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 (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 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?×?10⁴ and 3.3?×?10⁶ 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.     

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.     

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.     

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.     

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.     

Associations between Theileria orientalis Ikeda type infection and the growth rates and haematocrit of suckled beef calves in the North Island of New Zealand

AIMS: The principle aim of this study was to examine the association between infection with Theileria orientalis Ikeda type and growth rates of suckled beef calves on four beef farms. In addition, associations between calf sex, sampling time, and individual farm and T. orientalis Ikeda type infection intensity and haematocrit (HCT) were investigated.

METHODS: The study was a prospective longitudinal study in which 240 calves from four purposively selected beef farms in the North Island of New Zealand were blood sampled and weighed in late spring, mid-summer and early autumn. Two farms were from high-risk (A and B) and two from low-risk (C and D) tick areas. Blood samples were analysed to determine HCT, and the number of T. orientalis Ikeda type organisms/µL of blood (infection intensity) using a quantitative PCR assay. A calf was defined as infected if >415 organisms/µL were detected in a blood sample. Linear mixed models were used to examine associations between infection intensity, mean daily liveweight gain (MDG), HCT, calf sex and time of sampling on the four farms.

RESULTS: On Farms A and B nearly all calves were infected at each sampling time, on Farm C <30% were infected at any sampling and on Farm D infection prevalence increased from 32 to 79% between late spring and early autumn. On Farms C and D, from mid-summer to early autumn, mean MDG was 0.127 (95% CI=0.072–0.183) kg/day less for infected than uninfected calves (p<0.001). On all farms MDG was negatively associated with infection intensity for mid-summer and early autumn sampling times (p=0.037). The relationship between time of sampling and infection intensity varied between farms (p<0.001), and between male and female calves (p=0.018). Females had a higher infection intensity than males at the mid-summer and early autumn samplings. The association between HCT and infection intensity varied with sampling time and farm (p=0.018). There was a strong negative association between infection intensity and HCT at the late spring sampling, but in mid-summer there was no association, and in early autumn only a weak association.

CONCLUSIONS AND CLINICAL RELEVANCE: This study has shown that beef farmers in the North Island of New Zealand should be concerned about the welfare effects and economic impacts of T. orientalis Ikeda type infection in suckled beef calves.     

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.     

Clinicopathological features of 11 suspected outbreaks of bovine adenovirus infection and development of a real-time quantitative PCR to detect bovine adenovirus type 10

CASE HISTORY: A retrospective study was conducted to investigate 11 outbreaks of presumptive fatal adenovirus infection diagnosed through two New Zealand diagnostic laboratories during 2014 and 2015. Outbreaks occurred in 6–12-month-old Friesian or Friesian cross cattle during autumn, winter and spring. Individual outbreaks were short in duration, with mortality rates ranging from 3/250 to 20/600 (1.2 to 3.3%).

CLINICAL AND PATHOLOGICAL FINDINGS: Clinical signs included severe diarrhoea, depression, recumbency, and death. Post-mortem examination revealed congestion and oedema of the alimentary tract and fluid to haemorrhagic intestinal contents. Histopathological lesions were characterised by congestion and haemorrhage of the alimentary tract mucosa, oedema of the submucosa, and mild interstitial inflammation in the kidneys. Large basophilic intranuclear inclusion bodies were identified in vascular endothelial cells of the alimentary tract in 11/11 cases and of the kidney in 8/9 cases.

MOLECULAR TESTING: A real-time quantitative PCR (qPCR) assay was designed to detect bovine adenovirus type 10 (BAdV-10) using hexon gene sequences available in GenBank. DNA extracted from a field case and confirmed by sequencing was used as a positive control. The qPCR had a reaction efficiency of 101% (R²=0.99) and the limit of detection was <10 DNA copies/reaction. The qPCR detected BAdV-10 in formalin-fixed paraffin-embedded (FFPE) tissue from 10/11 cases. DNA sequencing of PCR products from nine of these cases showed them to be identical to BAdV-10 sequences in GenBank. For the PCR-negative case, the PCR product had a hexon sequence 99% similar to bovine adenovirus Wic isolate Ma20-1, a close relative of BadV-10.

DIAGNOSIS: Bovine adenovirus type 10 was identified in FFPE tissues from cattle with histopathological evidence of adenovirus infection.

CLINICAL RELEVANCE: Bovine adenoviruses, and especially BAdV-10, should be considered in the differential diagnosis for acute enteric disease and death in young cattle. The qPCR detected BAdV-10 from FFPE tissue of cattle with suspected adenoviral infection diagnosed by histopathology. However results should be interpreted in light of clinical and pathological findings due to the possibility of adenovirus shedding by healthy cattle and the presence of pathogenic adenoviruses other than BAdV-10.     

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.