The effect of month, farm type and latitude on the level of anaemia associated with Theileria orientalis Ikeda type infection in New Zealand cattle naturally infected at pasture.

Commencing in 2012, an epidemic of infectious bovine anaemia associated with Theileria orientalis Ikeda type has been present in New Zealand. The aims of this study were to analyse the temporal and spatial effects of T. orientalis Ikeda type infection on the sample submission rates and haematocrits of infected cattle over the first two years of the New Zealand epidemic. The data were collected from 30/08/2012 to 28/11/2014 and included all samples that met the case definition for Theileria associated bovine anaemia (TABA) and tested positive for T. orientalis Ikeda type by PCR. The sample submission rates by month and farm type were highly seasonal with dairy farm submissions peaking in September a month before beef farm submissions peaked. A second lesser peak of dairy farm submissions in April was absent for beef farms. A mixed effects model was fitted to the data and showed a significant interaction between farm production type (dairy or beef) and month of sampling (p=0.006) and between latitude and month of sampling (p=0.024). The estimated haematocrit, adjusted for month and latitude, for dairy cattle=0.125 (95%CI 0.121-0.129) and for beef cattle=0.151 (95% CI 0.138-0.165), p<0.0001. This research shows that infected beef animals tend to be less severely affected than dairy animals and that the month of sampling and latitude of the sampled farm have significant and interacting effects on the level of anaemia associated with T. orientalis Ikeda type infection.

Clinical haematology and biochemistry profiles of cattle naturally infected with Theileria orientalis Ikeda type in New Zealand.

AIMS: To present the haematology and biochemistry profiles for cattle in New Zealand naturally infected with Theileria orientalis Ikeda type and investigate if the results differed between adult dairy cattle and calves aged <6 months. METHODS: Haematology and biochemistry results were obtained from blood samples from cattle which tested positive for T. orientalis 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. 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). CONCLUSIONS AND CLINICAL RELEVANCE: The haematology and biochemistry changes observed in cattle infected with T. orientalis 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.

Prevalence studies for Theileria orientalis conducted during the early stages of the 2012 New Zealand epidemic of Theileria associated bovine anaemia.

Since 1982 there have been two epidemics of bovine anaemia associated with Theileria orientalis infection (TABA) in New Zealand. The latest and more serious epidemic started in 2012 and is associated with the T. orientalis (Ikeda) type. In 2013 the Ministry for Primary Industries (MPI) carried out several epidemiological investigations on the TABA epidemic, which included a series of on-farm herd prevalence studies. The aims of this paper are to present and summarise the results of these herd prevalence studies. The data were collected from March 2013 to September 2013 and comprised 26 farms. For 21/26 of these farms, a T. orientalis (Ikeda) positive index case of TABA precipitated the follow-up herd prevalence study. Each herd prevalence study involved a random sample of 10 cows for haematocrit, for piroplasm counts (number of Theileria infected RBCs per 1000 RBCs) and for PCR molecular diagnosis. Animals were diagnosed anaemic if the haematocrit ≤0.24. The results showed that for the index cases, the average haematocrit = 0.10 L/L and the average piroplasm count = 10.9 Theileria infected RBCs per 1000 RBCs, and for the herd prevalence studies the average haematocrit = 0.26 and the average piroplasm count = 6.9. The average herd prevalence of T. orientalis (Ikeda) PCR positive cattle and of anaemic cattle was 87% and 26% respectively. For dairy herds (n = 7 herds) it took, on average, 80 days for the within herd prevalence of anaemia to return to zero after the initial diagnosis. In conclusion the prevalence studies showed that infection rates and anaemia rates were already high in herds when the index case of TABA was observed. At the time, these prevalence studies significantly added to our understanding of the epidemiology of the evolving T. orientalis (Ikeda) epidemic and furthermore met the New Zealand MPI objective of being rapid and applied, fit for purpose and completed at a relatively low cost.

Monitoring Theileria orientalis (Ikeda)-associated bovine anaemia in affected cattle over time.

