title: Application of quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types to examine associations between severity of anaemia and parasitaemia in bovine anaemia outbreaks.
abstract: OBJECTIVE
  To use quantitative PCR assays to detect Theileria orientalis Ikeda type in cattle presumed infected with T. orientalis, to examine the relationship between theilerial piroplasm count and haematocrit (HCT), and the relationship with quantification cycle threshold (Cq) values.
  
  
  METHODS
  Blood samples in EDTA (n=1,024), derived from herds affected by anaemia associated with T. orientalis infection (TABA) between April and October 2013, were submitted for testing using quantitative PCR (qPCR) assays for T. orientalis and Ikeda type. Nucleotide sequencing of the major piroplasm surface protein (MPSP) gene was performed on 16 samples to identify T. orientalis types. Blood smear and/or HCT results were supplied with most samples. For data analysis, the number of theilerial piroplasm per 1,000 erythrocytes counted was categorised as negative (0), low (1-9), moderate (10-100) or high (>100). HCT was categorised as severely anaemic (<0.15 L/L), mildly anaemic (0.15-0.24 L/L) or not anaemic (>0.24 L/L). Differences between categories in proportion of samples positive for Ikeda type or mean Cq value were examined using χ(2) tests or analysis of variance, respectively.
  
  
  RESULTS
  Of 1,022 samples containing amplifiable DNA, 916 (90%) were positive for T. orientalis and 789 (77%) were positive for Ikeda type. Nucleotide sequencing of MPSP amplicons also identified the presence of Chitose and Buffeli types in 11 samples without Ikeda. Ikeda was detected in a greater proportion of severely anaemic (288/302; 95%) than mildly anaemic (227/252; 90%) cattle (p=0.02). In non-anaemic cattle, 344/406 (85%) were positive for T. orientalis and 247/406 (60%) were positive for Ikeda type. In samples from cattle that were piroplasm-positive, a greater proportion of anaemic (483/505, 96%) than non-anaemic (211/307; 69%) cattle were positive for Ikeda type (p<0.001). In piroplasm-negative cattle, 20/37 (54%) anaemic and 25/78 (32%) non-anaemic cattle were Ikeda-positive (p<0.05). The distributions of Cq values differed between piroplasm count and HCT categories (p<0.001). Mean Cq differed between high and negative, and low piroplasm categories (p<0.001), but not between high and moderate categories (p=0.81), and differed between severely anaemic and mildly anaemic (p<0.001), and non-anaemic categories (p<0.001).
  
  
  CONCLUSIONS
  The Ikeda type was found in a high proportion of cattle during outbreaks of TABA in New Zealand. Analysis of Cq values suggested a relationship of Ikeda parasitaemia with severity of anaemia, but further investigation is required to better understand the role of parasitaemia in the pathogenesis of TABA.
authors.affiliation: a Ministry for Primary Industries , Investigation and Diagnostic Centre , 66 Ward Street, Wallaceville, Upper Hutt , 5018 , New Zealand.
authors.forename: E
authors.initials: E
authors.lastname: Gias
authors.affiliation: a Ministry for Primary Industries , Investigation and Diagnostic Centre , 66 Ward Street, Wallaceville, Upper Hutt , 5018 , New Zealand.
authors.forename: D J
authors.initials: DJ
authors.lastname: Pulford
authors.affiliation: b Institute of Veterinary, Animal and Biomedical Sciences , Massey University , Palmerston North , 4410 New Zealand.
authors.forename: K
authors.initials: K
authors.lastname: Lawrence
authors.affiliation: a Ministry for Primary Industries , Investigation and Diagnostic Centre , 66 Ward Street, Wallaceville, Upper Hutt , 5018 , New Zealand.
authors.forename: Amj
authors.initials: A
authors.lastname: McFadden
mesh_terms: D000740:Anemia / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D000818:Animals; D002417:Cattle; D016054:DNA, Protozoan / Q000235:genetics; D004196:Disease Outbreaks / Q000662:veterinary*; D062665:Epidemiological Monitoring / Q000662:veterinary; D018512:Parasitemia / Q000469:parasitology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D016794:Theileria / Q000145:classification / Q000302:isolation & purification*; D013801:Theileriasis / Q000150:complications / Q000175:diagnosis / Q000469:parasitology*
chemical_list: D016054:DNA, Protozoan
keywords: Ikeda type; Theileria associated bovine anaemia; Theileria orientalis; haematocrit; piroplasms; quantitative PCR