WO2010051055A2 - Detection of spiroplasmosis and transmissible spongiform encephalopathies - Google Patents

Abstract

A method is disclosed for detecting spiroplasmosis and transmissible spongiform encephalopathies, including Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy, scrapie, and other TSEs. Spiroplasma are present in substantial numbers in the vitreous humor, the aqueous humor, corneal endothelia, and other locations. Samples from these locations can be used to detect spiroplasmosis or TSE, either before or after clinical symptoms arise. The method is only moderately invasive, and is suited for use in living human patients. In livestock the method may be used for diagnosis of live animals, or of animals post-slaughter, either for individual animals or for pooled samples taken from many animals.

Description

DETECTION OF SPIROPLASMOSIS AND TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Frank O. Bastian, William J. Todd

FiIe No. Bastian 07A44W

[0001] The benefit of the 12 March 2008 filing date of United States provisional patent application serial number US 61/035,946 is claimed under 35 U. S. C. § 119(e) in the United States, and is claimed under applicable treaties and conventions in all countries.

[0002] The development of this invention was partially funded by the

United States Government under grants R01 NINDS 0044001 , R01 NS 44000-04, and R01 NS044000-03S1 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

TECHNICAL FIELD

[0003] This invention pertains to the detection of spiroplasmosis and transmissible spongiform encephalopathies (TSEs)₁ particularly by the detection of Spiroplasma in tissue or fluid from the eye and other locations, including the vitreous and aqueous humors, the corneal endothelia, and other locations including tears and saliva. BACKGROUND ART

[0004] It is imperative to develop improved tests for the transmissible spongiform encephalopathies, particularly tests that may be used for pre-clinical screening.

A. The Diseases

[0005] Creutzfeldt-Jakob disease (CJD), a rare disease with a world-wide occurrence, is characterized by rapidly progressive dementia, neurological deterioration, myoclonus, and a triphasic encephalographic pattern. Historically, CJD has been a sporadic disease seen primarily in older individuals, with a peak incidence (-1-2 per million per year) around age 60 years. CJD is uniformly fatal, with 90% of patients dying within one year. A rarer familial form of the disease (5% of CJD cases), appears to be transmitted as an autosomal dominant trait. The histopathology of CJD is characterized by widespread spongiform alteration of gray matter, proliferation of hypertrophic astrocytes, and occasionally by focal tissue deposits with tinctorial properties of amyloid (this last is seen in -15% of all CJD cases, but is consistently observed in familial CJD).

[0006] Amyloid plaques surrounded by focal vacuolization (floccular plaques) of the neuropil are abundant in brain tissues from patients afflicted with new variant CJD. The nvCJD cases typically occur in younger individuals (typically 16 to 35 yrs), and are clinically distinct from sporadic CJD cases in that they present with psychiatric symptoms and have a longer clinical course.

[0007] CJD is one of the so-called transmissible spongiform encephalopathies (TSEs). TSE has long been recognized in both domestic and wild animal populations. Scrapie has been documented in British sheep herds for over 200 years, and is also endemic in other countries. The disease occurs in approximately one third of English flocks, and persists in the United States in scattered flocks. A similar disease is seen in farmed mink populations (transmissible mink encephalopathy, TME), and in cattle in England and continental Europe (Bovine spongiform encephalopathy, BSE, sometimes popularly called "mad cow disease"). Chronic wasting disease (CWD) is prevalent in deer and elk in the United States, by some estimates present in ~20% of wild populations. CWD is characterized by floccular amyloid plaques similar to those characteristically seen in nvCJD cases. TSEs in animals show a variable distribution of lesions, depending on the strain of the agent. Pathologic studies of experimental scrapie in mice have shown evidence for multiple strains, whereas BSE in cattle or in experimental animals shows a uniform neuropathological pattern suggesting the presence of but a single strain of TSE, and a single strain is believed to be responsible for CWD in deer. Amyloid deposition is not prevalent in scrapie in sheep, but is abundant in some experimental scrapie mouse models. BSE has occurred in epidemic proportions since the disease was recognized in 1986 in English cattle. BSE-infected animals have been banned from human consumption since 1988. There have also been reports of TSEs, prions, or both in exotic animals, American coots, domestic cats, pumas, and cheetahs.