The aim of the study was to observe changes in haematocrit (HCT) over time in a New Zealand South Island dairy herd affected by an outbreak of Theileria-associated bovine anaemia (TABA; Ikeda). A secondary aim was to relate individual cow HCTs to the amount of Theileria orientalis Ikeda DNA present in the blood, as measured by cycle threshold values, using a quantitative PCR (qPCR). Over a 6 month period, blood samples from 19 randomly selected cattle were monitored from a herd of 600 dairy cows. The sampling interval was approximately fortnightly for the first six weeks, followed by sampling at between four and six week intervals. At the initial report of the outbreak, two from six cattle were anaemic (HCT<0.25L/L). Blood collected from 14 cattle 11 days later showed that 57% (95% CI 33-77%) of the cattle sampled were anaemic. Of the 19 cattle that went on to be monitored, 12 (63% 95% CI=41-81%) developed anaemia at some point during the period of monitoring. One of the anaemic animals did not meet the case definition for TABA Ikeda. For individual cattle, the average number of days between when cattle were first detected as anaemic and when HCT returned to normal was 53days (median=47 days, range=6-92 days). At the point of notification the amount of T. orientalis Ikeda DNA in the blood of the six cattle tested was low (Cq median=36), but 11days later the amount of DNA in blood of 14 additional cows tested was relatively high (Cq median=24). Levels of all 19 cows monitored continued to remain moderately high through the period of testing (Cq median=29). This was despite a general improvement in the HCT of affected cattle. In four of the 15 cattle positive to T. orientalis Ikeda where blood fractions (plasma and whole blood) were tested, it appeared that T. orientalis Ikeda (as measured by qPCR) dropped more rapidly in plasma fractions than in whole blood at the point that HCT started to return to normal levels. Despite the assumption that tick populations were low in the Canterbury region of the South Island the impact of TABA (proportion of herd affected and the average period that animals remained anaemic) on the case herd was still relatively high.

Cluster analysis of the clinical histories of cattle affected with bovine anaemia associated with Theileria orientalis Ikeda type infection.

AIM To determine the most commonly used words in the clinical histories of animals naturally infected with Theileria orientalis Ikeda type; whether these words differed between cases categorised by age, farm type or haematocrit (HCT), and if there was any clustering of the common words in relation to these categories. METHODS Clinical histories were transcribed for 605 cases of bovine anaemia associated with T. orientalis (TABA), that were submitted to laboratories with blood samples which tested positive for T. orientalis Ikeda type infection by PCR analysis, between October 2012 and November 2014. χ2 tests were used to determine whether the proportion of submissions for each word was similar across the categories of HCT (normal, moderate anaemia or severe anaemia), farm type (dairy or beef) and age (young or old). Correspondence analysis (CA) was carried out on a contingency table of the frequency of the 28 most commonly used history words, cross-tabulated by age categories (young, old or unknown). Agglomerative hierarchical clustering, using Ward's method, was then performed on the coordinates from the correspondence analysis. RESULTS The six most commonly used history words were jaundice (204/605), lethargic (162/605), pale mucous membranes (161/605), cow (151/605), anaemia (147/605), and off milk (115/605). The proportion of cases with some history words differed between categories of age, farm type and HCT. The cluster analysis indicated that the recorded history words were grouped in two main clusters. The first included the words weight loss, tachycardia, pale mucous membranes, anaemia, lethargic and thin, and was associated with adult (p<0.001), severe anaemia (p<0.001) and dairy (p<0.001). The second cluster included the words deaths, ill-thrift, calves, calf and diarrhoea, and was associated with young (p<0.001), normal HCT (p<0.001), beef (p<0.001) and moderate anaemia (p<0.001). CONCLUSIONS AND CLINICAL RELEVANCE Cluster analysis of words recorded in clinical histories submitted with blood samples from cases of TABA indicates that two potentially different disease syndromes were associated with T. orientalis Ikeda type infection. One was consistent with the affected cattle suffering from a severe regenerative extravascular haemolytic anaemia, the second displaying as ill thrift and diarrhoea, particularly in young beef cattle.

Using a rule-based envelope model to predict the expansion of habitat suitability within New Zealand for the tick Haemaphysalis longicornis, with future projections based on two climate change scenarios.

Haemaphysalis longicornis is the only species of tick present in New Zealand which infests livestock and is also the only competent vector for Theileria orientalis. Since 2012, New Zealand has suffered from an epidemic of infectious bovine anaemia associated with T. orientalis, an obligate intracellular protozoan parasite of cattle and buffaloes. The aim of this study was to predict the spatial distribution of habitat suitability of New Zealand for the tick H. longicornis using a simple rule-based climate envelope model, to validate the model against published data and use the validated model to project an expansion in habitat suitability for H. longicornis under two alternative climate change scenarios for the periods 2046-2065 and 2081-2100, relative to the climate of 1981-2010. A rule-based climate envelope model was developed based on the environmental requirements for off-host tick survival. The resulting model was validated against a maximum entropy environmental niche model of environmental suitability for T. orientalis transmission and against a H. longicornis occurrence map. Validation was completed using the I-similarity statistic and by linear regression. The H. longicornis climate envelope model predicted that 75% of cattle farms in the North Island, 3% of cattle farms in the South Island and 54% of cattle farms in New Zealand overall have habitats potentially suitable for the establishment of H. longicornis. The validation methods showed an acceptable level of agreement between the envelope model and published data. Both of the climate change scenarios, for each of the time periods, projected only slight to moderate increases in the average farm habitat suitability scores for all the South Island regions. However, only for the West Coast, Marlborough, Tasman, and Nelson regions did these increases in environmental suitability translate into an increased proportion of cattle farms with low or high H. longicornis habitat suitability. These results will have important implications for the geographical progression of Theileria-associated bovine anaemia (TABA) in New Zealand and will also be of interest to Haemaphysalis longicornis researchers in Australia, Japan, Korea and New Zealand.

