2. Maintaining Biosecurity

2.1 MBS 301: DNA diagnostic procedures for the identification of selected species and populations of Lymantria and Orgyia moths from intercepted egg masses

Programme Title: DNA diagnostic procedures for the identification of selected species and populations of Lymantria and Orgyia moths from intercepted egg masses
Programme Leader: Karen Armstrong
Institution: Lincoln University

Summary

A molecular method was required for the routine species identification of Lymantria egg masses that are intercepted at the border and which are difficult to distinguish morphologically. Seven Northern Hemisphere lymantriid species, Lymantria dispar, L. mathura, L. monacha, Orgyia antiqua, O. leucostigma, O. pseudotsugata and O. thyellina, have been selected as high risk to the New Zealand forest industry based on their favourable biology, life history strategies and the availability of transport pathways.

DNA extraction used a solid matrix method modified for use with small insects. DNA variation was determined by PCR and sequence analysis of ribosomal DNA regions. Polymorphic restriction sites were identified from the DNA sequence data and restriction analysis carried out to define species-specific patterns. Additional, flanking DNA sequence data was collected for PCR primer design.

Specimens were obtained for all species and the DNA PCR amplified from all except O. thyellina. Appropriate nucleotide variation was revealed in the rDNA ITS1 and lymantriid-specific PCR primers were designed for this region. Diagnostic restriction site patterns of the rDNA ITS1 were identified that could readily distinguish the six target species, as well as an additional low-risk species, L. xylina. To date there is not evidence of intra-species variation in these patterns. The method has been optimised for use with egg mass material and early instar larvae; identifications are achieved in about one working day. The final procedure has been documented in a manual for routine use by non-specialist laboratories. Data used to develop this low-tech approach could also be used in more sophisticated high-throughput procedures. Recommendations from this research are to validate the method using material intercepted at the border and to continue testing populations, as they become available. The method could also be extended to include additional Australian and South American lymantriid species. The similarity of this approach to that currently in use for fruit flies, and under-way for mealybugs, demonstrated its flexibility and the potential to streamline molecular diagnostic procedures for a number of plant pests through a series of common techniques and kits.

Background

The goal of this project was to develop a DNA method for the species-level identification of morphologically indistinguishable Lymantria egg masses that are intercepted at the border.

Seven Northern Hemisphere lymantariid species, Lymantria dispar, L. mathura, L. monacha, Orgyia antiqua, O. leucostigma, O. pseudotsugata and O. thyellina, require accurate identification. All are considered high risk to New Zealand forestry, based on the likelihood of their arrival and establishment here. Key factors are favourable biology, life history strategies and availability of transport pathways. Different molecular markers, to those already published for L. dispar, are required for this range of species.

Approach & Outcomes

Positively identified specimens were obtained through contacts in the USA, Canada, Europe and Asia. The approach for extracting and analysing DNA was based on that used in previous MAF Operational Research for the identification of fruit fly immature life stages. This included a modified Prep A Gene (BioRad) extraction procedure, specific polymerase chain reaction (PCR) amplification of ribosomal DNA (rDNA) and sequence and restriction site analysis of the PCR products.

DNA from single legs of adults and single eggs were successfully analysed for six species. For O. thyellina, one source was misidentified and the other, museum specimens, would not PCR-amplify reliably. Sequence analysis using the other species revealed the first internal transcribed spacer (ITS1) to be sufficiently variable and a suitable source of diagnostic restriction sites. Lymantriid-specific PCR primers were designed and restriction fragment length polymorphism (RFLP) patterns for the ITS1 PCR product were established. Each of the six species could be readily distinguished from all others by between three and seven of the restriction enzymes Acc I, Cfo I, Hinf I, Mse I, Rsa I, Sau 3A, and Taq I; a species identification RFLP matrix was constructed. The most similar species were O. leucostigma and O. pseudotsugata. An additional species, L. xylina, provided very distinct RFLP patterns. This species is native to Taiwan and considered low risk to New Zealand, but was useful for further testing the species-specificity of the markers. A preliminary population analysis did not reveal any intra-specific variation in the restriction patterns. The diagnostic procedure is documented in a manual for use egg mass material, typically matted with abdominal hair, and also early instar larvae. Diagnosis takes about one working day. The sequence data used to develop this low-tech approach could also be used to develop more sophisticated high-throughput procedures.

Publications

Armstrong, K.F., McHugh, P.C. and Frampton, R. (2000): A molecular-based quarantine procedure for the identification of selected lymantriid species from egg masses intercepted at the border. To be submitted to the Agricultural and Forest Entomology.

Armstrong, K.F. and McHugh, P.C. (2000): Review of the molecular methods available for insect species identification and their suitability for routine diagnostic procedures. To be submitted to Bulletin of Entomological Research.

2.2 MBS 302: Assessment of contamination soil as a risk pathway

Programme Title: Assessment of contamination soil as a risk pathway
Programme Leader: Dr John Marshall
Institution: Crop & Food Research

Summary

Goal: To identify pathways, and determine relative risks to New Zealand, of pest and disease entry as a result of soil introductions into New Zealand with particular reference to the container trade.

Methods: External surfaces of 1484 containers were examined for soil or mineral contaminants. All soil samples collected were examined for the presence of, and species composition of, fungi, animal pathogens and plant parasitic nematodes.

Results and conclusions:

  1. Most soil samples contained potentially pathogenic organisms.
  2. Most soil samples were sand and gravel's with a small number of organic soils detected.
  3. Organic soils pose a greater risk of carrying potentially damaging pathogens.
  4. Surveys should be continued to identify organic contaminants on containers, and the contaminated containers should be cleaned at the shippers cost.
  5. Other forms of container contamination pose less risk and containers should only be treated, as a matter of consistency, if they are grossly contaminated.
  6. A focussed survey for specific organisms should be set up to target the most likely vector or host source of contamination.
  7. The range of species present in the samples analysed soil is less than would be found in New Zealand soil samples.
  8. The genetic make-up of the organisms found in samples and their potential to introduce new genotypes was not examined, but if a widely divergent gene pool was entering New Zealand by this route the need for routine decontamination of all containers may be indicated.
  9. The presence of human pathogens was consistently recorded, indicating that there may be health risks for inspectors and container workers if they are exposed to this material for long periods without adequate protection.