[0008] BSE has been linked to human nvCJD cases. The unique neuropathological pattern associated with nvCJD human cases has been seen in Macaques inoculated with the BSE agent. The pattern in which sugar groups bind to the proteins differs distinctly for PrP^rΘS (an infection-specific protease-resistant protein-see below) from nvCJD-infected humans and from BSE-infected cattle, on the one hand, versus PrP^res associated with sporadic human CJD, on the other hand. The ability of the BSE agent to infect humans suggests that a transformed BSE agent (the nvCJD strain) has acquired increased virulence, which likely evolved from the practice of feeding sheep offal to cattle, and from feeding offal from diseased cattle to other cattle (leading to the possibility of serial passage). This possibility is consistent with observations that experimental, serial passage of the CJD agent in hamsters reduces the incubation period from 467 to 216.5 days. A virulent CJD agent strain has emerged that poses a serous threat of a future epidemic in humans, through the blood supply or otherwise. B. Transmission and Pathogenesis of the Disease

[0009] CJD was first recognized as a TSE in 1959, when the pathology of scrapie was compared with that of CJD and kuru (a fatal degenerative brain disease among the Fore people of eastern New Guinea). Both kuru and CJD were subsequently passaged to chimpanzees. Because the Fore people practiced funerary cannibalism, and because incidence of the disease declined dramatically once this practice had been stopped, kuru was presumed to be caused by oral transmission, particularly the consumption of the brain. Subsequently, kuru was transmitted experimentally to nonhuman primates via the oral route. Further support for the oral route as a significant portal of entry for the TSE agent(s) was found in the persistence of scrapie infectivity in lymphoid tissues of sheep along the Gl tract, including the tonsils.

[0010] The reticuloendothelial system is involved in the pathogenesis of

TSEs. Following experimental inoculation of scrapie into rodents, the agent has been reported to replicate in the spleen, and to show a hematogenous phase before eventually localizing in the brain. Pathology was only evident in brain tissue, although a depletion of B lymphocytes in the spleen has also been observed. Tissues from scrapie- or CJD-infected animals generally show no evidence of a gross inflammatory reaction. However, in mouse scrapie-infected brains, long before the onset of clinical symptoms, there is significant microglial proliferation and T-lymphocyte recruitment. The immune system is itself apparently involved in the pathogenesis of scrapie infection; it is surprisingly difficult to induce scrapie infection in severe combined immunodeficient (SCID) mice.

C. Nature of the Agent

[0011] A few of the biological and physical properties of the TSE agent are known from TSE transmission experiments in rodents. The transmissible agents of scrapie and nvCJD are relatively large, the size of a medium-sized virus (> ~35 nanometers). On sucrose gradients, TSE infectivity sediments primarily within the microsomal fraction. The TSE agents show marked resistance to radiation, although not necessarily beyond that of a conventional virus. The scrapie agent retains infectivity following exposure to high temperatures. However, heating to 100⁰C kills 99% of the scrapie agent, and complete sterilization of scrapie-contaminated material can be achieved by steam autoclaving at 132⁰C for 60 minutes, mimicking properties of thermophilic bacteria. Furthermore, only a small sub-population of the agent shows a high level of heat resistance, a property that is not inconsistent with bacterial spore formation. S. mirum and the scrapie agent both show resistance to radiation and heat. Both the scrapie agent and S. mirum survive heating to ~87.5°C with little effect on infectivity, which then gradually declines for both as temperatures increase to 100⁰C and above. The scrapie agent is susceptible to tetracycline, as has been shown by inhibition of infection when tetracycline is administered concurrently with inoculation. Likewise, Spiroplasma have demonstrated resistance to all antibiotics we have tested to date except for tetracycline (unpublished data), further suggesting a correlation between Spiroplasma and the TSE agent.

