand.
authors.forename: S S
authors.initials: SS
authors.lastname: Syed-Hussain
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand.
authors.forename: L
authors.initials: L
authors.lastname: Howe
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand. Electronic address: W.Pomroy@massey.ac.nz.
authors.forename: W E
authors.initials: WE
authors.lastname: Pomroy
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand.
authors.forename: D M
authors.initials: DM
authors.lastname: West
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand.
authors.forename: S L
authors.initials: SL
authors.lastname: Smith
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand.
authors.forename: N B
authors.initials: NB
authors.lastname: Williamson
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000915:Antibody Affinity / Q000276:immunology*; D003048:Coccidiosis / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards / Q000662:veterinary*; D005260:Female; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D018688:Neospora / Q000276:immunology*; D011247:Pregnancy; D015203:Reproducibility of Results; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D007074:Immunoglobulin G
keywords: Avidity IgG; ELISA; N. caninum; Serology; Sheep