Background

The goal of this project was to identify pathways, and determine relative risks to New Zealand, of pest and disease entry as a result of soil introductions into New Zealand with particular reference to the container trade.

Soil is present on many objects entering New Zealand, including containers. To evaluate the potential for new organisms to establish in New Zealand as a result of undetected soil introductions on containers we therefore need to access likely pathways and define possible risks using a selected range of indicator organisms.

Approach & Outcomes

We examined the external surfaces of 1484 containers, of which 33.7 percent yielded sufficient soil samples for a range of analyses for a selected range of organisms.

Organisms detected in soil samples:

Pseudomonads: The most common species of Pseudomonas in the soil samples was Pseudomonas putida and P. fluorescens. Other species detected included Aeromonas, Alcaligenes, Burkholderia and Chryseomonas.

Pythium: 74 percent of all samples yielded at least one Pythium species, but only six species in total were identified, the most frequent being P. aphanidermatum and P. intermedium. There is a wider diversity of Pythium species in NZ than those detected in the container soil samples.

Fusarium: Of the 98 soil samples examined 20.4 percent yielded Fusarium species; 12 Fusarium species were identified and of these 25 percent were F. oxysporum.

Animal pathogens: E. coli, faecal coliforms and Enteroccocus were present in most samples. Although faecal organisms do not represent a major quarantine risk they can present a health risk to workers associated with the container trade.

Cyst nematodes: Only one positive was found, an undescribed Heterodera.

Freeliving nematodes: Aphelenchoides was present in 38 percent of the samples while Meloidogyne was present in only 9.5 percent. This was a low frequency compared to the 75 percent and 50.5 percent representation of these species in the NZ soil samples analysed to provide a comparison.

The results of our analysis showed that soil attached to shipping containers is a potential pathway for pests and diseases to enter NZ. In most cases the soil consisted of inorganic sands and shingle, most likely derived from hard loading surfaces. Organically derived soils were collected infrequently. The containers from which these samples were collected may have been loaded on soft soil or on undeveloped loading areas as the material was pushed up onto the rails and jammed into forklift holes. Organic soil is likely to be picked up by containers in the same locations that are primary sites for diseases, and this type of contamination is likely to present a higher quarantine risk.

Publications

A paper will be prepared for submission to a refereed journal at the conclusion of this year's contract.

2.3 MBS 303: Evaluation of broad-spectrum attractants for use in monitoring and surveillance of beetles of quarantine interest in New Zealand

Programme Title: Evaluation of broad-spectrum attractants for use in monitoring and surveillance of beetles of quarantine interest in New Zealand.
Programme Leader: Roger Sands
Institution: University of Canterbury, School of Forestry

Summary

Beetles of quarantine interest to New Zealand were trapped in a mixed coniferous forest in California, USA during the summers of 1989-91 and 1999 to test the most effective attractant for use in detection. Attractant lures evaluated included five common monoterpenes: -pinene, -pinene, myrcene, limonene and 3-carene; two bark beetle pheremones (frontalin and ipsdienol; and ethanol in various combinations. Treatments were tested using Lindgren funnel traps. -pinene+ethanol attracted the most species. -pinene+ethanol attracted the greatest number of individual beetles.

It is recommended that Lindgren funnels baited with -pinene+ethanol be used for detection in ports and port warehouses because of its broad-spectrum attraction.

Background

The goal of this project was to set out to identify an attractant trap method that would catch the greatest number of species per unit effort.

Host plants compounds (monoterpenes, ethanol) were our primary focus for evaluation because of their universality. They are important volatile components of most plants and thought likely to be important cues for host selection by many phytophagous insects (Visser, 1986).

Bark beetles are attracted to a variety of plant volatiles e.g. -pinene, -pinene, and ethanol. In recent years these products have been shown to be effective in detecting a broad spectrum of wood-dwelling beetles (wood 1982, Hobson 1989, Humble 1999).

In this project we set out to set these products singly and in combination to identify the best performing attractant. The study was carried out in California where many beetle species of quarantine interest are easily detected. The fauna trapped in the study site forest includes representative species of most world families of wood boring insects.

Approach & Outcomes

Experimental treatments varied over the years of study (1989-1991 and 1999). However the study beetles were trapped with funnel Lindgren type traps from PheroTech Inc. Attractant lures also varied over the years of the study.

All treatments caught quite large numbers of beetles of quarantine interest for New Zealand. Even unbaited traps had high accidental catches. In 1999 -pinene+ethanol stood out as the best performing product. It has a high catch rate when compared with the other treatments when Dendroctonus valens was excluded. It attracted and caught D. valens and also caught more species than the other treatments. Both -pinene+ethanol and -pinene+ethanol caught the most common species but -pinene+ethanol caught many others.

2.4 MBS 304: Identification of fungal infections in imported timber

Programme Title: Identification of fungal infections in imported timber
Programme Leader: Professor Roberta Farrell
Institution: University of Waikato

Summary

This project was undertaken to assess the range of fungi being imported on solid wood packaging material in to New Zealand. Maintaining New Zealand's biosecurity is of acute significance for its inhabitants as a factor in its economic well-being. In order to minimise the threats posed by exotic organisms, sound principles of risk assessment and management need to be developed, based on good science and cost-effectiveness in terms of the benefit-risk ration. Samples of wood from imported wood packaging were collected at different ports, which were then classified in the laboratory and statistical analyses were carried out assessing the diversity of fungi. The fungi identified represent some of the more ubiquitous Genera, some not specific to the source i.e. the wood, rather they are widespread and will be incidentally carried on the packaging, not having "infected" it per se. Other Genera include forest plant and forest products' pathogens. Sample of visually compromised wood did not necessarily contain more fungal type than "clean" wood. The implications for the MQS, even if not significant in all cases, is far reaching. Although this study sheds light on the range of fungi coming in to New Zealand, collecting accurate information will not be feasible without a more focussed approach, i.e. by directly probing wood with target orientated DNA probes against pathogens predetermined as having major potentially detrimental effects on New Zealand's commercial agroforestry and its indigenous forests.

