Sub-project 1: Johne’s disease pathogenesis and proteomics.

‘Johne’s Disease pathogenesis and proteomics’, represents efforts by collaborating institutes to characterize virulent strains of pathogenic bacteria that are of economic and social importance to the Victorian dairying community.

Mycobacterium avium ssp. paratuberculosis (MAP) has long been recognized as the causative agent of Johne’s disease (JD), a chronic intestinal disease affecting cattle, sheep, goats and other ruminants. Figure 1 shows a cow in the advanced stage of Johne’s disease. MAP is difficult to reliably detect and treat, and is a pathogen of enormous economic significance to agriculture world-wide.

Researchers from CSIRO Livestock Industries, Novel Products and Proteomics Stream are applying proteomic methodologies to elucidate not only the molecular mechanisms underlying MAP pathogenesis in animals but also proteins that might represent biomarkers of JD infection.

Mycobacteria have a thick and waxy external coating which makes them robust and persistent during infection. This characteristic necessitates novel analysis strategies that maximize extraction of cellular proteins.

The researchers compare cellular disruption methods suitable for proteomic analysis (Figures 2,3 & 4) that are used to investigate MAP isolated directly from JD-infected cattle and goats. In this research they use:

• high-resolution, analytical and preparative-scale 2-dimensional gel electrophoresis formats;
• preparative isoelectric focusing apparatus;
• capillary-flow HPLC and ion-trap mass spectrometer;
• various aspects of protein biochemistry.

References

1. Lanigan MD, Vaughan JA, Shiell BJ, Beddome GJ and Michalski WP, “Mycobacterial proteome extraction: comparison of disruption methods”, Proteomics (2004), 4, 1094-1100.
   
2. Egan S, Lanigan MD, Beddome G, Shiell B, Stewart D, Vaughan JA, Doran T and Michalski WP, “Isolation and purification of intact Mycobacterium paratuberculosis from intestinal sections from cattle and goats”, in proceedings of the 8 th International Colloquium on Paratuberculosis (2005) Copenhägen, Denmark, 14 th-18 th August p. 73.
   http://www.paratuberculosis.org/proc8/abst4_m1.htm
   
3. Lanigan MD, Tsen L, Vaughan JA, Shiell BJ and Michalski WP, “Detergent enrichment of mycobacterial envelope proteins”, in proceedings of the 8 th International Colloquium on Paratuberculosis (2005) Copenhägen, Denmark, 14 th-18 th August, p. 34. http://www.paratuberculosis.org/proc8/abst2_p27.htm

Sub-project 2: Antibiotic resistance and bacterial virulence factors

Survival & persistence of Salmonella in dairy processing – a genomic approach
Despite the high standards of food safety observed in Australia, an estimated 5.4 million cases of food poisoning occur in Australia each year. Furthermore, the potential for foodborne illness is increasing as a result of factors such as expanding food trade with other countries and new food production methods. Salmonella is the focus of this research due to its importance as a food-poisoning organism in dairy manufacturing and because there is a large amount of information already available about the Salmonella genome. The genome of an organism such as Salmonella contains the full set of its hereditary information, encoded in the form of genes. Recent advances in genomic technology mean that researchers can now determine precisely which genes are important to the bacterium’s survival under particular conditions and this provides a powerful tool to understand the way bacteria can potentially adapt to harsh conditions and survive during dairy production and manufacturing. The new information obtained can be used to enable better design of dairy processing procedures and the hygiene and sanitation practices employed in dairy production and manufacturing.

Enterococci in the dairy production & processing chain – their potential role in emerging antibiotic resistance
Concern over the emergence and spread of antibiotic resistant pathogens in human medicine has led to studies to investigate sources of these organisms including the food supply. As antibiotics are used to treat disease in dairy cattle, bacteria (enterococci) isolated from raw milk are being examined to determine their susceptibility to human and veterinary antibiotics. The potential ability of the isolates to receive antibiotic resistance genes  from and to transfer these genes  to other bacteria under milk production conditions is also being studied. The data obtained will provide information on the prevalence of antibiotic resistance in dairy bacteria and an indication of the involvement of these bacteria in the spread of antibiotic resistance in bacterial populations.

Sub-project 3: Detection of enteric viruses in milk and dairy products

Background
The biosecurity of food supply is a major global concern and the emergence of new and virulent pathogens is considered to pose a major risk to food safety. Foodborne illnesses are a widespread problem. Understanding the risks associated with emerging pathogens in dairy products and the dairy production system, is vital to ensure safe products for human consumption and to maintain confidence in Australian produce.