[0012] It is widely (though not universally) accepted that the TSE agent does not possess a nucleic acid component, based on a single set of experiments that showed the scrapie agent to be resistant to psoralen with UV radiation in doses that would normally be expected to cause irreversible damage to nucleic acids. Some viruses (e.g., polio) and some spore-forming bacteria (e.g., Bacillus subtilis) are resistant to the penetration of psoralen and subsequently are far less susceptible to UV radiation. Indeed, the biological properties of the transmissible TSE agent, such as exponential replication, strain variation, mutation, and long or evasive persistence in the host are consistent with the possibility that it possesses its own genome. In fact, TSE infectivity has never been demonstrated in preparations that are scrupulously devoid of nucleic acids. (Laura Manuelidis, Yale University, personal communication) Despite years of effort, the prion protein alone has not been shown to fulfill Koch's postulates, the "gold standard" for showing cause and effect for infectious disease. D. Search for a Marker of the Agent

[0013] The apparent lack of a nucleic acid component in the transmissible agent led to the widely-accepted hypothesis that an infection-associated protein or "prion" is the causal agent of TSEs. A protease-resistant, low-molecular-weight protein, p_rp^res _ancj ^g p_roc|_uct _of jt_s limited proteolysis, PrP 27-30, or prion, can be seen in Western blots of TSE brain homogenates following detergent extraction. PrP^res is produced from PrP^c, which is a normally occurring, protease-sensitive host isoform that is encoded by a chromosomal gene. PrP^c and PrP^r8S both have molecular weights of 33 to 35 kDa. PrP⁰ can be transformed to PrP^res by a post-translational process occurring in the Golgi. PrP^res then accumulates intracellular^ in secondary lysosomes. The transition from PrP^c to prion involves significant conformational changes, including the acquisition of increased β-sheet structure.

[0014] Some association certainly exists between prions and TSEs.

Knockout mice lacking a functional PrP gene otherwise appear normal. When these knockout mice are inoculated with scrapie, they do not develop clinical or neuropathological features of scrapie, nor do they propagate prions.

[0015] However, there are substantial data to suggest that PrP itself is insufficient to transmit CJD or other TSEs. Infectivity has never been unequivocally demonstrated with purified, recombinant, or transgenic PrP protein, i.e., protein that is scrupulously free of nucleic acids.

[0016] Amphotericin B treatment of hamsters infected with scrapie significantly delays buildup of PrP^r8S, but does not inhibit the increase in infectivity, a finding that appears to dissociate the prion protein from infectivity. Also, the absence of prion accumulation in the brains of over 55% of mice inoculated with the BSE agent suggests that PrP^res is actually a product of the infection, a consequence of a TSE, but is not itself the causal agent.

[0017] S. Sakaguchi et al., "Kinetics of infectivity are dissociated from PrP accumulation in salivary glands of Creutzfeldt-Jakob disease agent-inoculated mice," J. Gen. Virol., vol. 74, pp. 2117-2123 (1993) reported that the salivary glands of mice inoculated with scrapie were infectious, and yet did not appear to contain prion deposits.

[0018] C. Mathiason et ai, "Infectious prions in the saliva and blood of deer with chronic wasting disease," Science, vol. 314, no. 5796, pp. 133-136 (2006) reported finding infectious prions in saliva and blood from CWD-affected cervids. Although the authors reported finding "infectious prions" in saliva, the data presented only showed infectivity in saliva; no evidence was presented to show that prion protein was actually present in saliva.

[0019] One possibility is that the prion may be a receptor onto which the

TSE infectious agent binds. It is interesting to note that a strain of Brucella spp. uses the normal prion on the cell surface as a receptor protein to initiate infection, and that removal of the normal prion will prevent Brucella abortus infection.