Background

The goal of this project is to assess the range of fungi being imported on wood packaging into New Zealand.

Maintaining New Zealand's biosecurity is of acute significance for its inhabitants as a factor in its economic well-being. Today, however, its borders are incessantly tested by organisms having potentially detrimental effects on its Biodiversity, economy, environment and quality of life. In order to minimise the threats of exotic organisms, sound principles of risk assessment and management need to be developed, based on good science and cost-effectiveness in terms of the benefit-risk ratio. International commitments to free trade, however, have placed a strain on national priorities as set out in recent legislation regarding aspects of biosecurity. In spite of the fact that the MAF Quarantine Service (MQS) inspects more containers than any other country, case histories of exotic disease introductions abound.

Approach & Outcomes

Chisel samples of wood from imported wood packaging were collected at Auckland airport, Auckland and Lyttelton wharves during summer and winter 2000. These included both visually compromised (overt mycelial growth, sporangiophores, fruiting bodies evidence of mycostaining, bacterial growth/slime or algal growth, and "clean" wood. Samples were classified in the laboratory and statistical analyses were carried out assessing the above objectives.

The fungi identified represent some of the more ubiquitous Genera, some not specific to the source i.e. the wood, rather they are widespread and will be incidentally carried on the packaging, not having "infected" it per se. Some of the Genera are typical wood saprophytes. Other include both forest plant and forest products' pathogens. A significant decrease was observed in the number of different isolates taken from Auckland Airport, probably due to the generally better quality wood used in airfreight, often kiln dried or custom wood. Custom wood was shown to have significantly fewer fungi, of those isolated most were obligate saprophytes. Significantly higher isolate counts were observed in the summer as compared to the winter sampling. The sources of the samples could not be definitively ascertained due to the phenomenon of transhipping and non-specific manifests. Comparisons with the New Zealand fungal databases have not revealed any Classes or Genera not known to exist locally. Clearly, this does not indicate whether within these Genera are species, subspecies or formae speciales etc., which may contain pathogens not found in New Zealand. Isolates from "clear" wood carry an increased range of incidental spores, leading to the greater diversity observed. The implications of this finding for the MQS, even if not significant in all cases, is far reaching. The natural tendency of the MQS to pick out visually compromised wood for quarantine procedures (incineration, fumigation or identification) may be in fact negatively selecting against the wood harbouring potential disease.

Although this study sheds light on the range of fungi coming into New Zealand, a more encompassing study requires a focussed approach. This can be achieved by directly probing the wood with target orientated DNA probes against pathogens predetermined as having major potentially detrimental effects both on New Zealand's commercial agroforestry and its indigenous forests. In the future gene probes specific to pathogens of consequence will be used to probe wood in situ allowing rapid diagnoses for MQS operations. We now have DNA probes for one of the greatest threats to New Zealand forestry, the casual agent of pitch canker of P. radiata.

Publications

Janson, R., Ram, A., Duncan, S. and Farrell, R. (2000): Survey of Fungal Biodiversity on Wood Packaging Imported into New Zealand. Proceedings of the XXI IUFRON World Congress. Forest and Society: The Role of Research. Kuala Lumpur, Malaysia.

Janson, R. .and Farrell, R. (2000): Wood packaging imported into New Zealand: A threat to Biosecurity ? Presented to the conference on Cellular and Molecular Mycology. Bulls, New Zealand.

2.5 MBS 305: Validation of Pest - Host Associations

Programme Title: Validation of Pest - Host Associations
Programme Leader: Dr Brett Alexander
Institution: National Plant Pest Reference Laboratory (NPPRL), Lincoln

Summary

  • Validated pest-host association records for six horticultural genera (Asparagus, Capsicum, Fragaria, Olea, Vaccinium, and Zea) and four forestry genera (Eucalyptus, Populus, Quercus, and Ulmus) were obtained from MAF NPPRL archives and collections, the literature, Landcare Research national collections, and the Forest Health Database.
  • Landcare Research New Zealand Ltd also provided validated pest-host association records for Agaricus, Lilium, Narcissus, Paeonia, Ribes, Rubus, Sandersonia, and Tulipa.
  • To date, a total of 7,737 validated pest-host association records have been provided to the Plant Pest Information Network (PPIN) Administrator for entry into PPIN.
  • The majority of the records were obtained from the Forest Health Database (53 percent of total) and MAF archives and collection (30 percent). Landcare Research provided 15 percent of the records, while 2 percent of records were obtained from the literature.
  • Pathology (69 percent) and entomology (29 percent) records comprised the bulk of the total number of validated records.
  • Approximately 500 MAF entomology records remain to be validated. It is anticipated that these records (approx. 6 percent of total records) will be validated by 30 April 2001, if current rate of progress is maintained.

Background

The maintenance of New Zealand's horticultural security and the development of policies facilitating access to overseas horticultural markets requires ready access to current information defining New Zealand's plant health status. Unfortunately, such information has not been available from a single source. Valuable pest-host association data, some of which is unpublished, is contained in the archives and specimen collections of NPPRL and other scientific institutions in New Zealand. The goal of this programme was to provide this information for six horticultural genera (Asparagus, Capsicum, Fragaria, Olea, Vaccinium, and Zea) and four forestry genera (Eucalyptus, Populus, Quercus, and Ulmus) specified by MAF Biosecurity Authority. The test groups of interest included arthropods, bacteria, fungi, nematodes, and viruses.

This programme was divided into three objectives:

  1. The retrieval and validation of MAF data (for all ten host genera) and records from the literature (for six horticultural genera);
  2. The retrieval and validation of Landcare Research records (for all ten host genera plus Agaricus, Lilium, Narcissus, Paeonia, Ribes, Rubus, Sandersonia, and Tulipa); and
  3. The retrieval and validation of Forest Health Database records (for four forestry genera).

