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CASE NOTES

QUANTITATIVE PCR AS A TOOL FOR THE DIAGNOSIS OF BOVINE THEILERIOSIS

Cheryl Jenkinsa and Daniel Bogemaa,b

aElizabeth Macarthur Agricultural Institute, Camden NSW

bithree institute, University of Technology, Sydney, Broadway NSW

Posted Flock & Herd March 2015

Introduction

Typical symptoms of bovine theileriosis include a combination of pale and/or jaundiced mucous membranes, lethargy, ataxia, tachypnoea and abortion in pregnant cows. Clinical theileriosis is usually diagnosed by ruling out alternate causes of similar symptoms in combination with observation of piroplasms on blood film and/or a positive PCR result. From a laboratory perspective, the presence of both benign and pathogenic genotypes of Theileria orientalis complicates the diagnosis of clinical theileriosis. Diagnosis cannot be achieved via blood film examination alone because individual genotypes cannot be distinguished based on morphology and the presence of this parasite can represent an incidental finding. Development of conventional PCR assays for T. orientalis and its genotypes represented a further advance in laboratory diagnosis1. However, since the first outbreaks of bovine theileriosis in 20062, 3, the pathogenic genotypes (Ikeda genotype with or without the Chitose genotype) have become enzootic in herds over large areas of the eastern seaboard and are now commonly observed in subclinical as well as clinically-affected animals4, 5. Recent developments in quantitative PCR technology6 provide further aids to laboratory diagnosis of this disease.

METHODOLOGY

Clinical diagnosis using multiplex qPCR

EDTA blood samples (n = 214) from clinical diagnostic cases and surveillance studies were collected from cattle herds across four states (NSW, VIC, QLD and WA) and tested in a validated multiplex quantitative PCR6. Animals were divided into four groups with respect to their theileriosis status as follows: clinically-affected (n = 110), recovering (n = 25), in-contact (n =24) and subclinical animals from herds without clinical cases (n = 55). Animals were considered clinically affected if they displayed a combination of symptoms consistent with theileriosis at the time of sampling (anaemia, jaundice, depression, ataxia, lethargy, tachypnoea, tachycardia, pyrexia, or late-term abortion) and did not have a confounding diagnosis. Recovering animals had symptoms of theileriosis in the weeks prior to sampling and evidence of regenerative anaemia on blood smears. In-contact animals were those that had cohorts that were clinically-affected with theileriosis. The multiplex qPCR consists of a universal component (detects all T. orientalis types) which measured total parasite load, and a genotyping component which detected the Ikeda and/or Chitose genotypes. The presence of the Buffeli genotype was measured using a singleplex qPCR assay (in preparation). Total parasite loads (expressed as gene copies per microlitre of DNA extract (GC/μL) were compared using one-way analysis of variance (ANOVA).

Temporal dynamics of T. orientalis genotypes

EDTA bloods were collected from naive 2 yr old heifers (n = 10) which were introduced to a property with a history of theileriosis. Blood samples were collected weekly for a period of 11 weeks and packed cell volume (PCV) was recorded. The temporal dynamics of the T. orientalis genotypes was investigated using singleplex quantitative PCRs that specifically amplified each individual genotype (Ikeda, Chitose or Buffeli) as described previously7.

Subpopulation analysis of the Chitose genotype

Samples from clinical theileriosis cases (n = 89) or active surveillance studies (n = 48) testing PCR positive for the Chitose genotype were analysed. A genotyping assay7 was used to determine the allele frequency of two different subgroups of the Chitose genotype (Chitose A and Chitose B). This assay was also used to test selected samples from the temporal study.

Results

Clinical diagnosis using multiplex qPCR

Quantitative PCR revealed that subclinical animals from herds that were not experiencing any clinical cases had a significantly lower mean parasite load than animals from herds with clinical cases (P<0.001), regardless of whether the individual animals were clinically affected, recovering or in-contact with affected cohorts. Within herds with clinical cases, the mean parasite load of clinically-affected animals was significantly higher than that of recovering or in-contact animals P<0.001); however recovering and in-contact animals did not have statistically different mean parasite loads from each other.

The quantitative data derived from this study was used to define clinical thresholds to aid laboratory diagnosis. All subclinical animals from healthy herds tested in this study had parasite loads of <1.5-104 GC/μL of DNA extract. In contrast, the majority of animals (91%) displaying clinical symptoms consistent with theileriosis, had parasite loads >1.5-104 GC/μL. Furthermore, of the animals exhibiting a parasite load above 3.0-105 GC/μL, 95% displayed symptoms consistent with theileriosis.

In addition to providing information about total parasite load, the multiplex qPCR also identified whether the pathogenic Ikeda and/or Chitose genotypes were present in each sample. Animals testing positive for the Ikeda genotype alone, or the Ikeda genotype in combination with the Chitose genotype had significantly higher overall parasite loads than animals harbouring only the Chitose genotype, or only the Buffeli genotype (P<0.001). There was no statistical difference in total parasite load detected in animals infected with the Ikeda genotype compared to those infected with both the Ikeda and Chitose genotypes. Semi-quantitative data from the genotyping component of the qPCR revealed that in 58% of mixed Ikeda-Chitose infections, Ikeda was the dominant genotype.

Temporal dynamics of T. orientalis genotypes

Singleplex qPCR assays were used to monitor the genotype populations of mixed T. orientalis infections over time in ten naive animals introduced to a Theileria-endemic area. All ten animals were confirmed negative for T. orientalis upon introduction, and all ten animals subsequently became infected with the Ikeda, Chitose and Buffeli genotypes of T. orientalis during the course of the study. The Ikeda genotype was the first to be detected in the majority of animals (7/10) at 11 days post-introduction. The three remaining animals were all positive by day 20. The Chitose genotype was only detected in one animal at day 11, but all 10 animals by day 20. The Buffeli genotype was detected in nine animals by day 20 and the remaining animal by day 34.