Bacterial agents can be easily detected and quantified by well-established techniques. In contrast, reliable methods for the detection of viral contaminants in foods are not well developed.

Rotavirus is a major cause of severe gastroenteritis in young humans and animals, including diarrhoeal disease in calves. Bovine rotavirus (BRV) has been found in both dairy and beef cattle herds and associated prevalence rates have been documented from 7% to 94% in cattle farms in different regions of the world.

There is limited information on BRV’s in Australian cattle. The most recently published study on the occurrence of BRV in Australian calves was conducted in Victoria in 1988. Most studies report prevalence levels of BRV amongst calves with diarrhoea approximating 40%. Although interspecies transmission has not been directly documented, there are increasing reports of rare and atypical rotaviruses that were apparently derived from transmission between humans, cats and dogs; humans and cattle; humans and pigs; pigs and cattle; and pigs and horses. This has led to emergence of novel rotavirus strains due to genetic reassortment.

There are no studies describing the stability of BRV after food processing. Limited studies have investigated the inactivation of human and simian rotavirus by current and/ or alternative processing techniques, including high pressure processing (HPP).

Aims

The project aims to:

• Identify and characterise bovine rotavirus (BRV) and examine the role BRV plays in animal and human disease. A survey of bovine strains in local cattle herds will be used to determine the prevalence of BRV associated with diarrhoea in calves. Additionally, the mechanisms of genetic reassortment between rotavirus strains of human and bovine origins will be studied by identifying and characterising novel bovine strains associated with disease in children.
  
• Determine the effectiveness of conventional and novel food processing methods to inactivate rotavirus. Methods used to detect infectious and non-infectious rotavirus will be investigated. These studies will establish and validate leading edge techniques, including real-time polymerase chain reaction (PCR) assays, required to identify viral agents following dairy processing. The risk of rotavirus in dairy products will also be evaluated, with the information used to develop sound strategies for the future production of safe dairy foods.

References

1. Kirkwood C, Bogdanovic-Sakran, N, Barnes G and Bishop R, “Rotavirus serotypeG9P[8] and acute gastroenteritis outbreak in children, northern Australia”, Emerging Infectious Diseases – Centers for Disease Control and Prevention (September 2004), No. 9, 4, 1593-1600.
   
2. Kirkwood C, Bogdanovic-Sakran, Palombo E, Masendycz P, Bugg H, N, Barnes G and Bishop R, “Genetic and antigenic characterization of rotavirus serotype G9 strains isolated in Australia between 1997 and 2001”, Journal of Clinical Microbiology (August 2003), No. 8, 41, 3649-3654.

Sub-project 4: Communication and extension.

Aims

The project aims to:

• Form a network that maintains surveillance of emerging issues in dairy food safety and enables rapid communication to stakeholders of these issues.
  
• Communicate research outputs from the sub-projects of MP1/016 to better facilitate uptake of scientific discoveries and manage potential risks.

Key Outputs

Project outputs will include:

1. Communication of the key messages from the project MP/016 through the development of a dedicated project webpage.
   
2. Communication of food safety information on best practice risk management. This will be delivered by a range of information material such as guidelines, notes and technical standards, as well as through industry forums and seminars.

Progress to Date:

To date the following progress has been achieved:

1. The National Guidelines for food safety on dairy farms were endorsed by the Australian New Zealand Dairy Authorities’ Committee (ANZDAC) in October 2005 and will be formally launched in 2006.
   
2. Dairy Food Safety Victoria has hosted several industry forums involving dairy companies’ senior dairy field staff.
   
3. Dairy Food Safety Victoria has conducted a series of seminars with Victorian dairy manufacturers on a range of topics including: the Food Standards Code, Pathogen Management, Chemical Residues and the Dairy Food Safety Scheme.

