- Refinement of diagnostic test methods for Johne’s disease in deer.
- Determination of the prevalence of paramyxovirus serotypes 1, 2, and 3 in caged birds, wild birds and poultry in New Zealand.
- Validation of bluetongue PCR test.
- Strategies for the monitoring and management of tropical grass webworm (Herpetogramma licarsisalis) in Northland.
4. Maintaining agricultural security
4.1 MAS 311
Programme Title: |
Refinement of diagnostic test methods for Johne’s disease in deer. |
| Programme Leader: | Dr Geoff de Lisle |
| Institution: | AgResearch, Wallaceville |
Summary
This programme aimed to investigate the use of the liquid culture system MGIT, for the isolation of Mycobacterium paratuberculosis and to evaluate a PCR test to distinguish between the “ovine” and “bovine” strains of this organism. The study was carried out using deer samples that had previously been cultured using the conventional method employing Herrold’s egg yolk medium. The MGITs were a rapid method for culturing M. paratuberculosis from tissues. Isolation times of M. paratuberculosis for MGITs were approximately half those for Herrold’s egg yolk medium. A PCR test was developed to distinguish between “ovine” strains of M. paratuberculosis and “bovine” strains. Primers were developed from IS900 and evaluated on purified DNA from a range of different isolates of M. paratuberculosis. “Ovine” and “intermediate” strains of M. paratubeculosis gave a product that was not present when “bovine” strains were examined. Using this PCR test the researchers demonstrated that some “ovine” strains could be cultured using the MGIT system. However, while this PCR test worked well for examining cultures of M. paratubeculosis, it was not suitable for identifying these strains in clinical samples.
********************************************************
Background
The goal of this project was to develop improved methods for the isolation and identification of Mycobacterium paratuberculosis for the post-mortem diagnosis of Johne’s disease in deer.
Johne’s disease is an emerging problem in farmed deer. Currently, Johne’s has been diagnosed in over 100 deer farms. In a proportion of the infected deer, the lesions caused by M. paratuberculosis are grossly and microscopically indistinguishable from those caused by M. bovis. Bacteriological isolation of M. paratuberculosis is the “gold standard” for the diagnosis of Johne’s disease. However, the current culture procedures using specialised solid media are slow. Furthermore, some strains of M. paratuberculosis, especially those infecting small ruminants, are not readily isolated using Herrold’s egg yolk medium.
Recently, the BACTEC 12B radiometric culture system developed for isolating human tuberculosis organisms, has been adapted to successfully isolate both “ovine” and “bovine” strains of M. paratuberculosis. This system has the disadvantages of requiring an expensive instrument to detect growth and the use of needles to inoculate the culture media. In order to overcome these deficiencies Becton Dickinson developed a new liquid culture system MGIT (Mycobacterial growth indicator tubes) based on the use of a fluorescent indicator. In this project the researchers have evaluated the use of MGIT for the isolation of M. paratuberculosis and developed a PCR method for distinguishing between “ovine” and “bovine” strains of this organism.
Approach & Outcomes
A total of 81 tissue homogenates from deer and sheep were cultured using Herrold’s egg yolk medium and MGITs. The homogenates were all from cases that had been previously cultured for mycobacteria, including M. paratubeculosis and had been stored at –20oC for up to 24 months. Prior to culturing, the tissue homogenates were decontaminated, centrifuged and then inoculated into a MGIT and onto three slopes of Herrold’s egg yolk medium supplemented with mycobactin. The MGIT was supplemented with mycobactin and the antibiotic supplement, PANTA. A PCR test was developed for distinguishing between “ovine” and “bovine” strains of M. paratuberculosis. Primers were developed from IS900 and evaluated on purified DNA from a range of different isolates from M. paratuberculosis. “Ovine” and “intermediate” strains of M. paratuberculosis gave a product that was not present when “bovine” strains were examined.
The isolation rate with MGITs was slightly less than that achieved with Herrold’s egg yolk medium. In four samples, no results were recorded with MGITs due to the over-growth of cultures with bacterial contaminants. Importantly, the mean time to detection of positive cultures with MGITs was less than half that observed with Herrold’s. The time to detect positive cultures was related to the number of M. paratuberculosis present in the lesions. MGITs on average, detected M. paratubeculosis growth 17.7 days from samples containing large numbers of organisms compared with 34.2 days from the samples that contained few organisms.
