If one were to paraphrase this statement for caliciviruses, the following true statement can be made:
"every calicivirus that has been adequately studied has been found to be pathogenic and to infect more than one animal species. These epidemiologically important properties could be predicted for any newly identified virus once it was shown morphologically to be a calicivirus."
Of all the caliciviruses known, those that infect rabbits and hares causing hemorrhagic disease are considered by some to be exceptions but these agents have simply not yet been adequately studied.
The CSIRO-AAHL animal infectivity experiments were said to address this issue of cross species transmission into non-rabbits. Instead this was a series of experiments that were designed to give a negative result. It is already known that the virus dosages were held so low as to not "force a response." These are Dr. Westbury's words quoted in Science 12 April 1996. What this means is that virus counts were purposefully kept below immunogenic levels and purposefully kept below the infectivity threshold of many disease agents. It is widely accepted that if we intend to test a new host animal's resistance to a given agent it cannot be said that the agent did not infect that animal until after administering extremely high doses, millions to billions of viruses given by multiple routes of entry using young, old, normal, immuno-compromised, lactating, gestating, and nutritionally or otherwise stressed animals with pen-contact animals of the same species to test horizontal virus transfer. Put simply, one must attempt to "force a response" which would mimic that occurring under conditions of natural exposure and these experiments should be repeated. There are thousands to millions of non-rabbit individual animals being exposed to RHD in Australia, not just four as occurred at AAHL. If none of the experimental animals become infected under these real-life conditions of high exposure (they do not have to exhibit disease they only have to replicate virus because the disease will often surface after heating up by passage in the new found host), then we become more confident that this species has an effective natural barrier to the particular virus being tested. If animals do become infected then the question must be how easy or difficult are they to infect, and what are the dangers of this animal becoming a carrier (common with caliciviruses) where other contact animals of the same species or a new species become exposed in new ways?
Such experiments appear not to have been carried out or at least publicly reported even in a single species. If the public health agencies of Australia and New Zealand have any concern for the potential threat of this new hemorrhagic disease to human health, one must assume that experiments such as those described above have been carried out in some higher primate species. Has this essential step for addressing the human risk taken place?
What about the experiments that were carried out on twenty eight Australian species and two New Zealand species? Other than the purposefully low virus doses and only four test animals per species what else was wrong?
a. Each species was tested once for the presence of RHD virus 14 days post-exposure at
necropsy, when most virus infections would be expected to have run their course and been
cleared from the body.(3,6,8) These results were negative but who is surprised? Why were
not some animals necropsied at 24, 48, 72, and 158 hours when virus would be expected
to be replicating in the tissues of an infected animal?
b. One and two day blood tests for virus were carried out in some species using PCR, but
some caliciviruses do not go through a viremia (virus in the blood stream) stage.
Furthermore, for these PCR tests to be validated with proper "range finding" protocols,
the virus should have been given then sequential blood samples taken at 2, 4, 8, 24, 48,
and 72 hours to determine if viable virus was detectable in the blood stream of each test
species at anytime using PCR.
c. The competitive ELISA was never said to have been standardized for each test species.(1)
d. The competitive ELISA test does not detect RHD antibodies in infected rabbits until at
least 20 days after infection. All 28 Australian species plus the short tailed-bats were
tested at 14 days and less, when it was known that the test should give a result that
could be called negative even if infection occurred. The mice were said to have been
antibody positive but there has been no mention of infectivity tests being repeated in mice.
Instead the antibody information was seemingly disregarded along with the possibility that
RHD could infect mice and it was then said that RHD would not infect any other species
except rabbits.
e. The competitive ELISA test was given an arbitrary cut-off of 30%. Specific data using
serum samples over time were made available only on the tests from Kiwi and short-tailed
bats.(6) The Kiwis all had ELISA test scores of 10 to 19% interference even before they
were given the virus. This suggested that some antibodies in their system bound to the
same RHD virus antibody binding sites as the RHD monoclonal antibody used in the
competitive ELISA. However, this possibility was discounted as non-specific reactivity
because the test cut-off had been bumped up to 30%. By day 14, all the Kiwis had to be
scored positive because they had increased to above the 30% cut-off and by day 35 all
were even higher with one Kiwi scoring a 78% interference. These levels were all called
"low" by the Australian and New Zealand spokesmen promoting RHD release, when in
fact the one Kiwi had a score nearly 10% higher than that of an experimentally infected
rabbit. (1,6) Furthermore, great numbers of tests were carried out to prove that the Kiwi
were never infected. This could be viewed as a possible smoke screen because negative
tests for vial presence 35 days after exposure does not address the possibility of a previous
infection that has long since passed. Kiwis exhibited RHD antibody activity that should
have cleared their systems of the virus infection long before the 35 days, making it doubly
safe to look diligently and with much fan-fare for a virus that should have been long gone.
f. The short-tailed bats are a slightly different story. There were seven of them, not four.