E. Spiroplasma as the Cause of CJD and other TSEs

[0020] Prior research by us and our colleagues has pointed to a

Spiroplasma as the most likely candidate for the causal agent of CJD and other TSEs. Spiroplasma have a helical morphology and are motile. Spiroplasma are present in the hemolymph of most insects, and they abound in the salivary glands of vector insects that transmit plant diseases. Spiroplasma contain both DNA and RNA, and possess all the machinery of protein synthesis. Their genome size is ~10⁹ Dalton. Spiroplasma are fastidious, and for in vitro culture require enriched medium with exogenous cholesterol, and high osmolality. Many, perhaps most Spiroplasma that have been observed microscopically have not been successfully cultured. Nevertheless, at least 26 serologically distinct groups of Spiroplasma have been successfully grown in vitro. Different strains of Spiroplasma show considerable diversity. For example, some strains show restricted growth over a small temperature range, while others will grow at temperatures from ~5°C to -45⁰C. Spiroplasma typically produce poorly-defined, inverted, umbilicated colonies on solid agar. They show vigorous whirling and flexing movements in liquid culture, and a helical morphology during at least a portion of their growth cycle in liquid or solid medium. Our research has consistently shown that Spiroplasma infection is associated with TSE-affected brains, while it is absent from control brain tissues. Despite the work that has been previously published by us and our colleagues, the role of Spiroplasma in TSEs is not widely accepted. The consensus in the scientific community continues to be that prion protein alone is the infectious agent in TSEs. See F. Bastian, "Spiroplasma as a candidate agent for the transmissible spongiform encephalopathies," J. Neuropathol. Exp. Neurol., vol. 64, pp. 833-838 (2005).

[0021] Our published work to date, in support of our hypothesis that

Spiroplasma is the causal agent for TSEs, is consistent with Koch's postulates. Two gaps remain before Koch's postulates will be completely satisfied for this hypothesis. We expect that further research will successfully fill the two remaining gaps: Koch's Postulate 1. Established. Spiroplasma is found in all organisms suffering from TSEs. Spiroplasma spp. related to S. mirum have been isolated from ruminant brains affected with scrapie, from cervids affected with chronic wasting disease (CWD), and from human brains affected with Creutzfeldt-Jakob disease (CJD) via passage in embryonated eggs. Additionally, PCR evidence has confirmed the presence of Spiroplasma 16S rDNA in the brains of sheep affected with scrapie, and in the brains of deer affected with CWD. See Bastian et al. (2004). Koch's Postulate 2. Established. Spiroplasma have been isolated from diseased organisms and grown in pure culture. We have isolated and cultured Spiroplasma from CJD, scrapie, and CWD. See Bastian et al. (2007). Koch's Postulate 3. Established-in-Part. The cultured Spiroplasma induce TSE-like illness when inoculated into previously-healthy ruminants. We say "TSE-like" because the symptoms of disease are generally quite similar to those of naturally-occurring TSEs, except that we have not to date recovered prion protein from the inoculated animals. Recovery of prion protein from Spiroplasma- inoculated animals is the first of the two gaps mentioned above in satisfying the four Koch's Postulates. However, neither is prion protein universally present in naturally- occurring cases of TSE. Further, it may be the case that serial passage of the organism may cause the organism to adapt to the new host, and that prion protein may then be produced after several passages. Further research is needed on this point. Koch's Postulate 4. To Be Demonstrated. Spiroplasma must be re-isolated from the inoculated, diseased experimental hosts in #3, and identified as Spiroplasma. We have not yet made this confirmation; this is not due to any experimental difficulties, but is simply an acknowledgment that this work has not yet been done. We expect that confirmation of Postulate 4 will be straightforward when this work is performed in the future. This is the second of the two gaps mentioned previously.

[0022] Some publications have been strongly critical of our hypothesis that

Spiroplasma is the causal agent for TSEs. See, e.g., I. Alexeeva et a/., "Absence of Spiroplasma or other bacterial 16S rRNA genes in brain tissue of hamsters with scrapie," J. Clin. Microbiol., vol. 44, pp. 91-97 (2006).

