In vivo growth and genomic characterization of rickettsia-like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand.

A rickettsia-like organism, designated NZ-RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ-RLO2 was able to grow in CHSE-214, EPC, BHK-21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF-89T grew in all but BHK-21 and Sf21. NZ-RLO2 grew optimally in EPC at 15°C, CHSE-214 and EPC at 18°C. The growth of LF-89 T was optimal at 15°C, 18°C and 22°C in CHSE-24, but appeared less efficient in EPC cells at all temperatures. Pan-genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p-value = 94%). NZ-RLO2 was genetically different from previously described NZ-RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p-value = 88%), but were closely related to each other. TaqMan and Sybr Green real-time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ-RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.

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.

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.

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.

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.

An investigation of the genetic basis of increased susceptibility to neutralization by anti-fusion glycoprotein antibody arising on passage of human respiratory syncytial virus in cell culture.

Human respiratory syncytial virus isolates have previously been shown to exhibit resistance to neutralization by anti-fusion glycoprotein antibodies that is lost on passage in cell culture. Early passage resistant and late passage susceptible stocks of two virus isolates from different epidemics were cloned by plaque purification. Early passage stocks of both isolates yielded predominantly neutralization resistant clones while late passage stocks yielded predominantly susceptible clones. On further characterization of resistant and susceptible clones, resistant virus yields were lower and they were relatively resistant to both neutralization and fusion inhibition by anti-F murine monoclonal antibodies and were also resistant to neutralization by human sera and by Palivizumab. The full genome of resistant and susceptible clones from one of the isolates was sequenced. Four differences, confirmed by sequencing sister clones, were found between resistant and susceptible clones, one in each of the SH, G, F, and L genes.

New Zealand juvenile oyster mortality associated with ostreid herpesvirus 1-an opportunistic longitudinal study.

During the 2010-11 summer outbreak of ostreid herpesvirus 1 (OsHV-1) in New Zealand, an opportunistic longitudinal field study was conducted. OsHV-1 PCR-negative oyster spat (Crassostrea gigas) were relocated to an OsHV-1 PCR-positive area of the North Island of New Zealand that was experiencing juvenile oyster mortalities. Over a period of 13 d, spat were monitored for mortality, sampled for histopathology, and tested for the presence of OsHV-1 using real time PCR and Vibrio culture. Histopathology showed some evidence of tissue pathology; however, no consistent progressive pathology was apparent. Field mortalities were evident from Day 6 on. After 5 and 7 d of exposure, 83 and 100% of spat, respectively, tested positive for the virus by real time PCR. Vibrio species recovered during the longitudinal study included V. splendidus and V. aestuarianus. This study offers insight into the rapidity of onset and virulence of the virus in naïve oyster spat in New Zealand waters.

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish.

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient.

Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO(4) as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20-36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20-52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20-52% continuous gradient, the virus was recovered in the region of density 1.15-1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.

Increased susceptibility of human respiratory syncytial virus to neutralization by anti-fusion protein antibodies on adaptation to replication in cell culture.

Subgroup A respiratory syncytial viruses present in respiratory secretions and low passage level cell culture isolates were found to be markedly less susceptible to neutralization with monoclonal antibodies (MAbs) to the F glycoprotein than the cell culture adapted A2 virus strain. Low passage virus isolates collected over a 20 year period and belonging to several sub-group A lineages were refractory to neutralization with antibodies recognizing two major neutralizing antigenic sites located sub-terminally at opposite ends of the F(1) glycoprotein sub-unit. On further passage in cell culture, virus isolates exhibited both increased infectivity titers and increased susceptibility to neutralization by antibodies to both antigenic sites. The consensus nucleotide sequence of the membrane associated proteins M and of the SH, G and F glycoprotein genes, and their intergenic regions were compared for neutralization resistant and susceptible stocks of one virus strain, R17532. No changes were observed in the known monoclonal antibody epitopes on the F glycoprotein. In line with this, the increase in susceptibility was not found to be associated with any increased binding of monoclonal antibody to isolated F glycoprotein in a BIAcore assay, thus excluding the possibility that passage in cell culture selected for viruses with mutations in the antibody binding sites. M and SH genes were conserved but a number of sites in the G and F glycoprotein genes were found to vary on adaptation to cell culture suggesting that change in susceptibility to neutralization was associated with a change in the prevalent quasispecies present in the virus population. The genetic basis of phenotypic change in susceptibility remains to be determined.