Victory in sight on war against Johne's disease

It's a Gollum of a pathogen: sneaky, tough, persistent, and able to remain hidden from its host's immune system as it goes about its nefarious business.

Mycobacterium avium ssp. paratuberculosis, the agent of Johne's disease, a chronic wasting condition in ruminants, has defied efforts to subdue it with conventional control measures. A recently introduced vaccine is the next great hope.

Melbourne company CSL Animal Health (currently being taken over by Pfizer) markets the vaccine for sheep. It is also being considered for use in cattle, principally dairy, but Johne's disease is only a minor problem in the beef industry.

The killed-cell vaccine works, but antibody tests will not be able to distinguish between the animals' response to the vaccine and the live pathogen.

Johne's disease researcher Dr Mark Tizard, of CSIRO Livestock Industries' Australian Animal Health Laboratory in Geelong, says the problem with current vaccines for Johne's disease and bovine tuberculosis, caused by Mycobacterium bovis, is that they ameliorate the clinical symptoms of infection and reduce shedding of bacteria, but do not provide full, sterilising immunity.

He says there is considerable debate about whether the main economic impact of the disease results from reduced animal productivity, or to the control measures imposed on properties to prevent its spread.

Tizard says it's clear Johne's disease has a detrimental effect on animals' health as it affects the gut, impairing nutrient absorption. On severely affected properties, annual mortality rates can reach 10 per cent. "When it gets into a herd or flock, there's no acute effect -- exposed animals that pick up the infection may take months or even years to show clinical symptoms. Only one or two animals may be infected, or more."

The farmer confronts a dilemma: to vaccinate, or not? By producing a counterfeit signature of chronic infection, the vaccine will prevent the farmer achieving disease-free certification.

Decisive phase

But the long war on Johne's disease reached a decisive phase late in 2002, when US research teams led by Dr John Bannantine, of the US Department of Agriculture's National Animal Disease Centre in Iowa, and Dr Vivek Kapur, at the University of Minnesota, completed sequencing the genome of M. avium ssp. paratuberculosis.

Bannantine arrived in Australia late last year to help the AAHL team establish a research project that will apply the latest functional genomics and proteomics technologies to the Johne's conundrum.

"We aim to unravel the infection process, and identify key genes involved in establishing and progressing the disease in the animal," Tizard said. "We will then evaluate the proteins from these genes as targets for diagnostics and therapeutics."

The project will also link the Australian team into a network of some 70 Johne's disease researchers in the US. Tizard's group is already involved in the International Association for Paratuberculosis, whose research interests extend to strains of M. avium that infect humans and birds.

As its scientific name implies, M. avium is believed to have originated in birds. Bannantine says Kapur's Minnesota team has extensively sampled M. avium ssp. avium serotypes in birds and other animals, which rarely show symptoms of infection.

Bannantine says subspecies paratuberculosis has much lower genetic variability than avium. "It's almost clonal, and we believe one particular serotype of avium was its progenitor -- either serotype 2 or 9. Subspecies avium appears to be spread by birds flying over waterways -- it can cause a mild infection in some young children, but it's a serious problem for immunosuppressed AIDS patients.

"They come into hospital for a routine examination and can end up being infected by avium in the hospital water supply -- it's quite resistant to normal decontamination measures."

'Hard to manipulate'

Bannantine says the genome project revealed that M. avium ssp. paratuberculosis has around 4500 genes, 3000 of which are homologous to genes in Mycobacterium tuberculosis, the agent of human tuberculosis.

The M. avium ssp. paratuberculosis genome is larger than that of M. tuberculosis -- 4.8 million bases compared with 4.4 million. Subspecies paratuberculosis seems to have discarded some genes present in its presumed progenitor, M. avium ssp. avium.

Bannantine says part of the problem of studying the host-parasite interactions involved in Johne's disease microbe is that it is "very refractory to analysis, slow-growing and hard to manipulate". For this reason, it may be some time before research yields an effective vaccine. "The more immediate aim is to identify some of the genetic sequences that mediate its survival in sheep, like genes for virulence factors," he says.

"With DNA microarrays, we can get whole-genome expression profiles, so that we know which genes are turned on or off in any condition that the bacterium is exposed to."

The microbe's ability to evade the immune system's defences is due to the fact that it colonises macrophages -- the giant scavenger cells that roam the body, engulfing and destroying microbial invaders. "Macrophages are specialised to kill microorganisms, but this microbe can survive for extended periods inside macrophages," Bannantine says.

Normally, when a macrophage engulfs a bacterial cell, it holds it within a pocket-like cavity called a phagosome, which subsequently fuse with bubble-like structures called lysosomes, containing a cocktail of enzymes that break down and digest the captive bacteria.

Bannantine says it appears that the Johne's disease microbe actively suppresses this fusion process. He will help train the CSIRO team in the use of DNA microarray technology, to identify how patterns of gene activity differ between macrophages colonized by the Johne's microbe, and those that have engulfed other species of bacteria.

Proteomic techniques, high-pressure liquid chromatography and mass-spectrometry will also be used to analyse protein-expression patterns in the microbe, to identify proteins involved in establishing the chronic infection.

Tizard says the long-term aim would be to develop antibody tests that would readily distinguish the antibodies generated by a genetically engineered, sub-unit vaccine, from those resulting from a chronic Johne's disease infection.

A new sub-unit vaccine might be administered orally or intranasally, instead of by injection, avoiding the risk of farmers of veterinarians suffering needlestick injuries.