[0023] Spiroplasma have not been studied extensively by molecular methods. Their 16S rDNA is distinct from that of other Mollicutes. The Spiroplasma genetic information is present on a circular chromosome. There is a wide range in Spiroplasma genome size, covering at least the range 780 to 2200 kbp. The Spiroplasma genome is AT-rich, with a GC content typically ~ 26% to 32%. Spiroplasma are characterized by several unique proteins, including spiralin, adhesin, and the 59 kDa fibril protein. The S. mirum fibril protein is morphologically identical with scrapie-associated fibrils. Hyperimmune rabbit serum against scrapie infection-specific proteins cross-reacts with Spiroplasma fibril proteins.

[0024] Among the many species of Spiroplasma, only one, Spiroplasma mirum, classified in serological group V, is known to flourish at mammalian body temperatures. The species S. mirum has several strains, including the suckling mouse cataract agent (SMCA), and the GT-48 strain. These two strains of S. mirum were originally isolated from rabbit ticks. They have been reported to experimentally induce persistent brain infection in mice and rats. The GT-48 strain has been reported to experimentally induce neurological deterioration and spongiform encephalopathy in suckling rats. The GT-48 strain localizes in the brain following subcutaneous or intramuscular inoculation in the rat model, demonstrated its neurotropic character. [0025] H. Kirchhoff et al., "Pathogenicity of Spiroplasma sp. strain SMCA in Syrian hamsters: clinical, microbiological, and histological aspects," Infection & Immunity, vol. 31 (1 ), pp. 445-52 (1981 ) reported experimental data in which the intracerebral inoculation of newborn Syrian hamsters with pure cultures of Spiroplasma sp. strain SMCA caused severe, prolonged disease involving the central nervous system, culminating in death. The disease was characterized by spasms, muscular tremors, disturbances in motor control, inability to feed, dramatic loss of weight, and runting. The effect was dose-related, with the larger numbers of viable Spiroplasma producing a higher incidence of disease and death in shorter times. Severe hemorrhaging developed throughout the brain, liver, and spleen, and Spiroplasma were readily recovered from each of these organs, indicating that the agent disseminated from the initial site of infection to distant host tissues. Newborn animals were susceptible, while adults were resistant; findings similar to those that have been reported for newborn mice and rats. Unlike mice and rats, hamsters did not develop cataracts visible to the unaided eye. The histopathological features of eye disease in hamsters differed from those in rats, and were characterized by microophthalmia (especially in runted hamsters), and abnormal proliferation, disorientation, and disorganization of corneal, lens, and retinal tissues.

[0026] R. Friedlaender et al., Ocular pathology induced by the suckling mouse cataract agent," Investigative Ophthalmology, vol. 15, pp. 640-647 (1976) describes eye disease induced by SMCA in rats. The authors suggested that the main pathological process was inflammation in the posterior eye tissues including the retina, and that pathology observed in the lens could be a consequence of primary inflammation occurring elsewhere in the eye.

[0027] J. TuIIy et al., "Pathogenic mycoplasmas: Cultivation and vertebrate pathogenicity of a new Spiroplasma," Science, vol. 195, pp. 892-894 (1977) reported experimental evidence suggesting that a Spiroplasma was the pathogen in so- called suckling mouse cataract agent (SMCA). Egg-passaged SMCA was reported as characteristically inducing cataracts, uveitis, and chronic brain infection in suckling mice and rats following intracerebral challenge at an early age, usually less than 96 hours after birth. Approximately 50-60 percent of mice or rats receiving undiluted SMCA cultures died within 5 to 10 days of challenge. Nearly all rats that survived challenge exhibited bilateral cataracts; however, surviving mice did not develop cataracts. The cultured GT-48 strain was also highly lethal to suckling rats, but produced no cataracts in survivors challenged with diluted cultures. Following autopsy, Spiroplasma were recovered from cultures from brain and eye tissue pools of infected rats, but not from control tissues. Isolates recovered from the eye and brain were found to be serologically indistinguishable from SMCA.