Approach & Outcomes

Pest-host association data for ten host general were retrieved from MAF NPPRL archives and collections, the literature, Landcare Research national collections, and the Forest Health Database. In addition, pest-host association data for Agaricus, Lilium, Narcissus, Paeonia, Ribes, Rubus, Sandersonia, and Tulipa were obtained from Landcare Research. These records were validated by scientists from NPPRL, Landcare Research, and Forest Research Associated with expertise and experience appropriate to the pest involved. Validated records were provided to the Plant Pest Information Network (PPIN) Administrator for entry into PPIN. Any records with quality or technical deficiencies were excluded from PPIN, pending resolution of the problem.

At the time of reporting, a total of 7,737 validated pest-host association records had been provided to the PPIN Administrator for entry into PPIN. The majority of the records were obtained from the Forest Health Database (53 percent of total) and MAF archives and collections (30 percent). Landcare Research provided 15 percent of the records, while 2 percent of records were obtained from the literature. Pathology (69 percent) and entomology (29 percent) records comprised the bulk of the validated records (data nor presented). Approximately 500 MAF entomology records remain to be validated. PPIN is available to MAF Biosecurity Authority (Plants and Forest groups), researchers, and the New Zealand public.

2.6 MBS 306: Development of diagnostic keys for Tortricidae

Programme Title: Development of diagnostic keys for Tortricidae
Programme Leader: Peter Holder
Institution: National Plant Pest Reference Laboratory (NPPRL), Lincoln

Summary

For many of the leaf-roller specimens collected in the execution of MAF's biosecurity obligations, determinations have hitherto not been possible or practical.

The project goal has been to develop and integrate a series of morphological and molecular diagnostic keys to economically important larvae and adult New Zealand Tortricidae, as well as the exotic torticid species most likely to be introduced to New Zealand.

This project has co-ordinated molecular and morphometric research focus into separating and defining the target species. Morphological keys have been produced for the species and life stages for which morphological characters are consistent; and these are integrated with molecular protocols that will enable the diagnoses are not reliable.

Background

The goal of this project has been to develop and integrate a series of morphological and molecular diagnostic keys to economically important larvae and adult New Zealand Tortricidae, as well as to the exotic tortricid species most likely to be introduced to New Zealand.

For many of the tortricid specimens collected in the execution of MAF's biosecurity obligations (Specific Crop Surveys, General Surveillance and MAF Quarantine Service interceptions), determinations have not been possible or practical. This was due to either: i) the current systematics knowledge being insufficient or ii) information was inaccessible and/or not in a format able to be employed for efficient use of diagnostic time. In addition, separation of many species has not been reliable or possible with traditional techniques because morphological characters are not consistent and/or unknown (especially for early life stages).

Accurate and rapid diagnosis of pests is essential to provide MAF with a technically sound basis for decisions on risk management and exclusion of exotic pests, as well as helping to establish the pest status of New Zealand. The research and keys that are the result of this project will enhance the ability of MAF to achieve efficient and reliable identifications of Tortricidae specimens.

Approach & Outcomes

This project builds on existing tortricid references, and extends our knowledge base by the co-ordinated focus of molecular and morphometric research effort into separating and defining hitherto indistinguishable species. The practical product of this research is an integrated diagnostic guide. Morphological keys have been produced for the species and life stages for which morphological characters are consistent; these are combined with molecular protocols that will enable the diagnoses of species and life stages for which morphological diagnoses are not reliable. The guide is being published.

It was necessary to follow a stepwise process. A systematic framework determined by the adult morphological and molecular research that was used to develop the morphological keys to larvae. In summary, the process followed was:

  • assemble tortricid reference material from New Zealand and international collections, plus collect specimens from the field and laboratory cultures (i.e. create life stage series - authenticated where possible);
  • compare taxa and derive robust diagnostic morphological character fields for adults, then establish what differences occurs in larvae;
  • sequence the mtDNA COI & II region (2.5kb). These data were aligned and analysed for restriction enzyme sites with potential to provide diagnostic restriction fragment length polymorphisms (RFLP's). Restriction maps for all species were assembled. The determination and testing of potential restriction sites to differentiate between species was conducted (i.e. we sought phylogenetically informative differences).
  • reconstruct a phylogenetic tree (using DNA distance data and using neighbour-joining method);
  • align morphologically differentiated taxa with molecular information;
  • molecular 'multiplex approach' protocols were incorporated with the late instar morphological larva key (i.e. the molecular `key' requires the determination be partially made by morphological examination). For other life stages, molecular determination is by way of DNA sequence and blast search for a sequence data match;
  • test the diagnostic characters that were derived, for accuracy, consistency and ease of interpretation (including testing across all life stages and various populations);
  • describe and photograph or illustrate diagnostic characters as well as whole insects for a pictorial key;
  • assemble and test keys and protocols.

We can now characterise the larvae and adults of leaf-rollers likely to be encountered by MAF. The species covered in this project include two of the three tortricid subfamilies (notes are given on the third). Sixty-four taxa are keyed out in the morphological aspect of the project; 46 species have been sequenced and 26 taxa are defined using molecular data within the integrated late instar larvae key.

Assessments of potential morphological characters confirmed that there is a high degree of structural uniformity within the Tortricidae family and many features investigated were either not reliable or of no diagnostic value. We defined 40 larval character states/characters and a comparison matrix was constructed. Diagnostic characters were tested for accuracy, consistency and ease of interpretation. Easily accessible features are not reliable for adult females, and so females are keyed on genitalia. Once the feasibility of using this method had been confirmed, the large task of preparing and photographing genitalia slides was undertaken.

Following phylogenetic analysis, a total of seven restriction enzymes were identified which discriminate all genera. The identified diagnostic markers were tested for reliability before larger-scale screening across all life stages and multiple specimens and populations. However, given how closely related many taxa are, the ability to discriminate taxa on the basis of restriction enzyme profiles was not possible. A stand-alone molecular key was attempted using a PCR multiplex approach, but the diverse range of taxa being encompassed in this key resulted in the most robust characters being the raw DNA sequence data. We established that the most effective means of molecular determination is to sequence a smaller region within the COI & II region, and to carry out a blast search for a sequence data match using the National Centre for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/). The sequence data from this project has been added to this international database.