The dominant genotype shifted in all animals over the course of the study, from the Ikeda to the Chitose genotype. The peak in the Ikeda genotype occurred at 35-40 days post-introduction, followed by the peak in the Chitose genotype at 45-50 days post-introduction. The Ikeda genotype re-emerged as the dominant genotype toward the end of the study period in most animals (around day 60). The Buffeli genotype dominated in only 1/10 cows ("Cow 1"); however the parasite load in this animal was lower overall than in the other nine cows.

All animals experienced a drop in PCV over the course of the study, approximately 8 days after the Ikeda genotype had peaked and around the time that the Chitose genotype was at a maximum. All cows became anaemic (PCV<24) apart from "Cow 1" in which the Buffeli genotype dominated.

Subpopulation analysis of the Chitose genotype

While the role of the Ikeda genotype in clinical theileriosis has been clearly established1, 5, 8, the role of the Chitose genotype is less clear. A real-time PCR assay aimed at determining the frequency of two phylogenetically distinct Chitose alleles (Chitose A and Chitose B) was used to test samples from clinical and non-clinical cases. The Chitose A allele was found to be significantly associated with both the Ikeda genotype (55/56 cases) (P<0.0001) and clinical disease (56/56 cases) (P<0.0001). In contrast the Chitose B allele was more often associated with subclinical infection (21/29 cases), unless it co-occurred with the Chitose A allele (35/52 cases).

Samples derived from the temporal study described above were also tested, at the time-point where the Chitose genotype was determined (by qPCR) to be at its peak. Of the ten cows tested, six contained the Chitose A allele and the remaining four contained a mix of Chitose A and Chitose B genotypes. Only one animal ("Cow 1") contained a greater concentration of the Chitose B allele relative to the Chitose A allele.

Discussion

Diagnosis of clinical theileriosis is complicated by the presence of multiple genotypes of differing pathogenicities and the widespread prevalence of this organism in subclinical animals. Multiplex quantitative PCR can simultaneously provide an estimate of T. orientalis parasite load as well as provide information on the presence/absence and relative abundance of the two pathogenic genotypes of the organism. Total parasite load as determined by qPCR is directly related to clinical status at both the herd and the individual level, and in combination with information on the genotypes present, can provide veterinarians with a powerful tool for diagnosis. The clinical thresholds described can be used as a guide for differentiating between subclinical carriers and animals that are clinically-affected, recovering or cohorts of clinically-affected cattle.

While the Ikeda genotype alone can cause clinical theileriosis, it is often reported as occurring as a co-infection with the Chitose genotype1, 5, 8. Reports of the Chitose genotype causing disease as the sole agent are much rarer9. The temporal dynamics of mixed T. orientalis infections indicates that the initial increase in parasitaemia is due to the Ikeda genotype; however the population of Ikeda parasites later declines while that of the Chitose genotype increases. While the Chitose genotype contributes significantly to the overall parasite burden over the course of the infection, this appears to apply largely to the Chitose A subtype, which is strongly associated with both Ikeda infection and clinical disease. The temporal switching of genotypes needs to be taken into account when considering clinical diagnosis of theileriosis, as depending on the time of sampling, the Chitose genotype may dominate over the Ikeda genotype.

Acknowledgements

This work was funded by Meat and Livestock Australia (Project B.AHE.0213) and the McGarvie Smith Trust. Technical assistance from Melinda Micallef, Sherin Alex, Ania Deutscher and Shayne Fell is gratefully acknowledged. We are also grateful to the private and district veterinarians who collected samples for this study.

References

  1. Eamens GJ, Gonsalves JR, Jenkins C, Collins D, Bailey G. Theileria orientalis MPSP types in Australian cattle herds associated with outbreaks of clinical disease and their association with clinical pathology findings. Veterinary Parasitology 2013;191:209-217
  2. Kamau J, de Vos AJ, Playford M et al. Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks. Parasit Vectors 2011;4:22
  3. Izzo M, Poe I, Horadagoda N, De Vos AJ, House JK. Haemolytic anaemia in cattle in NSW associated with Theileria infections. Australian Veterinary Journal 2010;88:45-51
  4. Eamens GJ, Bailey G, Gonsalves JR, Jenkins C. Distribution and temporal prevalence of Theileria orientalis major piroplasm surface protein types in eastern Australian cattle herds. Australian Veterinary Journal 2013;91:332-340
  5. Eamens GJ, Bailey G, Jenkins C, Gonsalves JR. Significance of Theileria orientalis types in individual affected beef herds in New South Wales based on clinical, smear and PCR findings. Veterinary Parasitology 2013;196:96-105
  6. Bogema RD, Deutscher AT, Fell S et al. Development and validation of a multiplexed hydrolysis probe qPCR assay for the detection and quantification of Theileria orientalis and differentiation of clinically-relevant subtypes. J Clin Microbiol 2015 (In press)
  7. Bogema DR, Micallef M, Alex SM et al. Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle. (Submitted) 2015
  8. Perera PK, Gasser RB, Firestone SM et al. Oriental theileriosis in dairy cows causes a significant milk production loss. Parasit Vectors 2014;7:73
  9. McFadden AM, Rawdon TG, Meyer J et al. An outbreak of haemolytic anaemia associated with infection of Theileria orientalis in naive cattle. New Zealand Veterinary Journal 2011;59:79-85

 

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