MGITs were used to culture 25 cervine tissues which contained acid fast organisms, but no mycobacteria were isolated when they were initially examined using conventional solid media, including Herrold’s egg yolk medium. M. paratuberculosis was only isolated from 8/25 of these samples using MGITs. The reason for the failure to isolate M. paratuberculosis from all samples was not determined. However, the “ovine” PCR demonstrated that 5 of these 8 positive were the “ovine” type of M. paratuberculosis. The PCR test based on IS900 could identify “ovine” strains of M. paratuberculosis when it was applied to purified DNA or cultures. This test was not suitable for identifying these strains in clinical samples.
Publications:
de Lisle, G.W.; Yates, G.F.; Cavaignac, S.; Collins, D.M. (1999) Evaluation of the MGIT system for culturing Mycobacterium paratuberculosis and characterisation of strains by polymerase chain reaction tests. Sixth International Colloquium on Paratuberculosis. 14-19 February, 1999. Melbourne, Australia. (Poster).
4.2 MAS 312
Programme Title: |
Examination of pasteurised milk for the presence of viable Mycobacterium paratuberculosis. |
| Programme Leader: | Dr Geoff de Lisle |
| Institution: | AgResearch, Wallaceville |
Further research is being undertaken to provide additional clarification to the results of this initial research. The results from this study will be published once the series of further experiments has been completed.
4.3 MAS 313
Summary
This programme aimed to provide information on the prevalence of avian paramyxovirus serotypes (PMVs) in New Zealand to allow confident decision-making regarding the importation of birds and poultry products and consequently protect the poultry industry and our bird population, including native birds.
Blood samples collected from caged birds, wild birds, and poultry were analysed for the presence of PMV1, 2, and 3 antibodies by HI test and cloacal swabs collected from caged and wild birds were inoculated into embryonated eggs to attempt PMV isolation. No virus was isolated from any of the 271 cloacal swabs and therefore prevalence of the PMVs under study can only be interpreted from the serology results. A very low prevalence of PMV1 antibody was found in caged birds (4.8%) (e.g., guinea fowl, Chinese quail) and in wild birds (1.7%); in poultry the researchers found 0.3% reactants to PMV3 which were assessed to be a cross-reaction to PMV1 antigen. The significance of the very low titres to PMV2 (1.7% in caged birds, 4.2% in wild birds, and no reactants in poultry) cannot be unequivocally interpreted as an indication of the presence of PMV2 in New Zealand and needs to be treated cautiously. Researchers can be more confident of the PMV3 results and this survey shows that PMV3 is unlikely to be present in the New Zealand bird populations under study. Only regular surveys will provide accurate information on the prevalence of PMVs in New Zealand.
********************************************************
Background
The goal of this project was to gain information on the prevalence of avian paramyxoviruses (PMVs) in New Zealand to allow confident decision-making regarding importation of birds and poultry products, and consequently protect the poultry industry and our bird population, including native birds. To comply with the new trading rules, New Zealand must develop importation protocols based on scientifically sound information to control the importation of live birds or poultry products. No information was previously available on the prevalence of the PMVs under study, as no similar surveys had been carried out in New Zealand.
Approach & Outcomes
Blood and cloacal swab samples were collected from a wide range of birds, to cover a variety of species with different habitats, and serological and virological approaches were used to determine the prevalence of the viruses.
To detect the presence of specific antibodies in the serum, the haemagglutination inhibition (HI) test with PMV1, 2 and 3 antigens was used. The results were expressed as the reciprocal of the highest dilution of serum inhibiting 4 haemagglutination units of the antigen with the first recorded dilution being 1:4. The cloacal swabs collected from caged and wild birds were investigated for the presence of PMV by inoculating samples into embryonated SPF chicken eggs. Two 4–6-day-long passes were performed and allantoic fluid collected from eggs was checked for haemagglutinating activity to confirm the presence of the virus.