Molecular protocols were integrated with the morphological-based key so that some taxa are identified to species group morphologically and then to species by molecular techniques; whereas species and life stages for which morphological determinations are reliable are given greater morphological emphasis. Adults of all significant species along with the genitalia preparations are represented by photographs. Diagnostic characters for larvae have been illustrated by line drawings. Diagnostic notes on each species are given.

The project has been more technically complex than anticipated. In addition to the extra effort needed develop reliable diagnostic features, phylogenetic inconsistencies required length focus before resolution. (North and South Island Planotortrix excessana are being treated as separate species; there appears to be paraphyly of Apoctena: both A. flavescens and A. orthropis are not monophyletic within the "genus", nor closely related to each other).

Publications

Gleeson, D., Holder, P.W., Newcomb, R., Howitt, R. and Dugdale, J. (2000): Molecular Phylogenetics of Leafrollers: Application to DNA Diagnostics. Proceedings of the NZ Plant Protection Conference 53: 157-162

Dugdale, J.S., Gleeson, D., Clunie, L.H., and Holder, P.W. (2001): A Diagnostic Guide to Tortricidae Encountered in Field Surveys & Quarantine Inspections in New Zealand: Morphological and Molecular Characters. MAF Publication

Some of the outputs from this project are to be included on the international collaboration "Tortricidae of Economic Importance" CD-ROM (co-ordinated by ETI, University of Amsterdam).

The sequence data from this project has been added to the National Centre for Biotechnology Information international database (http://www.ncbi.nlm.nih.gov/).

2.7 MBS 350: Distribution, seasonality and prevalence of the biting stablefly, Stomoxys calcitrans in New Zealand

Programme Title: Distribution, seasonality and prevalence of the biting stablefly, Stomoxys calcitrans in New Zealand
Programme Leader: Dr A.C.G. Heath
Institution: AgResearch, Wallaceville

Summary

Stomoxys calcitrans was found throughout New Zealand, but being more particularly widespread in the North Island, with only the volcanic plateau being free of the fly.

South Island distribution was mainly coastal and principally in the northern half of the island, with an isolated Southland pocket. The south central South Island was free of the fly.

Distribution of S. calcitrans in New Zealand closely matches the distribution of dairy cows in milk.

Stomoxys calcitrans is active throughout the year in the North Island, although numbers are lowest during June to November and there appears to be a shorter activity period in the south of the North Island. It is likely too that the activity period is much shorter in the South Island, although there were insufficient data to support this conclusion strongly.

A move away from ensilage stacks to polythene-wrapped ensilage may reduce the number of breeding sites available to S. calcitrans as will improved hygiene around stables where manure has, traditionally, been stockpiled.

A study such as this could be improved if undertaken more intensively in, say two climatically dissimilar areas and if a survey of breeding sites was also incorporated into the study.

Background

The goal of this project was to determine the distribution, seasonality and prevalence of Stomoxys calcitrans using a simple adhesive trap and employing principally the services of school children in rural areas.

Within New Zealand, the distribution of S. calcitrans is not known with accuracy, although it has been recognised as present since the 1840s. Only one previous study has been carried out and it determined, for the North Island only, factors affecting the distribution and prevalence of S. calcitrans. The current study was commissioned in order to provide data that would enable the development of a strategy for minimising risk of transmission of exotic diseases from imported livestock to disease-free stock already present in New Zealand.

Approach & Outcomes

Adhesive yellow traps were deployed by schools in rural areas and after a trapping period of 7 days, traps were returned to AgResearch, Wallaceville for analysis and detection of S. calcitrans. In areas where S. calcitrans was not trapped, veterinarians were telephoned and asked to provide anecdotal information on the presence or absence of the fly in their areas. Some farmers also returned a questionnaire on the presence of S. calcitrans on their farms.

2.8 MBS 351: Determining the relationship of non-specific amplicons of EFB PCR to the bacteria fauna of New Zealand honey bees

Programme Title: Determining the relationship of non-specific amplicons of EFB PCR to the bacteria fauna of New Zealand honey bees
Programme Leader: K.M.Tham (MAS320)
Institution: National Centre for Disease Investigation

Summary

A diverse microflora of microbes (56 bacteria and one yeast) was detected from 20 honey bee larvae. Tests using standard bacteriological biochemistry and advanced molecular techniques showed that the Bacillus sp. And the Lactobacillus sp were closely related to M pluton with respect to their bacterial 16S rRNA gene and could have been responsible for producing the non-specific amplicons seen in the EFB PCR.

Background

The goal of the project was to determine the relationship of the non-specific amplicons of the M pluton PCR with the bacteria microflora of the New Zealand honey bee larvae.

European foulbrood (EFB) is found on every continent, except in New Zealand, where honey bees are kept. A sensitive PCR assay for EFB developed and optimised at the former Central Animal Health Laboratory has been used on honey bee larvae in an EFB survey project. Preliminary results showed that all the bee larvae were free of Melissococcus pluton (M pluton), the organisms causing EFB. However, non-specific amplicons (PCR amplified fragments) of sizes different from the M pluton-specific amplicons were observed in some bee samples. Preliminary experiments using M pluton PCR assay and cycle sequencing of these non-specific amplicons were inconclusive as to the relationship between these amplicons and the template DNA of honey be larvae. This project aims to clarify the relationship of the non-specific amplicon with the honey bee larvae DNA by using molecular techniques such as PCR, cycle sequencing and phylogenetic analyses.

Approach & Outcomes

Bee samples

Twenty honey bee larvae collected from a previous M pluton survey project were selected for this study. Washed larvae were homogenised in sterile PBS and 50ul of the homogenate was plated on to two blood agar plates and incubated, one aerobically at 30oC or 37oC with 5 percent CO2 and the other anaerobically at 37oC. After one to three days of incubation, colonies were streaked out on to individual blood agar plates and subsequently single colony purified. Nine basic microbiological methods were used to identify the bacterial isolates. These were colony morphology, cell morphology, Gram reaction, catalase reaction, oxidase reaction, glucose metabolism, motility, presence of an endospore and the oxygen requirements. To further characterise the Gram negative bacteria, the Microbat system was employed.