Two were apparently not injected but were left in contact with the five that were; only one
of the seven had a pre-test antibody screen for RHD using the now infamous competitive
ELISA. That bat scored a 1% inhibition.(6) It was a fully legitimate negative. Fourteen
days later blood samples were taken from six of the bats (4 exposed and 2 in contact).
The bat that had a 1% baseline (negative) reading went up to 12% inhibition on the
competitive ELISA. In other words, this animal sero converted to the RHD exposure but
because it was below the (useful) 30% cut-off, this positive response was reported as
negative.(6) Oddly enough the remaining five bats tested had test results at 14 days ranging
from 10% to 16%. The two contact bats had readings of 10% to 11%. Presuming all to
have had little or no pre-infection reactivity such as was the case with the only bat tested
for pre-inoculation baseline antibody, these are rather startling results and certainly should
never be passed off as a negative finding without further testing of additional bats
particularly where several experimentally infected rabbits had lower competitive ELISA
test readings at fourteen days than the bats. Once again the available evidence argues
against this agent being rabbit specific while the agencies promoting RHD releases cite
these laboratory test and say RHD is species specific. This sounds like a rerun from
British Mad-Cow Disease play book.
g. Think for a moment what these results on the Kiwis, bats, and perhaps other test species
would look like if the arbitrary cut of the quirky competitive ELISA test had been set at
20% and all the test animals had been tested at 3-4 weeks post-infection instead of at 2
weeks when the test was known to give what was called a negative response even in
infected rabbits?
h. There is another test animal story that appears tainted. This involves the bird and seagull
testing. Please remember that uncultivatable caliciviruses (they are RHD like in this
regard) have been shown to cause disease in chickens, pheasants, and guinea fowls.
Furthermore, David Chasey suggested possible bird transport of RHD from France to
Britain and it was reported several years ago at an international meeting that a non-cultivatable
calicivirus had caused a blistering disease on a fairy tern chick sampled on
French Frigate Islands in mid-Pacific. This has subsequently been published (July 1996 J.
Wildlife Dis.) in a peer reviewed scientific journal. Having established that
uncultivateable caliciviruses from unknown sources infect birds, we find the following
remarkable statement on page 139 of the Import Impact Assessment prepared for the
import of RHD into New Zealand(6):
"the development of antibodies by the Kiwi parallels results from other species tested in Australia suggesting that birds in general are more likely to produce antibodies to RCD than other animals."
This statement becomes especially meaningful when evaluating the events involving the so called sentinel rabbits. Rabbits were placed in cages on the fences inside test compounds where RHD experimental infectivity studies were carried out. As shown by the Wardang Island experiments, these sentinel rabbits, and presumably there are about 30 of them, that is, one per animal species experiment, had little expectation of actually becoming infected but a "sentinel rabbit" gives a nice public relations touch. It sounds so safe and sure. Any serious study of this nature would have provided several "susceptible rabbits" per experiment and placed them in direct contact with excreta and body fluids. Even without direst contact, the unthinkable happened. In the pen with the sea gulls and other birds, the sentinel rabbit died of RHD. This was quickly explained away by a claim of failed experimental technique inside the maximum containment facilities at AAHL involving possible spreading of virus while doing sea gull injections. Who wants to believe that personnel and procedures inside such a high containment, high risk, "hot" facility are that loose? Why did the rabbits death have to be explained away using any reason except the most probably, that is, the seagulls and other birds becoming infected and shedding the virus? Once again all these birds were reported as negative but there is still the troubling statement regarding birds and antibodies in the import application document quoted above which suggests antibodies (infection) may have occurred but was called negative using competitive ELISA tests.
