Because RHD/RCD cannot be grown in cell cultures the assurances of having any pure cultures do not exist. The virus preparations in use in Australia and elsewhere are from processed ground-up organs taken from diseased rabbits; as a consequence there is confusion. Two camps of scientists have formed: U.S. scientists at Plum Island and Chinese scientists have published results demonstrating a parvovirus presence and they implicate that agent as the cause of RHD while the European scientists have demonstrated calicivirus presence and implicate that as the causal agent of RHD. As a result of not having grown pure cultures of either agent, Kochs postulates which have been used for over 100 years to identify the casual agent of diseases cannot be carried out with RHD. Koch's postulates require that a proven pure culture of the suspect agent (not available for RHD) must be isolated from a sick animal and inoculated into a healthy animal where typical disease must occur and then in step three the original suspect agent must be re-isolated from the sick test animal in pure form. Steps one and three of Koch's postulates have never been completed for RHD. Australian scientists may claim that purified virus has been separated from dead rabbit organs using physical means such as differential centrifugation but these methods do not absolutely purify. They can significantly alter the ratio of one virus type to another if there are known differences in the physical properties of the mixture of viruses. However, without knowing what all the virus types are that might be present this becomes a very blind and unrewarding game, especially if one remembers that even with ratios of millions of recognized virus to one unknown virus, the unknown agent can replicate and keep causing disease even when inoculated into an animal along with millions of copies of the known virus.

Without having pure virus grown in clean reproducible cell cultures even vaccines become crude preparations. They are made of ground-up liver from diseased rabbits with additives to kill the virus and stimulate antibody. Heinous anecdotal reports in Australia tell of "wasting" disease, hair loss and abcessation of injection sites and elsewhere. The only vaccines available worldwide for RHD are for rabbits only. Should disease occur in any other species the current vaccine would be unsatisfactory and/or dangerous, particularly for humans. Although the probabilities may be remote, how does the government of Australia and New Zealand propose to cope with such an event should it occur?

All of this was clearly understood by the Australian scientists when they wrote in the BRS booklet(3) that they would need to propagate the RHD agent in laboratory cell culture before serious animal inoculation experiments were carried out. Even though they failed in their efforts to adapt the agent to cell cultures the RHD release program was kept on it's predetermined schedule. Without first meeting their own stated essential prerequisites, program directors proceeded to do animal inoculations experiments which included all the inadequate testing of thirty non-rabbit species and the ill-fated Wardang Island affair. Thus began a phase of the RHD release program where needed research and troubling research data was not allowed to hold-up the intended release of RHD. The ANZRCDP/RBAG leadership position seems properly summarized as "steam roll all obstacles."

4. VIRUS PREPARATIONS: This section is provided because details are given in a peer reviewed Australian research report (1) that demonstrates reasons to question the purity of the virus preparations that were used to inoculate animals for disease and antibody production. These same virus preparations were also used in the competitive ELISA test, and understanding the nuances of this competitive ELISA test are essential to understanding the distorted science reporting that surrounds the RHD release program. During virus preparation, there appears to be two steps reported that could begin to separate out virus mixtures if more than one virus type occurred in the RHD infected rabbit liver samples used. The first was the treatment of infected liver hemoginates with lipid solvents (chloroform) which can inactivate groups of viruses having lipid envelopes. The second step was centrifugation in cesium chloride which can be prepared as a continuous concentration gradient thereby allowing viruses having differing specific gravity characteristics to be separated during ultracentrifugation as bands of particles having different weights. However, differential separation was not done. Cesium chloride was used as a single concentration having a specific gravity of 1.3. Therefore, all particulates (viruses) heavier than 1.3 would form a pellet in the bottom of the tube during centrifugation for a few hours at 100,000 times gravity. Caliciviruses have a buoyant density of 1.36 - 1.38 (approximately 1/3 again heavier than water) so they will form a pellet in the bottom of the tube. Parvoviruses have a buoyant density of 1.40 - 1.45 so they will also, if present, co-mingle with the caliciviruses (the U.S. Plum Island scientists and Chinese believe parvoviruses are present) to form a pellet in the bottom of the tube. This pellet (mixture?) was used as the seed stock for subsequent infections, virus stocks, the RHD typing sera production and the competitive ELISA standardization as well as the antigen in the competitive ELISA tests. This virus preparation was also used to stimulate mice for monoclonal antibody production. In other words, the RHD virus preparation used to make the diagnostic reagents and to infect rabbits remained, in terms of viral purity, a crude liver preparation that could have contained one, two, or more small non-enveloped viruses that could include several different caliciviruses, several different parvoviruses as well as varieties of enteroviruses and rhinoviruses.