Molecular analyses

Bacteria DNA was extracted using a modified procedure. Briefly, bacteria grown either in LB broth or blood agar plates were harvested after 24hrs of incubation, spun and the pellet was resuspended in sucrose buffer and incubated at 37oC for 10 min. 10ul each of mutanolysin (10,000U/mi) and lysozyme (10mg/ml) was added and the mixture was incubated at 37oC for 5 min. 50ul of lysis buffer, 18ul of 20 percent SDS, and 8ul of RNaseA (10mg/ml) were added, mixed by inversions and incubated at 37oC for 45min. DNA was extracted from the mixture using the commercial High Pure PCR Template Preparation kit. A pair of primers 5'-AGGAGGTGATCCAACCGC-3' (DG74) and 5'-AACTGGAGGAAGGTGGGGAY-3' (65ab), based on the published sequences of the bacterial 168S rRNA gene was constructed for use in a modified PCR. Briefly, a total volume of 50ul containing 5ul of bacterial DNA, 1U Taq DNA polymerase, 2.5mM MgCl2, 0.2uM each of dNTPs, 0.2uM of each of the sense (65ab) and antisense (DG74) primers in 1 x PCR buffer were prepared for each DNA. The mixture was then treated with 8-methoxypsoralen. With a programmable thermo-cycler, the mixtures were heated at 95oC for 2 min and amplified by 30 repetitive cycles of denaturation at 95oC for 30 sec, annealing at 55oC for 30 sec and extension at 72oC for 30 sec, with a final extension of 10 min. An aliquot of the PCR product was analysed by gel electrophoresis and visualised by ethidium bromide staining and UV transillumination. The remainder of the PCR product was purified and sequenced by cycling PCR using both DG74 and 65ab as the sequencing primers. Each nucleotide sequence was checked manually for readability. The nucleotide sequences were analysed using the WebAngis package (ANGIS, Australia).

All the 20 honey bee larvae yielded culturable microbes; 56 bacteria and one yeast were isolated. The nine standard bacteriological methods adequately identified the Bacillus sp., the Staphylococcus sp. and the Enterobacteriaceae. Bacillus sp. were the most commonly isolated microbes, being present in 40 percent of the honey bee larvae studied. Further identification of the Gram negative bacteria (36 percent of the bacterial isolates) using the Microbat system was inadequate as it is primarily designed for the identification of pathogenic bacteria.

Direct cycle sequencing of the six purified non-specific amplicons of the M pluton PCR yielded sequences that were unreadable and not suitable for the selection of new primers. However, the universal primers (65ab and DG74) for the bacterial 16S rRNA gene yielded a fragment of 400 bp which was purified and successfully sequenced. Using biochemical and molecular tests, all but one of the isolates were identified to the genus level, and 35 of the 56 bacterial isolates were identified to the species level. Bacillus sp. (23 percent) were found to be the most common of the 56 bacterial isolates, followed by Lactobacillus sp. (21 percent), Enterobacteriaceae (14 percent), Leuconostoc sp. (7 percent), Staphylococcus sp. (7 percent) and Psedomonas sp. (7 percent). The Enterobacteriaceae could not be identified to the genus level as six genera of this bacteria group exhibit 100 percent identity with the gene sequence. Only 18 of the 56 bacterial isolates yielded nucleotide sequences that are readable when sequenced. Nucleotide sequences from the other bacterial isolates contained high background noise and had to many N (undetermined) signals, thus making these sequences unacceptable for analyses. Multiple alignment analysis of the nucleotide sequences of the 16S rRNA gene of the 18 bacteria isolates and the M pluton shows that there are consensus sequences which are presumably evolutionarily conserved in all these bacteria. Phylogenetic dendrogram of the 16S rRNA gene sequences of all these bacteria provides evidence that certain isolates, notable the Bacillus sp. and to a lesser extent the Lactobacillus sp. isolated from the honey bee larvae, are closely related to M pluton. However, when the deduced amino acid sequences of the 18 bacterial isolates were analysed, the phyolgenetic tree shows that M pluton forms a distinct group. Taken together, these results suggest that there is a diverse microflora of bacteria present in the New Zealand honey bee larvae that could become the target template when DNA were extracted from these larvae and tested by PCR. The data obtained herein strongly indicate that the non-specific amplicons were due to the presence of bacterial DNA, extracted from the honey bee larvae. The most likely candidate bacteria that reacted non-specifically with the M pluton primers in the EFB PCR are the Bacillus sp., with the possible involvement of the Lactobacillus sp., as these two species of bacteria are closely related genetically to M pluton with respect to their 16S rRNA genes. This is supported by the findings that the six bee larval samples that yielded the non-specific amplicons contained predominantly the Bacillus sp. and the Lactobacillus sp. The main obstacles encountered in this study were (i) the failure to sequence directly the non-specific amplicons to obtain sequences for the construction of new primers (ii) the inability to clone and sequence the non-specific amplicons (iii) the availability of computer software for molecular analyses.

2.9 MBS 352: Development and validation of a serological assay for the detection of Echinococcus granulosus cysts in animals

Programme Title: Development and validation of a serological assay for the detection of Echinococcus granulosus cysts in animals
Programme Leader: Reinhold Kittelberger
Institution: National Centre for Disease Investigation (NCDI)

Summary

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 objective of this project was to develop an ELISA that could be used to test imported animals for the presence or absence of cysts of E. granulosus.