For the 231 caged birds, antibodies to PMV serotypes 1, 2, and 3 were detected in 11, 4, and 6 blood samples respectively and most of these were from "fancy" poultry breeds. In the 522 wild birds the highest number (22) of low titres were to PMV2 antigen and 59% of these were detected in passerine birds; only 9 samples had titres to PMV1 and 4 to PMV3 antigen. No virus was isolated from any of the cloacal swabs tested. Only 5 of the 1778 poultry sera samples had titres to PMV3 antigen and these were later found to be a cross-reaction to PMV1.
Publications:
Stanislawek et al.: Determination of the prevalence of paramyxovirus serotypes 1, 2, and 3 in caged birds, wild birds, and poultry in New Zealand. (In preparation.)
4.4 MAS 319
Programme Title: |
Validation of bluetongue PCR test. |
| Programme Leader: | Dr Kok-Mun Tham |
| Institution: | National Centre for Disease Investigation, Wallaceville |
Summary
This programmed aimed to validate RT-PCR assays for the detection of BTV VP7 gene. Sheep experimentally infected with two serotypes of BTV were sampled for buffy coat cells at intervals post-infection and spleen were collected from all the sheep at postmortem. These samples were tested for the presence of BTV by RT-PCR, virus isolation in embryonated chicken eggs and antigen ELISA.
Comparison of the three diagnostic tests showed that the BTV RT-PCR was as sensitive as the traditional virus isolation in embryonated chicken eggs and antigen ELISA for the detection of BTV in buffy coat cells and spleen of experimentally infected sheep. Moreover, the RT-PCR could detect BTV RNA two to three days before infectious BTV could be recovered from the buffy coat.
********************************************************
Background
Current diagnosis of BTV infection relies upon serology and costly, laborious and time-consuming virus isolation. In addition, serology may not identify an active BTV infection and may be complicated by cross-reactions at the serotype and serogroup level. Recently, a diagnostic PCR assay was developed in the former Central Animal Health Laboratory, Wallaceville for BTV using plasmid DNA carrying the VP7 gene of BTV as positive template. The goal of this project was to validate the BTV PCR using blood and spleen samples of sheep experimentally infected with two serotypes of BTV.
Approach & Outcomes
Four sheep were inoculated with BTV1 (Australian) and another four with BTV3 (South African). Two uninoculated sheep were used as negative controls. Bloods were collected into EDTA tubes from the animals on day 0 prior to BTV inoculation and at intervals post-inoculation. Spleen samples were collected from all sheep at post-mortem. Buffy coat cells were prepared from the EDTA bloods and stored at -70ºC, as with the spleen samples.
The presence of BTV in clinical samples (bloods and spleen) from experimentally infected and control sheep was detected by reverse transcription (RT)-PCR using four sets of primers specific for the VP7 gene of BTV. Standard diagnostic tests such as virus isolation in embryonated chicken eggs and antigen ELISA were performed by AAHL, Geelong.
Sheep infected with the Australian BTV1 and the South African BTV3 showed outstanding clinical and post-mortem signs, indicative of successful experimental infection. RNAs extracted from the spleen and buffy coat cells were RT by random primers with greater efficiency and this method was subsequently used to synthesise 1st strand cDNA from all the splenic and buffy coat RNAs. RT-PCR assays for the VP7 gene of BTV performed with the primer pairs B8.3/B8.8. B8.7/B8.8 and B8.1/B8.8 yielded amplified fragments of the expected sizes of 338, 414 and 659 bp respectively with buffy coat cell and spleen RNAs. However, RT-PCR using other primer pair combinations such as B8.1/B8.2, B8.3/B8.4 and B8.5/B8.6 showed that these primers were less efficient in amplifying the desired gene sequences. This is particularly so with the primer pair B8.1/B8.2 which failed to amply a VP7 gene fragment of 1156bp whereas the primer pairs B8.3/B8.4 and B8.5/B8.6 yielded the expected fragments of 770 and 859bp in some sample RNAs only. The failure of these primers to amplify satisfactorily the desired gene fragments could be attributed to the inefficiency in the RT reactions of synthesising long cDNA fragments which are essential for successful PCR amplifications.