5. COMPETITIVE ELISA: This test(1) appears to be the one relied upon to test for antibodies against the RHD agent following experimental infection studies in all thirty non-rabbit species. Presumably it is the test of choice for assaying human sera for RHD antibodies in Australia and is thought to be the test most relied upon in Australia to detect RHD antibodies in serums from any source. Although the test was adequately defined as published, there are several possible pitfalls in the way it was used.

a. It relies on a single monoclonal antibody, therefore a substitution of only one of the 7,000 bases in the RHD genome could cause a change in the antibody binding site such that this monoclonal would give a false negative test result (when testing sera from animals other than rabbits the virus replicating in a non-rabbit would be expected to have genetic changes concurrent with replicating in a non-rabbit host species). Thus a false negative (b) would be more likely to occur in humans or other non-rabbit species when using this test.

(b)    False negative is when the individual is positive but the test is negative.

b.   Certain parameters of the test were arbitrarily selected and could be used, and seemingly
     were used to eliminate unwanted or troubling test results.  The test is set up to work as
     follows: serum from a test animal is added to the RHD virus preparation.  If the test
     animal has antibodies to the virus, these antibodies bind to the virus.  Next comes the
     competition part: the monoclonal antibody is added and it will only bind to free sites, that
     is those not already occupied by the antibody in the test serum.  The more free sites
     available for the monoclonal the less positive the test sera.  The test scores are given as
     percent inhibition where 0% inhibition (no interference with the monoclonal antibody
     binding)  would be a negative test and 100% inhibition would be a positive test.  The
     trouble begins because results are rarely 0% or 100% so a determination has to be made
     that some value between 0% and 100% will be used as the cut-off between a negative and
     a positive test.  For competitive ELISA tests in general, these values are often set in the
     10% up to the 20% range and occasionally as low as 5%.  The competitive ELISA used in
     Australia was given a cut off of 30%.  In an impact assessment report(6), this value was
     referred to as the arbitrary absorbing cut-off for designation of unequivocal positive
     diagnoses.  In other words, test sera binding up to 30% of the monoclonal binding sites
     could arbitrarily be called negative and this percent inhibition was stated by the authors(1)
     page 91 as the value needed because there appeared to be pre-existing (perhaps
     calicivirus) antibodies in wild Australian rabbits that interfered with the monoclonal
     antibody binding.  These same rabbits had tested positive using an indirect ELISA test and
     perhaps although not stated a hemagglutination-inhibition test.  Even after "rigging" the
     test, two of the wild rabbits sera sampled prior to 1994 continued to register positive with
     readings up to 40% inhibition while five experimentally infected rabbits gave readings as
     low as 20% three weeks after infection and one of four gave readings of only 70%
     inhibition even at 50 days post exposure.  In other words, most unexplained and unwanted
     positive reactions could be eliminated by disregarding the sera that reacted at a 30%
     inhibition level or less by simply calling them negative and saying the reactivity was non-specific.  
  Please remember this point, it becomes extremely important when evaluating the
     experimental animal infectivity studies.

c.   Even in experimentally infected rabbits, the competitive ELISA test does not reliably
     detect antibody conversion until 20-30 days after animal infection.(1)  Other HI and
     Indirect ELISA tests were shown to detect antibodies at day 7-14 post-infection in
     rabbits.(1)  Please remember this point for the discussion of experimentally infectivity
     studies on 28 species.