[0028] B. Lorenz etal., "First evidence of an endogenous Spiroplasma sp. infection in humans manifesting as unilateral cataract associated with anterior uveitis in a premature baby," Graefe's Arch. Clin. Exp. Ophthalmol., vol. 240 (5), pp. 348-353 (2002) describes a 4-month premature baby having a progressive cataract associated with anterior uveitis. PCR detected Spiroplasma. Spiroplasma were seen in the lens fibers by transmission electron microscopy. However, serological testing and microbial cultures of the vitreous and the lens were negative.

[0029] R. Zeigel et a/., "Electron microscopy of the suckling mouse cataract agent: a noncultivatable animal pathogen possibly related to Mycoplasma," Infection and Immunity, vol. 9, pp. 430-443 (1974) described suckling mouse cataract agent as a noncultivable agent that grows to high titer in the brains and eyes of intracerebral^ inoculated mice, in which it induced cataract, uveitis, and chronic brain infection. Mycoplasma-like entities were observed in the retina. See also E. Olmsted et al., "Ocular lesions induced in C57 mice by the suckling mouse cataract agent (SMCA)," Investigative Ophthalmology and Visual Science, vol. 5, pp. 413-420 (1966).

[0030] In mice inoculated with SMCA, there is a prominent microcytic encephalitis, localized to the subpial and subependymal zones and deep gray matter. A mild mononuclear cell infiltrate may be present. There is prominent proliferation of astrocytes in some regions. Hydrocephalus has been observed in ~36% of rats inoculated with SMCA. Hydrocephalus has also been seen in experimental CJD infection in hamsters, and in experimentally-induced scrapie in mice. Similar spongiform encephalopathy in suckling rats inoculated with the GT-48 strain has also been reported. The GT-48 Spiroplasma has also been shown to be neurotropic. Other Spiroplasma strains have also been shown to be persistent and pathogenic in mice.

[0031] Spiroplasma spp. related to S. mirum have been isolated from ruminant brains affected with scrapie, cervids affected with chronic wasting disease (CWD), and from human brains affected with Creutzfeldt-Jakob disease (CJD) via passage in embryonated eggs.

[0032] Following intracranial inoculation into deer, sheep, and goats, the

SMCA strain induced neuropathology remarkably similar to that of naturally occurring TSEs. Neurological deterioration occurred in the SMCA-infected deer that was essentially identical to that seen in naturally-occurring CWD in cervids. Following intracranial inoculation into suckling rats, the GT-48 strain induced neurological deterioration and spongiform encephalopathy similar to that seen in the TSEs.

[0033] Spiroplasma spp. related to S. mirum have been identified in brain tissues of all CJD-, scrapie-, and CWD-affected brains that we and our colleagues have tested to date, as documented by one or more of the following techniques in each case:

PCR, DNA sequence analysis, electron microscopy, Southern blot, and immunohistochemistry. Antibodies directed against scrapie-associated fibrils (SAF), a consistent morphological marker for TSE, react with Spiroplasma fibril antigens.

Isolation of Spiroplasma from various animal species (including controls) were reported in Bastian et al. (2007) as follows:

Normal sheep brain 0/3

Normal deer brain 0/3

Normal human brain 0/3

CJD human brain 4/4

Scrapie sheep brain 2/2

CWD Deer brain 1/1 Electron microscopy and immunocytochemistry may be used to screen for Spiroplasma. We expect that for routine testing, monoclonal antibodies will become preferred, but to date the most reliable method employed has been passage in embryonated eggs. In embryonated eggs 100% of TSE brains and 0% of control brains tested to date have been positive for Spiroplasma. Spiroplasma have also been consistently isolated from scrapie sheep eyes by direct inoculation into M1 D media, followed by dark-field microscopy, or PCR/DNA sequence analysis. Negative stain EM is also expected to be useful, but has not yet been tested.