The RT-PCR assays used in this study could detect BTV RNA in the buffy coat cells of sheep experimentally infected with two serotypes of BTV as early as two days post-infection. Both serotypes of BTV RNA could be detected in all the infected sheep up to days 11 post-infection with the primer pair B8.7/B8.8. Comparison of RT-PCR for the detection of BTV in the buffy coat cells and spleen of experimentally infected sheep with the standard BTV detection tests such as virus isolation in embryonated chicken eggs and antigen ELISA showed that the RT-PCR was as sensitive as virus isolation in enbryonated chicken eggs. Moreover, the RT-PCR could detect BTV RNA two to three days before infectious BTV could be isolated from the buffy coat. The RT-PCR for the BTV VP7 gene was also more sensitive than both the virus isolation and antigen ELISA tests for the detection of BTV in the spleen samples. The RT-PCR assay was also tested on RNAs extracted from additional Australian BTV isolates and a BTV isolate originated from each of Indonesia and South Africa.
The results showed that the primer pair B8.7/B8.8 which yielded the 414bp fragment could amplify all the Australian, Indonesia and South Africa BTV isolates tested whereas the primer pair B8.3/B8.8 which yielded the 338bp fragment could amplify only the Australian BTV isolates. RNA extracted from the deer epizootic haemorrhagic disease virus (EHDV) was tested as a control negative for the specificity of the PCR assay and primer pairs failed to amplify the deer EHDV RNA, which clearly showed that the RT-PCR was specific for the VP7 gene of the BTV. These results validated that the RT-PCR assay developed in New Zealand is sensitive and specific for the detection of the VP7 gene of the BTV in the buffy coat cells and spleen of sheep experimentally infected with two serotypes of BTV.
The main obstacles encountered in this study were:
- the viraemic phase of a natural BTV infection was not determined by RT-PCR,
- the RT-PCR assays need to be further tested with the other 22 serotypes of BTV,
- the amplified products should be confirmed by sequence-specific internal probe(s) or by direct cycle sequencing of the amplicons to confirm their authenticity,
- the use of sequence-specific antisense primers for the RT reactions was not thoroughly investigated, particularly with the use of improved reverse transcriptases which can synthesise long cDNA,
- the sensitivity limit of detections was not investigated, although preliminary tests using dilution series showed that the RT-PCR could amplify BTV RNA at 10-5 dilution of a buffy coat sample,
- the specificity of the BTV RT-PCR assay requires further validation using larger number of samples from bluetongue-free sheep.
4.5 MAS 321
Programme Title: |
Development and validation of a serological assay for the detection of hydatid (Echinococcus granulosus) cysts in animals. |
| Programme Leader: | Dr Michael Reichel |
| Institution: | National Centre for Disease Investigation, Wallaceville |
Summary
This programme aimed to develop an ELISA that could be used to test imported animals for the presence or absence of cysts of Echinococcus granulosus. An 8kDa protein antigen was purified to homogeneity from sheep hydatid cyst fluid, and the conditions for an ELISA were optimised using this antigen. The 8kDa ELISA was assessed with a panel of sera: 23 sera from experimentally E. granulosus infected sheep, five sera from T. hydatigena infected and four sera from T. ovis infected sheep, and 60 sera from New Zealand sheep, considered free from E. granulosus infections. The sensitivity was 47.8% and the specificity 96.7%.
Because of the low sensitivity, an alternative ELISA was developed, named ProtELISA, which used a crude protoscolex antigen preparation. By applying the same panel of sera as for the 8kDa ELISA, the sensitivity of the ProtELISA was 82.6% and the specificity 98.3%. Both ELISAs were more extensively validated with sera from 1012 New Zealand sheep, considered free from E. granulosus infections and sera from 52 experimentally E. granulosus-infected sheep. Sera from 19 T. ovis and 18 T. hydatigena experimentally-infected sheep were used for the detection of cross-reactivities. Cut-off values were established at mean absorbance + 3x standard deviation, as 0.490 for the 8kDaELISA and 0.373 for the ProtELISA.
