Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

<div><p></p><p>AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of <i>Theileria orientalis</i> found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for <i>T. orientalis</i>.</p><p>METHODS: Nucleotide sequences aligned with <i>T. orientalis</i> Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of <i>T. orientalis</i> infection, other veterinary laboratory samples, as well as plasmids containing <i>T. orientalis</i> type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for <i>T. orientalis</i>. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.</p><p>RESULTS: The <i>T. orientalis</i> type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²=0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with <i>T. orientalis</i>, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively, compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with <i>T. orientalis</i> ranged between 5.6×10⁴ and 3.3×10⁶ genomes per µL of blood.</p><p>CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of <i>T. orientalis</i> Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.</p><p>CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced <i>T. orientalis</i> and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with <i>T. orientalis</i> and can be used to monitor the parasite load of Ikeda in blood.</p></div

Investigation of the index case herd and identification of the genotypes of <i>Theileria orientalis</i> associated with outbreaks of bovine anaemia in New Zealand in 2012

<div><p></p><p>CASE HISTORY AND CLINICAL FINDINGS: On 7 September 2012 the Ministry for Primary Industries was notified of a dairy cow with regenerative anaemia (haematocrit (HCT) 0.08 L/L) in a herd of 465 Jersey-Friesian cross cows (index case herd) in the Northland region of New Zealand. Organisms consistent with <i>Theileria</i> spp. were present in red blood cells on a blood smear. No other causes of anaemia were detected following examination of affected cows. Blood samples collected from 29 randomly selected cows on 26 September 2012 showed that 24 (83%) were anaemic (HCT≤0.24 L/L) and therefore fitted the case definition for bovine anaemia associated with <i>Theileria orientalis</i> infection.</p><p>LABORATORY FINDINGS: Using a <i>T. orientalis</i> type-specific PCR assay that targeted the single subunit rRNA gene, all of six animals tested were positive for <i>T. orientalis</i> type Ikeda. Blood samples collected from clinically affected cattle in 11 subsequent outbreaks from throughout the North Island showed that <i>T. orientalis</i> Ikeda type was a common finding, but mixed infections with Chitose type were also identified. In addition, using a PCR assay that targeted the major piroplasm surface gene, <i>T. orientalis</i> type 5 was detected in one cow from the Waikato region.</p><p>DIAGNOSIS: The presence of <i>T. orientalis</i> type Ikeda, as well as type 5, was confirmed in cattle from outbreaks of bovine anaemia in herds throughout the North Island of New Zealand.</p><p>CLINICAL RELEVANCE: Two new types of <i>T. orientalis</i> were identified in this investigation, that were associated with a sudden rise in cases of bovine anaemia. The body of evidence showed that the Ikeda type was implicated as the cause of disease observed in this epidemic.</p></div

Prevalence and spatial distribution of cattle herds infected with <i>Theileria orientalis</i> in New Zealand between 2012 and 2013

<div><p></p><p>AIM: To describe the prevalence and spatial distribution of cattle herds infected with Ikeda and non-Ikeda types of <i>Theileria orientalis</i> in New Zealand between November 2012 and June 2013.</p><p>METHODS: Pooled serum samples collected historically between November 2012 and June 2013 were obtained from cattle herds throughout New Zealand. Each pooled sample consisted of approximately 20 individual cattle samples from that herd, and was provided with details of the spatial location of the herd (n=722). DNA from all samples was tested using two quantitative PCR assays for the detection of <i>T. orientalis</i> (all types) and the Ikeda type. The proportion of herds that were positive for <i>T. orientalis</i> and Ikeda type, or that were positive for <i>T. orientalis</i> but negative for Ikeda type (non-Ikeda positive) was determined for different regions of New Zealand.</p><p>RESULTS: The highest prevalence of herds infected with Ikeda type was detected in the Northland (33/35; 94%) and Auckland and the Waikato (63/191; 33%) regions. Only 2/204 (1%) herds were positive for the Ikeda type in the South Island. A high percentage of herds that were positive for non-Ikeda types was detected in the Gisborne and Hawkes Bay (23 (95%CI=13–37)%), Auckland and Waikato (22 (95%CI=16–29)%) and Bay of Plenty (24 (95%CI=10–44)%) regions.</p><p>CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of Ikeda type detected in cattle herds in the Northland, Auckland and Waikato regions represents a risk to naive cattle being introduced into these regions. There is also the potential for resident cattle herds in the Gisborne and Hawkes Bay, Auckland, Waikato and Bay of Plenty regions to experience increased infection with the Ikeda type.</p><p>The overall impact experienced by regions will depend on other factors such as the number of herds present and the predominant type of farming, as well as the interplay between tick ecology, cattle immunity and movement patterns of cattle.</p></div

Clinical haematology and biochemistry profiles of cattle naturally infected with <i>Theileria orientalis</i> Ikeda type in New Zealand

<p>AIMS: To present the haematology and biochemistry profiles for cattle in New Zealand naturally infected with <i>Theileria orientalis</i> Ikeda type and investigate if the results differed between adult dairy cattle and calves aged <6 months.</p> <p>METHODS: Haematology and biochemistry results were obtained from blood samples from cattle which tested positive for <i>T. orientalis</i> Ikeda type by PCR, that were submitted to veterinary laboratories in New Zealand between October 2012 and November 2014. Data sets for haematology and biochemistry results were prepared for adult dairy cattle (n=62 and 28, respectively) and calves aged <6 months (n=62 and 28, respectively), which were matched on the basis of individual haematocrit (HCT). Results were compared between age groups when categorised by HCT. Selected variables were plotted against individual HCT, and locally weighted scatterplot smoothing (Loess) curves were fitted to the data for adult dairy cattle and calves <6 months old.</p> <p>RESULTS: When categorised by HCT, the proportion of samples with HCT <0.15 L/L (severe anaemia) was greater for adult dairy cattle than for beef or dairy calves, for both haematology (p<0.002) and biochemistry (p<0.001) submissions. There were differences (p<0.05) between adult dairy cattle and calves aged <6 months in the relationships between HCT and red blood cell counts, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentrations, lymphocyte and eosinophil counts, and activities of glutamate dehydrogenase and aspartate aminotransferase. In both age groups anisocytosis was frequently recorded. The proportion of blood smears showing mild and moderate macrocytosis was greater in adults than calves (p=0.01), and mild and moderate poikilocytosis was greater in calves than adults (p=0.005).</p> <p>CONCLUSIONS AND CLINICAL RELEVANCE: The haematology and biochemistry changes observed in cattle infected with <i>T. orientalis</i> Ikeda type were consistent with extravascular haemolytic anaemia. Adult dairy cattle were more likely to be severely anaemic than calves. There were differences in haematology and biochemistry profiles between adult dairy cattle and calves, but most of these differences likely had a physiological rather than pathological basis. Overall, the haematological changes in calves aged <6 months appeared less severe than in adult dairy cattle.</p

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand

<p>CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons.</p> <p>LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D₇₅₂ that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak.</p> <p>DIAGNOSIS: Equine herpesvirus myeloencephalopathy.</p> <p>CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.</p