[0034] The prevailing means currently used for diagnosing TSE is screening for the presence of abnormally-folded, protease-resistant prion protein in brain tissues from suspect cases; or screening for spongiform abnormalities in the brain. These methods can normally only be used post-mortem, or in some cases with highly invasive brain biopsies. Furthermore, the prion protein has been reported to be absent in -10% of TSE cases. The misfolded prion protein may be a byproduct of spiroplasmosis. The misfolded prion protein byproduct may itself be highly neurotoxic, without being infectious. There is evidence that some prion proteins are not protease- resistant, and they would therefore would not be detected by standard methods currently in use. There is also some evidence suggesting that a significant fraction of human patients who have been diagnosed with Alzheimer's Disease, the most common form of progessive dementia, may instead have CJD or nvCJD. There is an unfilled need for a reliable method to differentiate CJD from Lewy body disease, which presents clinically with a similar pattern of rapidly progressing dementia. The most widely used current technique for the detection of prions in humans and other mammals, immunohistochemistry, lacks sensitivity and objectivity. One test that has been used for diagnosis of CJD is the 14-3-3 spinal fluid test; but this test only detects -55% of CJD cases, and is prone to both false positives and false negatives. Also, the CSF test becomes positive just prior to onset of clinical disease, and is therefore of limited practical value. Similarly, the prion itself is not well-suited for development of a preclinical test for TSE, since it is a modified host protein that does not usually generate any antibody response. [0035] The clinical diagnosis of CJD can be problematic, in that other degenerative brain processes may present with similar clinical histories or physical findings. Lewy body dementia is an example of an unrelated degenerative brain disease that is often confused with CJD. CJD often presents with psychiatric manifestations suggestive of psychotic mental illness; and such patients are often interred in a psychiatric institution until manifestations of organic brain disease are recognized. Brain tumors such as gliomas or lymphomas may present a similar diagnostic problem, especially when they are not readily diagnosed from brain scans. Also, physicians can be reluctant to order a brain biopsy or necropsy on such patients because of fear of the unknown nature of the transmissible agent of TSE. For these and other reasons, most diagnoses of CJD are based on detection of the 14-3-3 proteins in cerebrospinal fluid (CSF). However, that test has low reliability. Currently, the only other tests available for CJD are invasive tests involving brain biopsy or tonsil biopsy; or necropsy. Tissue diagnoses rely on the presence of spongiform degeneration of the brain. Prion can be recognized by Western blot, or by immunohistochemistry using monoclonal antibodies, but prion is not always detected in all TSE cases. There has been much effort to expand detection of prion to a non-invasive pre-clinical test for TSE. However, prion has not been reported in peripheral blood, although blood on occasion can be infectious. Prion is not reliably found in urine or CSF. Prion is found in tonsil but requires an invasive procedure. Tonsil biopsy has only been used for diagnosis in non-human animals. CJD in humans is instead diagnosed by brain biopsy, looking not only for prion but also for spongiform changes in the neuropil. Brain biopsy may be done through a burr hole under local anesthesia and simple sedation. Brain biopsy in the frontal lobe does not ordinarily cause any neurological deficit.

[0036] There is an unfilled need for simpler, more reliable, and more consistent techniques to test for TSEs. There is an unfilled need for simpler, more reliable, and more consistent techniques that may easily be administered to living patients to test for TSEs. There is especially an unfilled need for simpler, more reliable, and more consistent techniques to test for TSEs before clinical symptoms have developed. F. Significance

[0037] Definitive diagnosis of CJD and nvCJD in humans, and other TSEs in other animals can presently only be made by histologic examination of brains or PrP determinations of infected brain tissues, and typically only at necropsy

[0038] F. Bastian etal., "Spiroplasma spp. from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants," J. Med. Microbiol., vol. 56, pp. 1235-1242 (2007) report experimental data supporting the association of Spiroplasma with TSEs in ruminant animal models, and questioning the validity of the conclusion that misfolded prion protein is the sole cause of TSEs. See also F. Bastian etal., "Neuropathology of Spiroplasma Infection in the Rat Brain," AJP, vol. 114, pp. 496-514 (1984).