Sub-project 5A: Microbial Ecosystems of Milk - and the Influence of Natural Milk Inhibitory Systems on the Prevalence of Spoilage Organisms and Pathogens in Raw Milk

Background

• Raw milk harbours a complex and dynamic microbial ecosystem, including bacteria, yeasts and moulds, that is influenced by regional and climatic factors, the health status of animals, cow feeding regimes, the hygienic practices of farmers in addition to storage and transport conditions.
• The dominant microflora of milk include Lactococcus, Lactobacillus, Pseudomonas, Micrococcus and Staphylococcus species. Among the organisms that may constitute a health risk are Eschericia coli, Staphylococcus aureus along with species of Bacillus, Clostridium and Listeria.
• Milk has numerous natural bacterial inhibitory systems such as iron binding metalloprotein, lactoferrin, immunoglobulins and lysozyme. Many lactic acid bacteria used for the production of cheese and other fermented dairy products can also inhibit pathogens through reduction of pH, the production of bacteriocins and the generation of hydrogen peroxide which is a component of the bacterial inhibiting lactoperoxidase system.
• Recently (September 2005) Food Standards Australia New Zealand (FSANZ) gazetted an amendment to the Food Standards Code permitting the sale of Roquefort raw milk cheese made under specific conditions in France. FSANZ is also preparing to examine the issue of allowing local raw milk cheeses to be sold.

Project Outline

• Identify microbial ecosystem components of raw milk from varying regions of Australia based on genetic profiling by denaturing gradient gel electrophoresis (DGGE).
• Examine the growth dynamics and survival of potential spoilage organisms and pathogens in raw milk.
• Investigate the effect of natural milk and lactic acid bacteria mediated inhibitory systems on potential spoilage and pathogens in milk.
• Identify indicators, based on genetic profiling techniques, for assessing the potential of raw milk to contribute to the presence of spoilage organisms and pathogens in dairy products derived from raw milk.
• Examine the survival of spoilage organisms and pathogens in products manufactured from raw milk.

Project Outcomes

• The establishment of systems to assess the suitability of raw milk for direct use in dairy products manufactured in Australia.
• The establishment of production and manufacturing protocols to minimise the potential for contamination of products that are derived from raw milk.
• Evaluation of natural inhibitory systems of milk for control of spoilage organisms and pathogens in products derived from raw milk.

Sub-project 5B: Australian specialty cheese - Mycotoxin risk profile (This is a proposed project that has been put on hold)

Background

The specialty cheese sector of the Australian cheese industry supermarket sales is currently valued at 210 million dollars per annum and this is increasing at approximately 10 % annually. A significant proportion of specialty cheeses are also marketed through delicatessens, specialty cheese stores and markets. Many of these cheeses are manufactured by small independent producers with mould ripened cheeses a large percentage of the cheeses produced.

There are two major categories of mould ripened cheeses -

• blue-veined cheeses typically represented by varieties such as Stilton, Roquefort and Gorgonzola; and
• white surface-mould cheeses which include Camembert and Brie.

The moulds used in the production of these cheeses are Penicillium roqueforti which produces green-blue growth and Penicillium camemberti which produces grey-white growth. Both these moulds are able to produce substances toxic to humans and animals. These are known as mycotoxins. However, there is disagreement over the extent of mycotoxin production in cheese and there are indications that some toxins break down in cheese.

Toxin levels appear to be related to the strain used and the maturation conditions under which the cheese is stored.

There are also a number moulds that cause spoilage on cheese including species of Cladosporium, Penicillium and Phoma. Cladosporium cladosporioides and C. herbarum are not known to produce mycotoxins but others do, for example cyclopiazonic acid from some isolates of Penicillium commune.

The Proposed Study

Due to the general lack of knowledge on mycotoxin production in cheese it is proposed that a study be carried out to research these issues. The objective will be to determine the extent of mycotoxin development in both Australian and imported mould ripened cheeses and define the conditions under which mycotoxin production is both inhibited and promoted under laboratory and cheese maturation conditions. In addition the extent of mycotoxin formation by wild type and spoilage moulds will be investigated.

Strategic collaborative partnerships are in the process of being established, with potential participants being the Australian Starter Culture Research Centre, The University of Melbourne, CSIRO Animal Health and The Key Centre for Applied Nutritional Toxicology of RMIT University.

References

1. Australian Centre for International Agricultural Research, Mycotoxin Newsletters
   http://www.aciar.gov.au/web.nsf/doc/ACIA-5MDA7R
   
2. European Mycotoxin Awareness Network
   htttp://193.132.193.215/eman2/index.asp
   
3. United States Department of Agriculture, Food Safety and Inspection Service
   http://www.fsis.usda.gov/Fact_Sheets/Molds_On_Food/index.asp
   
4. United Nations Food and Agriculture Organization
   Mycotoxins and Food Supply
   http://www.fao.org/docrep/U3550t/u3550t0e.htm
   
5. ComBase food microbiology database of microbial responses to food environments
   http://www.combase.cc