Based on the results from 52 sera from experimentally infected sheep, diagnostic sensitivities for both ELISAs were relatively low at 46.2% and 55.8% for the 8kDaELISA and the ProtELISA, respectively. Larger numbers of sera from animals naturally infected with E. granulosus and of which the disease status is known, will be used for further validation of both assays. Nevertheless, results from 23 of the experimentally infected sheep with known cyst numbers, it can be concluded that animals with heavier infections will show up in these tests but not animals with light infections. Therefore, these ELISAs are useful for detecting infections on a flock basis, rather than in every individual animal. Research will be undertaken during 1999/2000 to increase sensitivities, for example by using oncosphere antigens, which are known to cause antibody responses during early stages of E. granulosus infections.
********************************************************
Background
Hydatid disease is about to become an exotic disease to New Zealand. Imported live animals will have the potential to re-establish the disease, because it is common in most other countries, especially in Australia, the most likely source country for imported livestock. The goal of this project is to develop an ELISA that could be used to test imported animals for the presence or absence of cysts of E. granulosus.
Approach & Outcomes
The objective for the agreement period was to optimise the parameters of an ELISA for the detection of E. granulosus infections in sheep. An 8kDa protein antigen was purified to homogeneity from sheep hydatid cyst fluid by concentration, heat treatment, separation of heat stable components by centrifugation, ethanol precipitation and gel filtration separation under denaturing conditions. An immunoblotting technique was applied to monitor the purification progress. The conditions for an ELISA were optimised using this 8kDa antigen. This 8kDaELISA was assessed with a panel of sera: 23 sera from experimentally E. granulosus infected sheep, five sera from T. hydatigena infected and four sera from T. ovis infected sheep, and 60 sera from New Zealand sheep, considered free from E. granulosus infections. The sensitivity was 47.8% and the specificity 96.7%. None of the T. hydatigena and T. ovis sera were positive in this 8kDaELISA. Because of the low sensitivity, an alternative ELISA was developed, named ProtELISA, which used a crude protoscolex antigen preparation. By applying the same panel of sera as for the 8kDaELISA, the sensitivity of the ProtELISA was 82.6% and the specificity 98.3%. None of the T. hydatigena and T. ovis sera were positive in this ProtELISA.
Both ELISAs were more extensively validated with sera from 1012 New Zealand sheep, considered free from E. granulosus infections and sera from 52 experimentally E. granulosus-infected sheep. Sera from 19 T. ovis and 18 T. hydatigena experimentally-infected sheep were used for the detection of cross-reactivities. Cut-off values were established at mean absorbance + 3x standard deviation, as 0.490 for the 8kDaELISA and 0.373 for the ProtELISA. These values were chosen because no cross-reactivity with sera from sheep infected with other taeniid species were observed, while at cut-offs of mean absorbance + 2x standard deviation some of these sera were positive. Specificities were good at 99.5% and 98.2%, respectively.
Based on the results from 52 sera from experimentally infected sheep, diagnostic sensitivities for both ELISAs were relatively low at 46.2% and 55.8% for the 8kDaELISA and the ProtELISA, respectively. Larger numbers of sera from animals naturally infected with E. granulosus and of which the disease status is known, could only be obtained very recently and will be used for further validation of both assays. Nevertheless, results from 23 of the experimentally infected sheep with known cyst numbers, it can be concluded that animals with heavier infections will show up in these tests but not animals with light infections. Therefore, these ELISAs are useful for detecting infections on a flock basis, rather than in every individual animal. Attempts will be made during 1999/2000 to increase sensitivities, for example by using oncosphere antigens, which are known to cause antibody responses during early stages of E. granulosus infections.
4.6 MAS 322
Programme Title: |
Strategies for the monitoring and management of tropical grass webworm (Herpetogramma licarsisalis) in Northland. |
| Programme Leader: | Bruce Willoughby |
| Institution: | AgResearch, Ruakura |
Summary
The aim of this programme was to provide information allowing a creditable assessment of the impacts of recently established tropical grass webworm in New Zealand.
Aspects of the biology of Herpetogramma licarsisalis were investigated by monitoring over-wintering populations of tropical grass webworm, determining temperature tolerance and undertaking screening of pasture species.
It was concluded that under the current climatic conditions H. licarsisalis was surviving on the edge of its ecological limits in northern Northland. Spread of this pest into areas immediately south of Kaitaia may occur but the evidence suggests that it is unlikely that these infestations would form permanent breeding populations. Further work would be necessary to determine whether infestations of H. licarsisalis are going to be sporadic in occurrence or will become a regular feature in pastoral farming systems in northern Northland.