Parameters of three ELISAs were optimised with a small number of sera, using an 8kDa hydatid cyst fluid protein (8kDaELISA), a protoscolex detergent extract (ProtELISA) and a recombinant oncosphere protein (OncELISA) and various microtitre plates, blocking reagents and conjugates. These ELISAs were then extensively validated with 52 sera from experimentally and 275 sera from naturally E. granulosus infected sheep, with 1012 sera from non-infected sheep and 39 sera from sheep infected with T. ovis and 19 sera from sheep infected with T. hydatigena. Signal to noise ratios were good and were within the range expected for ELISAs, i.e. 6.7, 5.0 and 5.3 for the 8kDaELISA, the ProtELISA and the OncELISA, respectively. Plate to plate variations for the positive control serum, were within the OIE-recommended range of about 15 percent and less than 20 percent. The cut-off absorbencies were 0.41, 0.30 and 0.42 (mean + 2SD) and 0.49, 0.37 and 053 (mean + 3SD) for the three ELISAs. Diagnostic specificities were good to excellent, ranging from 95.8 percent to 99.5 percent, depending on the cut-off value. A low degree of cross-reactivity with animals infected with T. ovis or with T. hydatigena was detected. Diagnostic sensitivities varied markedly between the three ELISA types. For the OncELISA they were poor, excluding this ELISA as a useful tool for the identification of animals with E. granulosus infections. Interesting results were obtained for the sensitivities of the 8kDaELISA. While the sensitivity for experimentally infected sheep was 48.1 percent at a cut-off of mean + 2xSD, it was only 10.2 percent with naturally infected sheep. A possible reason for this may be immunological differences in E. granulosus strains in various countries. Some evidence for this could be presented by comparing hydatid cyst fluids from experimentally and naturally infected sheep with a panel of sera.

The ELISA with the best diagnostic sensitivity was the ProtELISA, which was able to identify 63.5 percent of experimentally infected sheep and 58.9 percent of naturally infected sheep with cysts, at a cut-off mean of + 2xSD. With such sensitivities it will be possible to identify sheep flocks with hydatid disease, but it is not possible to identify every individual animal infected with E. granulosus. Further test development work is planned, which will examine the cellular immune response to E. granulosus (antigen-dependent gamma interferon test) and its usefulness for the detection of animals infected with E. granulosus.

Background

Hydatid disease is about to become an exotic disease in New Zealand. However, all live animals imported into the country will have the potential to re-establish the disease, In a recent case, a hydatid cyst was found in a cow, which was imported from Australia five years before discovery of the cyst at slaughter. Hydatid disease is common in most other countries, especially Australia, the most likely source country for imported livestock. For example, from 1995 to 1999, on average 90 sheep per annum have been imported into New Zealand, all from Australia. There is no established test to detect hydatid disease in livestock, except of necropsy after slaughter. The objective of this project was to develop and to validate extensively an ELISA that could be used to test imported animals for the presence of the cysts of E. granulosus.

Approach & Outcomes

Promising results reported by others on serological methods in human hydatid cases, applying a specific 8 to 12kDa hydatid cyst fluid antigen, prompted us to use a similar approach for livestock. In a previous MAF Policy funded project, such an 8kDa protein was purified and an ELISA developed (8kDaELISA), using this protein as antigen. Furthermore, an alternative ELISA was developed (ProtELISA), which used a crude protoscolex antigen preparation. Such antigen had been applied successfully for the serodiagnosis of E. granulosus infections in dogs. Both ELISAs were validated with a limited number of sera from E. granulosus experimentally infected sheep.

In the current project, these two ELISAs were extensively validated by using large numbers of sera from naturally and experimentally infected sheep and from sheep free from E. granulosus infections. A third ELISA, using a recombinant oncosphere antigen (OncELISA), was developed in addition to these assays and was also extensively validated.

2.10 MBS 353: Tissue Salmonella

Programme Title: Tissue Salmonella
Programme Leader: Gary Clark
Institution: LABNET Invermay Ltd

Summary

To access if cull ewes are a likely source of infection and contamination of S. Brandenburg in meat plants. A group of 10-12 ewes from confirmed S. Brandenburg farms were killed at a meat plant between early December and early April and samples were collected for Salmonella culture from the uterus. From ewes examined in February, March and April liver and mesenteric and internal iliac lymph nodes were also cultured for Salmonella species.

From this study, there appear to be a low risk of S. Brandenburg contamination at meat plants from ewes that aborted 3 or more months previously, as the organism was not isolated from any of the ewes sampled. Admittedly the December sampling did not include samples from the liver and mesenteric and internal iliac lymph nodes. The gross observations of extensive adhesions involving the fallopian tubes and, in a small number of ewes, focal pyometra indicates that uterine damage associated with abortion could have affected the ewe's ability to conceive. Twenty-four percent of the ewes in this study had these lesions but the degree of damage varied between farms, range 10 to 50 percent.

Background

The goal of this project is to assess if cull ewes are a likely source of infection and contamination of S. Brandenburg in meat plants. Recently there has been an increase in the isolation of Salmonella organisms from sheep and bobby calf meat products sampled at meat plants. This is creating concern in relation to market access, as a great number of countries do or may implement port of entry monitoring programmes, which invariably include Salmonella acceptance standard. Consignments of meat that fail to meet these standards are invariably rejected.

Approach & Outcomes

A minimum of 40 ewes that aborted from confirmed S. Brandenburg farms were killed at meat plants between early December and early April. Uterus, liver, mesenteric and internal iliac lymph nodes were taken from the ewes for culture for Salmonella. All Salmonella positive isolates were to be serotyped and, if identified as S. Brandenburg, DNA profiled using pulsed-field gel electrophoresis. Uteri were also examined for gross pathological changes.

The results of this study indicates that ewes that aborted 3 or more months previously have a low risk of contaminating meat plants with S. Brandenburg as the organism was not isolated from any of the ewes sampled. Admittedly the December sampling did not include samples from the liver and mesenteric and internal iliac lymph nodes. Results from a previous study showed that 36 percent of ewes that aborted as a result of S. Brandenburg were excreting the organism in faeces in September but at weaning and in March the figures were 3 percent and 2 percent respectively (Clark et.al., 2000). Therefore there is some risk of spread of infection from these ewes.

Twenty-four percent of the ewes examined had extensive adhesions involving the fallopian tubes and/or a focal pyometra that could have affected the ewes ability to conceive. However, the degree of damage varied widely between farms.