7. RHD ANTIBODIES IN SPECIES OTHER THAN AUSTRALIAN FERAL RABBITS: This discussion is intended to show that there is a considerable body of evidence which argues rather forcefully for RHD infectivity in species other than the European rabbits. The AAHL mice, Kiwis and bats have already been discussed and in addition antibodies to RHD in hares was reported in August 1995 in Poland.(7) Three test sera out of 100 were positive and 2 of these also had antibodies to the European Brown hare virus. These data clearly report that hares became exposed and presumably infected with RHD. Others in Germany and China also report possible RHD infection in hares.(2,5) Naturally and experimentally infected dogs and foxes developed RHD antibody and dogs shed the virus in their feces(9,10). These studies did not test for replicating virus. One paper from China, heavily cited by those promoting RHD introduction and release, cites an impressive list of animals that were thought not to be susceptible and then the paper cited clearly states that only natural contact took place and that RHD clinical disease was all that was looked for, but serology and virus isolation were not carried out.(2,4) Another paper reports inability to infect a series of laboratory animals, serologies were negative but virus isolation was not attempted. What is not being reported from that same paper is the account of a hare dying with RHD signs (European Brown Hare virus was not reported as a factor in China). Furthermore, tissues from this hare were injected into several rabbits and they all died of RHD. The liver had high HA titers that were blocked by RHD serum. Wild presumably asymptomatic hares in China also had elevated HA titers. Instances such as this report cannot be disregarded.(2) The failed attempts reported elsewhere to infect hares with RHD do not negate this finding. If it is legitimate, and it appears to be, then any number of similar but negative experiments could not erase this single finding of RHD occurring in a non-European domestic rabbit species. Again if this single report is correct then RHD cannot be declared a biological agent because it would not infect just a single species. Also, note that the most likely reason for any animal to have antibody against a pathogen they have had exposure to is because they were infected. Infection need not result in clinical disease or other outward manifestations of infection. Antibody presence is strong evidence for but not proof of infection.
8. POSSIBLE HUMAN INFECTION: The agencies and individuals promoting the release of RHD use the "there is no evidence" statement as an argument when discussing possible public health risks. They cite lack of reports in forty countries without qualifying whether any testing actually took place, how many, and which populations. In reality those forty countries appear to not be looking for RHD infection in people nor does it seem that they want to find evidence of such infection. Otherwise, there would be ample published results of such surveys to help make the "no human infection" arguments more credible. Only a few serologies have been run on humans in Australia and Mexico and if the same "slip scale" competitive ELISA that was previously discussed is used for humans in Australia, there is little chance of meaningful testing occurring in that county. For more insight into this problems of international testing, please refer to the June 1, 1996 letter to Dr. Jean Blaneou at the Office of International Epizootics which is attached but has never been answered.
With only a few human sera having been tested, there is the report of one Mexican worker developing antibodies. That finding has been discarded by the agencies committed to introducing RHD when they have absolutely no data supporting their position. They simply decree that the RHD antibodies in the exposed worker did not arise from an infection. When individuals are exposed to disease agents the most probable reason for them to have antibodies against that agent is that they did become infected. For that reason the assumption has to be made that one human was infected with RHD but did not develop any symptoms that were reported. With this finding, the ball has simply changed courts. It now has to be proven by the agencies committed to RHD introduction that humans cannot become infected with RHD and that the RHD antibodies which have occurred in humans are not the result of infection with the RHD agent.
9. WARDANG ISLAND: Without examining possible motives a very brief account of the Wardang events can bring unsettling insights. The following is a paraphrase of B.J. Coman's account in his Environmental Impact Draft report of February 1996.(8) A large compound was enclosed with an electrified rabbit proof fence. A 300 meter perimeter around that fence was cleared of rabbits. Inside that fence two additional compounds were fenced and within these two areas seven double-fenced test colonies of 10 rabbits each were established. On 23 March 1995 in the first pen two rabbits were given RHD and turned loose with their eight pen mates. These eight pen-contact rabbits did not develop RHD. On 27 April 1995 this protocol was repeated in two more pens. In one pen two rabbits contracted RHD but none contracted the disease in the other pen. A third trial on 20 June 1995 in two more pens resulted in seven of eight contact rabbits getting RHD in one pen but only a single rabbit becoming infected in the second pen.