Predicting the potential environmental suitability for Theileria orientalis transmission in New Zealand cattle using maximum entropy niche modelling.

The tick-borne haemoparasite Theileria orientalis is the most important infectious cause of anaemia in New Zealand cattle. Since 2012 a previously unrecorded type, T. orientalis type 2 (Ikeda), has been associated with disease outbreaks of anaemia, lethargy, jaundice and deaths on over 1000 New Zealand cattle farms, with most of the affected farms found in the upper North Island. The aim of this study was to model the relative environmental suitability for T. orientalis transmission throughout New Zealand, to predict the proportion of cattle farms potentially suitable for active T. orientalis infection by region, island and the whole of New Zealand and to estimate the average relative environmental suitability per farm by region, island and the whole of New Zealand. The relative environmental suitability for T. orientalis transmission was estimated using the Maxent (maximum entropy) modelling program. The Maxent model predicted that 99% of North Island cattle farms (n=36,257), 64% South Island cattle farms (n=15,542) and 89% of New Zealand cattle farms overall (n=51,799) could potentially be suitable for T. orientalis transmission. The average relative environmental suitability of T. orientalis transmission at the farm level was 0.34 in the North Island, 0.02 in the South Island and 0.24 overall. The study showed that the potential spatial distribution of T. orientalis environmental suitability was much greater than presumed in the early part of the Theileria associated bovine anaemia (TABA) epidemic. Maximum entropy offers a computer efficient method of modelling the probability of habitat suitability for an arthropod vectored disease. This model could help estimate the boundaries of the endemically stable and endemically unstable areas for T. orientalis transmission within New Zealand and be of considerable value in informing practitioner and farmer biosecurity decisions in these respective areas.

An observational study of the vertical transmission of Theileria orientalis (Ikeda) in a New Zealand pastoral dairy herd.

Although only recently recognised, Theileria orientalis (Ikeda) is now the most important infectious cause of anaemia in New Zealand cattle. The aim of this study was to test if vertical transmission of T. orientalis (Ikeda) from dam to calf across the placenta occurs in naturally infected New Zealand dairy cattle and to also test whether the infection status of the dam at calving affects the future susceptibility of its offspring to T. orientalis (Ikeda) infection. Dairy cows (n=97) and their calves were sampled at calving; and the calves again at 4 months of age. All samples were measured for haematocrit and screened for T. orientalis genotypes using a multiplex Buffeli, Chitose and Ikeda specific TaqMan assay. Ikeda positive samples were further tested by singleplex PCR in triplicate to calculate the Ikeda infection intensity as genomes/µl of blood from each infected animal. No T. orientalis (Ikeda) infected calves were born to either T. orientalis (Ikeda) infected or uninfected dams. There were 56/97 dams positive for T. orientalis (Ikeda) infection at calving and 79/90 calves positive for T. orientalis (Ikeda) infection at 4 months of age but no effect on calf susceptibility of dam infection status at calving. There was a significant negative effect of infection intensity on haematocrit after controlling for whether the infected animal was a dam or a 4 month old calf. Vertical trans-uterine transmission of T. orientalis (Ikeda) infection is unlikely in chronically infected dairy cows and thus not a factor in the epidemiology of T. orientalis (Ikeda) infection.

Monitoring an epidemic of Theileria-associated bovine anaemia (Ikeda) in cattle herds in New Zealand.