Objective 1: Monitoring of over-wintering tropical grass webworm
Objective 2: Temperature tolerance
Objective 3: Pasture species screening
********************************************************
Background
The presence of established breeding populations of tropical grass webworm (Herpetogramma licarsisalis) in northern Northland was confirmed in March 1999. A delimiting survey was undertaken by AgResearch for MAF Regulatory Authority in March 1999 confirming two loci of establishment, both on the Aupouri Peninsula; one centred at Pukenui (37,000 ha including 15,000 ha forestry), the second of 30 – 40 ha on Te Paki Station (Willoughby and Hardwick 1999).
The goal of this project was to provide information allowing a creditable assessment of the impacts of tropical grass webworm in New Zealand.
Approach & Outcomes
Aspects of the biology of H. licarsisalis investigated included:
- Three distribution surveys to determine its ability to over winter in northern Northland.
- Laboratory studies to determine temperature tolerance and initial screening of pasture species for feeding preference.
- Literature and laboratory studies, and field observations into the role of natural enemies controlling H. licarsisalis populations.
Populations of H. licarsisalis were sampled three times over the winter of 1999. During this monitoring period population densities of H. licarsisalis decreased from approximately 1500 m-2 in March to between 2-5 m-2 in July. In early September population densities were below quantifiable levels. During the same period the number of sites at which H. licarsisalis was found decreased from 64% of sites sampled in March to 6% of sites sampled in September. The sites where H. licarsisalis was found in September 1999 were typically well drained northerly facing steep slopes.
Experiments investigating the effect of temperature on various life stages showed that temperature has a large influence on the survival and development of H. licarsisalis. At 15°C or lower adults did not lay eggs or emerge from pupal cases. Below 10°C eggs did not develop or hatch. Newly hatched larvae could survive short periods at 0°C, indicating that they may be able to survive light frosts. The results of these trials indicated that larvae would be the life stage to over winter. Field observations confirmed this result.
Laboratory choice tests were used to investigate the feeding preferences of H. licarsisalis larvae. Early instar H. licarsisalis larvae were found to prefer kikuyu to ryegrass, tall fescue or meadow fescue. However, late instar larvae showed no distinct preference when presented ryegrass and kikuyu but did prefer kikuyu to tall fescue. Endophyte infection of ryegrass or tall fescue did not deter the feeding of early instar H. licarsisalis larvae. However, after 5 days there was less feeding on endophyte infected than on non-endophyte meadow fescue.
The role of endemic parasitoids, predators and diseases was assessed by the following methods.
- Field observations.
- Rearing field-collected H. licarsisalis larvae (450) and pupae (68) to screen for the presence of both pathogens and parasitoids.
- Laboratory bioassay in which larvae and pupae were exposed to the endemic parasitoid Apanteles ruficrus.
- Literature review.
The investigations indicated that existing parasitoids and pathogens were having little influence on populations of H. licarsisalis in northern Northland. A literature review showed that at least 19 species of parasitoids and predators are responsible for keeping H. licarsisalis populations in check in other parts of the world.
Publications
Willoughby, B.and. Hardwick, S. 1999. Survey to determine the infestation limits of Herpetogramma licarsisalis (Tropical Grass Webworm) in pastures in northern Northland. Report to MAF Regulatory Authority.
Willoughby, B. E and Hardwick, S. 1999. Tropical grass webworm (Herpetogramma licarsisalis (Walker) (Lepidoptera: Pyralidae)): A short-term establishment in Northland, New Zealand? Proceedings of the 7th Australasian Conference on Grassland Invertebrate Ecology.
Hardwick, S.; Baltus, J.; and Willoughby, B. 1999. Strategies for the initiation of monitoring and management of tropical grass webworm (Herpetogramma licarsisalis) in Northland. Report to MAF Policy.
| © MAF 1999 |  |
|
| MAFnet Help | Important Disclaimer |
Contact for Enquiries
Farm Monitoring Programme Manager
Monitoring and Evaluation
MAF Policy
PO Box 2526
Wellington
NEW ZEALAND
Phone: +64 4 894 0623
Fax: +64 4 894 0741
Contact this person