d.   The monoclonal antibody used in competitive ELISA was not (at least at the time of
     publication(1) tested to determine if the specific epitope represented is present on any
     calicivirus other than the single strain of RHD used in the Geelong laboratory including
     other RHD strains, the forty plus known cultivatable calicivirus types, the human
     Norwalk-like calicivirus types, and the multiple Hepatitis E types.  It appears therefore
     that competitive ELISA was not evaluated for the possibility of it detecting antibodies of
     multiple calicivirus types either closely or distantly related to RHD.  Such detections
     would give false positive tests such as a positive test for caliciviruses other than RHD
     when RHD infection had not occurred.  Conceivably this was the reason for the positive
     wild rabbit sera tested and discussed previously.  Furthermore, the specificity of the
     monoclonal for RHD was not reported to have been evaluated by testing it against a
     battery of non-calicivirus agents to be sure that the epitope in question (bound by the
     monoclonal antibody) was calicivirus specific and not shared by other viruses.  If the
     epitope was not calicivirus specific, the test would give false positive results.  Without
     those assurances, one wonders how this test passed the stringent in-house review expected
     of a test reagent to be used in an international referral laboratory having the status of
     AAHL.  The critical questions are how specific is the test?  Does the test detect only RHD
     antibody?  And how sensitive is it?  Will it always detect RHD antibodies even in minute
     amounts.  And is the specificity/sensitivity good enough even if optimum parameters are
     set to reliably detect RHD specific antibodies in all the test animal species.  We simply
     don't know. Please remember this point for the following discussion of animal
     infectivity studies in 28 species.

6. ANIMAL INFECTIVITY TESTS: The various experimental RHD doses used were given as follows: 103 RLD50 or more quoted by Coman(8), 1000 RLD50 or more quoted by Westbury and many others and they then add the 100,000 RLD said to have been given to Kiwis and short-tailed bats. This information on the Kiwi dosage surfaced after there was a need to explain away the Kiwi antibodies. This purported dosage error meant confessing to having made one of the most fundamental and inexcusable blunders in biomedical research, that is, giving a dosage that was 100 times greater than the calculated dose. The inadequacy of the infectivity doses used have all been extensively covered by Dr. Matson's submission to the NRA and other documents attached to this letter.

A short discussion on differences between host-range, host-switching and host-specificity would seem to be in order because virtually every document released by the agencies promoting RHD approval focus on this red herring as if it were meaningful. It is a non-issue. It makes little difference either to the animal or the virus which infects that animal for the first time whether the virus is a new variety adapted by mutation or a virus that could have always infected this species but just didn't have the opportunity. The end result is the same for all the Australian species now being exposed. In a very bottom line sense any virus that for the first time ends up in a new species "always could have infected that species", that is, the inherent genetic mechanisms or their precursor had to have been present for this event to occur. Previous expression of these innate mechanisms either did not have time, opportunity, and selective pressure to do this, or they did it and the infection went unrecognized. In the end, a "new" virus and a "new" disease in a "new" species such as has popped up repeatedly with the caliciviridae is a notable event and one that is expected to continue repeating itself. New host availability, new virus strains, high virus numbers, new ecologic relations and time all play into this formula. In his writings entitled "RCD and Mutations" which was released as a RBAG public information pamphlet, one Australian scientist with CSIRO affiliation commits numerous informational errors regarding the caliciviruses. For example it is stated that caliciviruses are generally host specific (they are not). There is implication that feline caliciviruses infect only cats and that too is patently wrong. Finally, there is a protracted discussion which becomes totally lost in host-range and host-switching as he attempts to explain away the obvious, which is that in all likelihood the so-called rabbit calicivirus did not originate in rabbits and this virus can be expected to infect other non-rabbit species. Dr. Colin Wilks, perhaps unwittingly, adds credence to this last statement by saying in his RBAG public information pamphlet "Viruses and Infectious Agents" that

"every herpes virus that has been adequately studied has been found to produce lifelong latent infections of the host with intermittent periods of viral shedding. This epidemiologically important property could be predicted for any newly isolated virus once it was shown morphologically to be a herpes virus."