To summarize at this point, in just one of the five trial pens did real transmission occur (7 of 8 rabbits at risk died) in two other pens RHD was transmitted to only 1 and 2 rabbits respectively, while two pens had no contact transmission at all. However, even though there were repeated failures to spread the disease within enclosures with direct contact exposure, a rabbit outside the experimental area was found dead of RHD. A strange finding indeed when one realizes that concentrations decline logrhythmically as they radiate out from a point source. The idea of some mechanical vector accidentally carrying the virus in higher concentrations across the various enclosures and through the barrier fences to specifically target and infect some distant rabbit instead of rabbits in direct contact with other dead and dying rabbits is a mystery and the implication could be ominous.
At this point the trials were stopped and rabbits in the pens killed. The Island underwent ten weeks of surveillance for additional RHD cases, none were reported, so on 13 September 1995 an additional two rabbits in each of two pens were injected and 2 of 8 contact rabbits were infected in one pen, none in the other. One might ask why was this second round of tests ever started when the circumstances of the first escape of the virus was still unknown or unreported? Within nine days (22 September 1995) a second RHD rabbit outside the contact pens was detected. Again all experiments were stopped and the rabbits killed. One week later a dead rabbit was detected on the Island outside all the fences and then two days later more were found.(8) Amid a great show of daily reports of things being under control, RHD spread. The rest is history.
Several questions arise. If investigators couldn't reliably transmit the disease by direct contact between rabbits in fenced warrens what value was the so-called sentinel rabbit with no direct contact in the experimental infectivity trials? Remember in three direct contact trials on Wardang Island no transmission occurred; in two, 25% of the contacts acquired disease, and in another two, only 13% became infected. Also remember, the "lone sentinel" rabbit died in the experimental bird cage but this finding has been discounted as meaningless. Why should this be so easily discounted, and shouldn't the bird trials have been repeated?
Next, one wonders why the Wardang Island experiments were re-started after it became known that either the agent could not be contained or the experiments were being sabotaged. In a sense, if the overriding objective (perish the thought) had been to surreptitiously introduce RHD into Australian feral rabbits, then the final Wardang Island trial provided the needed cover for that success.
Finally, there have been repeated confessions of bad laboratory procedures which have been used to explain away results not supporting a position of officially introducing and releasing RHD in Australia and New Zealand. Laboratory tests are adjusted in ways which excludes results that would complicate the pro-RHD introduction agenda. Twenty nine experimental animal species were tested at time intervals (up to 14 days) that would virtually ensure the desired negative test results. Compelling evidence of host non-specificity for RHD such as antibodies in mice, Kiwis, bats, dog, foxes, hares and a human are rejected as meaningless and a documented and reported case of the disease in a hare is overlooked. The only easily recognized success coming from an otherwise ill conceived Wardang Island field project was to provide a mechanism whereby the RHD virus could be removed from the high level biocontainment at AAHL and loosed into feral rabbits in Australia without first passing through all the regulatory and societal approval processes.
RHD introduction and spread presents a potentially dangerous disease situation for the livestock, wildlife, and people of Australia and one that they have needlessly been exposed to. RHD should not be given official status because of compelling evidence against host specificity. The most responsible position that the New Zealand government could take at this time would be to block the import and release of RHD and disassociate themselves from the twisted data being reported by Australian and New Zealand agencies promoting RHD release.
List of Appended Documents:
1. Letter to OJE
2. Letter to Pete Hodgson
3. Letter to Science
4. Letters published in Science
BIBLIOGRAPHY
1. Collins, BJ. et.al. 1995 Veterinary Microbiology 43:85-96
2. Xu, WY. 1991 Review Sci Tech Off. Int. Epiz 10:393-408
3. Munro, RK. and Williams, RT. 1994 Bureau of Resource Sciences, Rabbit Hemorrhagic Disease: Issues in Assessment for Biological Control
4. Wang, YK. 1988 Sci. Agric. Sinica, 21:6-11
5. Loliger, HC. and Eskeus, U. 1991 Review Sci. Tech. Off. Int Epiz, 10: 393-408
6. Ministry of Agriculture, Import Impact Assessment 1996 prepared applicants to import RHD
7. Frolich, K. et.al. 1995 Proceeding Abstract Joint Conference American Association of Zoo Veterinarians, Wildlife Disease Association, American Association of Wildlife Veterinarians p. 500
8. Coman, BJ. 1996 Environmental Impact Prepared for ANZRCDP (draft)
9. Leighton, FA. et.al. 1995 Journal of Wildlife Dis. 31:541-544
10. Simm, M. Del, CR. et.al. 1994 Recueil Medecine Veterinaire 170:841-845
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