Monitoring an epidemic of an emerging vector-borne disease can be problematic; particularly in a country where vector-borne disease has previously had minimal impact on livestock. This paper describes methods of past and current surveillance of the Theileria-associated bovine anaemia (Ikeda; TABA) epidemic in New Zealand, and the resulting inferences made. Over the three year period of the TABA epidemic a portfolio of surveillance methods has been used: case reporting (with subsidised PCR testing), syndromic surveillance, sentinel surveillance, testing convenience samples for herd infection, as well as specific active surveillance initiatives to understand the tick vector distribution. Surveillance data have shown that the number of affected cattle herds has continued to increase over time with seasonal peaks in spring and autumn coinciding with peak activity of nymph and adult ticks respectively. In spring 2014, the epidemic extended south into areas that were previously considered to be unsuitable for the tick vector. As a result a survey was initiated that showed that ticks were present in areas outside of the known distribution. Testing pooled blood samples from cattle herds across New Zealand showed there still remained a significant percentage of herds where only non-Ikeda type infections were present, indicating that these herds were at risk of future TABA (Ikeda) outbreaks. For some regions there had been a noticeable increase in the percentage of herds infected, yet with only a small increase in the number of outbreaks compared with the previous year. Thus, outbreaks had either gone unobserved or had not been confirmed by testing. In these regions extensive low-input beef farming could explain the non-detection observed. There was a close relationship between the number of syndromic reports of anaemia and the number of confirmed cases of TABA (Ikeda), (P<0.01, adjusted R-squared=0.74). Active monitoring of the epidemic for a three year period has provided valuable insight into seasonal nature of the disease and its continuing impact. Information from multiple surveillance sources can help build up an understanding of the epidemiology, even when data from each individual surveillance stream are limited. The TABA (Ikeda) epidemic in New Zealand represents a useful case study of long term monitoring where disease is caused by an emerging pathogen.

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand.

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. 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. DIAGNOSIS: Equine herpesvirus myeloencephalopathy. 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.

Prevalence and spatial distribution of cattle herds infected with Theileria orientalis in New Zealand between 2012 and 2013.

AIM: To describe the prevalence and spatial distribution of cattle herds infected with Ikeda and non-Ikeda types of Theileria orientalis in New Zealand between November 2012 and June 2013. 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 T. orientalis (all types) and the Ikeda type. The proportion of herds that were positive for T. orientalis and Ikeda type, or that were positive for T. orientalis but negative for Ikeda type (non-Ikeda positive) was determined for different regions of New Zealand. 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. 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. 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.

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

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 Theileria 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 Theileria orientalis infection. LABORATORY FINDINGS: Using a T. orientalis type-specific PCR assay that targeted the single subunit rRNA gene, all of six animals tested were positive for T. orientalis type Ikeda. Blood samples collected from clinically affected cattle in 11 subsequent outbreaks from throughout the North Island showed that T. orientalis 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, T. orientalis type 5 was detected in one cow from the Waikato region. DIAGNOSIS: The presence of T. orientalis 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. CLINICAL RELEVANCE: Two new types of T. orientalis 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.

Epidemiology of the epidemic of bovine anaemia associated with Theileria orientalis (Ikeda) between August 2012 and March 2014.

AIMS: To describe the epidemiology of the epidemic of bovine anaemia associated with Theileria orientalis infection (TABA) in New Zealand between 30 August 2012 and 4 March 2014. METHODS: Blood samples and associated data were obtained from cases of TABA. The case definition for TABA was met when piroplasms were present on blood smears and the haematocrit was ≤0.24 L/L. Samples were analysed using quantitative PCR (qPCR) assays for the detection of T. orientalis Ikeda type. Only cases that were positive in the qPCR assays were included in the analysis. A case herd was defined as a herd that had ≥1 animal positive for T. orientalis Ikeda. Movement records for farms were accessed through the national animal identification and tracing scheme. The OR for cattle movements onto a case farm compared to a non-case farm was estimated using a generalised estimating equation model and the geodesic distance for movements onto case and non-case farms compared using Student's t-test. The kernel-smoothed risk of disease at the farm level was calculated using an extraction map and the clustering of diseased farms in time and space was measured using the spatial temporal inhomogeneous pair correlation function. RESULTS: In the first 18 months there were 496 case herds; 392 (79%) were dairy and 104 (21%) beef herds. Of 882 individual cases, 820 (93.0%) were positive for T. orientalis Ikeda in the qPCR assays. Case herds were initially clustered in the Northland, then the Waikato regions. The OR for a case farm compared to a non-case farm having ≥1 inward cattle movements was 2.03 (95% CI=1.52-2.71) and the distance moved was 26 (95% CI=20.8-31.3) km greater for case farms. The risk of disease was highest in a north, north-eastern to south, south-western belt across the Waikato region. The spatial-temporal analysis showed significant clustering of infected herds within 20-30 days and up to 15 km distant from a case farm. CONCLUSIONS: Theileria orientalis Ikeda type is likely to have been introduced into regions populated with naïve cattle by the movement of parasitaemic cattle from affected areas. Local spread through dispersed ticks then probably became more important for disease transmission between herds once the disease established in a new area. CLINICAL RELEVANCE: Dairy and beef farming in the North Island of New Zealand will be significantly changed in the coming years by the incursion of this new disease.