2.11 MBS 354: Spread of Salmonella organisms in sheep yards and effect on lambs and ewes at meat plants

Programme Title: Spread of Salmonella organisms in sheep yards and effect on lambs and ewes at meat plants
Programme Leader: Gary Clark
Institution: LABNET Invermay Ltd

Summary

A study was carried out to compare the prevalence of S. Brandenburg in the dust of sheep yards of farms where S. Brandenburg abortions have occurred and those where no S. Brandenburg abortions were thought to have occurred.

Sixty-four farms were selected for study. On 32 there had been laboratory-confirmed S. Brandenburg abortion outbreaks, and on 32 there had been no evidence of S, Brandenburg and no significant abortion problems. The farms selected did not clean their yards down with disinfectants or 5 percent formalin or dampen the yards during drafting of lambs. Form these farms, at least 50 grams of dust was collected from the yards in late January and March/April and before scanning (end of May to late July). Twenty-five grams of the dust was cultured for salmonellae using the MIRNZ method.

The study showed that the percentage of S. Brandenburg isolates from farms where there had been S. Brandenburg abortions dropped from 39 percent in January to 16 percent in March and 12.5 percent at scanning. This indicates that the organism can remain viable in sheep yard dust for at least 5-6 months. All farms from which S. Brandenburg was isolated from sheep yard dust on more than one occasion had covered yards. This would have provided shade and increased the organism's ability to survive. From 6 percent of the control sheep yards, S. Brandenburg was cultured from dust in January and March/April showing that the organism can be present on farms where no clinical disease is evident.

Although the organism in the dust could be a source of infection for sheep being yarded this was not proven in the trials above. This may have been due to lack of infective organisms, as from two of the three affected farms Salmonella could not be isolated from sheep yard dust at the time of the trial, and or the sheep spent insufficient time in the affected yards.

Background

The goal of the project was to compare the prevalence of S. Brandenburg in the dust of sheep yards of farms where S. Brandenburg abortions have occurred and those where no S. Brandenburg abortions were thought to have occurred.

Salmonella Brandenburg is a major cause of sheep abortion in Canterbury, Otago and Southland, occurring in the last 2 months of pregnancy. The disease is rapidly spreading in Southland and coastal south Otago with the disease being diagnosed on over 120 sheep farms in 1998 and 223 farms in 1999. In the Clutha Veterinary Association practice area it is estimated to have affected about 15 percent of sheep farms (John Smart, pers. comm.). Carrier ewes contribute to spread of infection between years and black-backed gulls have a role during the abortion season. With the enteric form of salmonellosis in sheep it has been shown that S. Typhimurium and S. Bovismorbificans can be isolated from sheep yards on farms where this disease occurs and that ewes kept for 24 hours in infected yards can become infected. Often outbreaks of S. Brandenburg abortions are reported to occur following yarding. This project has been designed to examine the role of sheep yard dust in the spread of S. Brandenburg in sheep.

Approach & Outcomes

Sixty-four farms were selected for study. On 32 there had been laboratory-confirmed S. Brandenburg abortion outbreaks, and on 32 there had been no evidence of S. Brandenburg and no significant abortion problems. The farms selected did not clean their yards down with disinfectants or 5 percent formalin or dampen the yards during drafting of lambs. From these farms at least 50 grams of dust was collected from the yards in late January and March/April and before scanning (end of May to late July). Twenty-five grams of the dust was cultured for salmonellae using a MIRNZ method.

Ten lambs and 10 ewes from 10 control farms, where no S. Brandenburg had been isolated from sheep yard dust, were kept for 4 to 6 hours in yards from three farms where S. Brandenburg was isolated from sheep yard dust in late January. These sheep were slaughtered 24 hours later at the meat plant and two or more mesenteric lymph nodes were collected from each and sent to the laboratory for culture, using routine selective media. Salmonella organisms have been recovered more frequently from mesenteric lymph nodes than from caecal contents.

All Salmonella positive isolates were serotyped at ESR Communicable Disease Group and if identified as S. Brandenburg sent to Stan Fenwick at Massey University for DNA profiling using pulsed-field gel electrophoresis.

The study shows that in January, S. Brandenburg was present in the sheep yard dust of 39 percent of farms on which there had been S. Brandenburg abortions in the previous lambing season. On about a third of these farms the organism was still present in the yard dust pre-scanning. This indicates that the organism can remain viable in sheep yard dust for at least 5-6 months. All farms from which S. Brandenburg was isolated from sheep yard dust on more than one occasion had covered yards. This would have provided shade and increased the organism's ability to survive. However, there was no significant difference in isolation rates of S. Brandenburg from sheep yard dust between farms with covered and open yards. The lack of a significant difference may have been due to their only being a small number of farms (12) with open yards in the study.

A farm that had been positive for S. Brandenburg in January and March and negative at scanning had been cleaned out and disinfected twice, once about 10-12 days before a sample was collected. Disinfecting yards has previously been reported as a control measure and this could be the reason for failure to isolate this organism.

Although the organisms in the dust could be a source of infection for sheep being yarded this was not proven in the trials above. This may have been due to lack of infective organisms, as from two of the three affected farms Salmonella could not be isolated from sheep yard dust at the time of the trial, and or the sheep spent insufficient time in the affected yards.

From 6 percent of the control sheep yards, S. Brandenburg was cultured from dust in January and March/April showing that the organism can be present on farms where no clinical disease is evident. In addition, S. Brandenburg was cultured from samples of wool collected by Alliance Group from the brisket of ewes and lambs on two of the 10 control farms before transport (Jayne Marshall, pers. comm.). Again highlighting the fact that bacteria can be present on farms that are thought to be free of the disease.

Publications

Clark, G., Fenwick, S., Boxall, N., Swanney, S., Nicol, C., (2000): Salmonella Brandenburg the 1999 season. Proceedings Sheep and Beef Cattle Society of Veterinarians NZVA, Palmerston North.

The Gore District Council, Seminar on Salmonella Brandenburg, 27 July 2000.

VetSouth Sheep Farmers Seminars, Gore 15 August and Winton 16 August, 2000.

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