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

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 Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. METHODS: Nucleotide sequences aligned with T. orientalis 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 T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis 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 T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 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 T. orientalis, 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 T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per µL of blood. CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target. CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle.

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types. LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR. A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%). DIAGNOSIS: Bovine haemoplasmosis. CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.

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.

AIMS: 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.

Variation in bull beef quality due to ultimate muscle pH is correlated to endopeptidase and small heat shock protein levels.

This study set out to determine if ultimate pH (pH(u)) affected the performance of intracellular small heat shock protein and endopeptidase dynamics in muscle during beef ageing. Longissimus dorsi muscles from 39 Angus or Limousin×Angus bulls were examined to see if pH(u) achieved at 22h post mortem (rigor) affected tenderness and water holding capacity of beef. Samples were segregated into three pH(u) groups termed high (pH>6.3), intermediate (5.73 days post mortem for intermediate pH(u) beef. High levels of alpha ß-crystallin (aßC) at 22h post mortem coincided with delayed muscle protein degradation for low pH(u) beef. Our results support the hypothesis that aßC shields myofibrils and buffers against endopeptidase degradation of beef structure during ageing.

The intracellular distribution of small heat shock proteins in post-mortem beef is determined by ultimate pH.

This study analysed Longissimus dorsi muscles from 39 Angus or Angus×Limousin bulls to determine the small heat shock protein (sHSP) dynamics in beef aged at 15°C. Using quantitative ELISA we determined that alpha ß-crystallin and HSP20 were present at higher levels in muscles from Angus bulls. sHSP levels peaked at 0.5 and 3h post-mortem for HSP20 and alpha ß-crystallin respectively. Intermediate pH (pH 5.7-6.3) beef had the highest level of alpha ß-crystallin expression at 3-6h post-mortem. Low pH (<5.7) meat had no detected soluble alpha ß-crystallin and had diminished HSP27 at 22h post-mortem. The transition of sHSP between a soluble to an insoluble phase was demonstrated by pH titration of a sarcoplasmic fraction. The effect of ultimate pH upon sHSP distribution in post-mortem muscle may influence final meat quality characteristics.

Sex-specific association between bipolar affective disorder in women and GPR50, an X-linked orphan G protein-coupled receptor.

GPR50 is an orphan G protein-coupled receptor (GPCR) located on Xq28, a region previously implicated in multiple genetic studies of bipolar affective disorder (BPAD). Allele frequencies of three polymorphisms in GPR50 were compared in case-control studies between subjects with BPAD (264), major depressive disorder (MDD) (226), or schizophrenia (SCZ) (263) and ethnically matched controls (562). Significant associations were found between an insertion/deletion polymorphism in exon 2 and both BPAD (P=0.0070), and MDD (P=0.011) with increased risk associated with the deletion variant (GPR50(Delta502-505)). When the analysis was restricted to female subjects, the associations with BPAD and MDD increased in significance (P=0.00023 and P=0.0064, respectively). Two other single-nucleotide polymorphisms (SNPs) tested within this gene showed associations between: the female MDD group and an SNP in exon 2 (P=0.0096); and female SCZ and an intronic SNP (P=0.0014). No association was detected in males with either MDD, BPAD or SCZ. These results suggest that GPR50(Delta502-505), or a variant in tight linkage disequilibrium with this polymorphism, is a sex-specific risk factor for susceptibility to bipolar disorder, and that other variants in the gene may be sex-specific risk factors in the development of schizophrenia.

Differential efficacy of vaccinia virus envelope proteins administered by DNA immunisation in protection of BALB/c mice from a lethal intranasal poxvirus challenge.

DNA vaccines might offer an alternative to the live smallpox vaccine in providing protective efficacy in an orthopoxvirus (OPV) lethal respiratory challenge model. BALB/c mice were immunised with DNA vaccines coding for 10 different single vaccinia virus (VACV) membrane proteins. After an intranasal challenge with the VACV IHD strain, three gene candidates B5R, A33R and A27L produced > or =66% survival. The B5R DNA vaccine consistently produced 100% protection and exhibited greatest efficacy after three 50 microg intramuscular doses in this model. Sero-conversion to these vaccines was often inconsistent, implying that antibody itself was not a correlate of protection. The B5R DNA vaccine induced a strong and consistent gamma interferon (IFNgamma) response in BALB/c mice given a single DNA vaccine dose. Strong IFNgamma responses were also measured in pTB5R immunised C57BL6 mice deficient for MHC class I molecules, suggesting that the memory response was mediated by a CD4+ T cell population.