[0039] U.S. Patent No. 6,033,858 discloses Sp/rop/asma-specific

16SrDNA, as well as PCR oligonucleotide primers useful in assaying samples for Spiroplasma and for diagnosing TSEs. Modified versions of these primers have been used to identify S. mirum in tissues with Southern blotting; see F. Bastian et al., "Linking chronic wasting disease to scrapie by comparison of Spiroplasma mirum ribosomal DNA sequences," Exper. Molec. Pathol., vol. 77, pp. 49-56 (2004).

[0040] H. Kirchhoff et al., "Pathogenicity of Spiroplasma sp. strain SMCA in rabbits: clinical, microbiological, and histological aspects," Infection & Immunity, vol. 33(1 ), pp.292-296 (1981 ) reported that newborn rabbits inoculated intracerebral^ with Spiroplasma strain SMCA either died or developed eye disease. SMCA could be re- isolated from brain, liver, and eyes (although the authors did not specify from which portions of any of these organs - brain, liver, eyes - the organism was re-isolated). Neither eye disease nor death was induced by subcutaneous injection in newborn rabbits. In adult rabbits, no disease occurred after intravenous or subcutaneous injection or after inoculation into the conjunctival sac. [0041] U.S. Patent Application Publication No. 2004/0175775 hypothesized that the scrapie-specific isoform of prion protein (PrP^Sc) might be present in the eye fluids (aqueous and vitreous humors), but provided no evidence in support of that hypothesis. The disclosure described an immunological method that might be used in detecting PrP^Sc in eye fluids. The immunological method was demonstrated using an artificial mixture containing human eye fluid obtained during ophthalmologic procedures, spiked with small quantities of brain tissue taken from a scrapie-infected hamster. However, no data were given to show whether PrP^Sc was present in the eye fluid of any actual TSE subjects, either human or animal. The disclosure also reports prior work by others that PrP^Sc had been detected in the retina of cattle with BSE, and pre-clinically in nictitating membrane lymphoid tissue from sheep.

[0042] By contrast, experimental data were reported in J. Wadsworth et al., "Tissue distribution of protease resistant prior protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay," The Lancet, vol. 358, pp. 171- 180. Among the findings of Wadsworth et al. were the following: "Eight component parts of the eye obtained from single neuropathologically confirmed cases of vCJD and sporadic CJD were analysed: proximal optic nerve; sclera; retina; vitreous humour; lens; aqueous humour; iris and cornea. PrP^Sc was readily detected in proximal optic nerve and retina (figure 7) but was undetectable in all other components from vCJD eye (table 2)." (p. 177) . . . "Of particular concern at present is the degree of deposition of PrP^Sc in the eyes of patients with vCJD and the possibility of iatrogenic transmission through ophthalmic surgical instruments. Experimental inoculation into the eye is recognised as an efficient route of disease transmission in rodent models and in classical CJD infectivity in the eye has been shown through transmission of disease to non-human primates after inoculation with retina, vitreous, lens, and cornea. The finding that the posterior segment of vCJD eye contains a high concentration of PrP^Sc suggests that ophthalmic surgical instruments used in procedures involving the retina might represent a potential risk for iatrogenic transmission of vCJD. Similarly, there may be risks associated with section of the optic nerve during enucleation and exenteration procedures. The optic nerve is also divided when the eye is removed for the purposes of corneal grafting. Most eye operations, however, involve anterior segment structures. PrP^Sc has not been identified in the components of the anterior eye using these methods. "Tonsil is the tissue of choice for diagnostic biopsy . . . ." (p. 171 )

[0043] International patent application publication WO/2005/115483 discloses the use of antibodies in serum samples that react with recombinant Spiroplasma-spectilc HspδO (heat shock protein 60) as a means for diagnosing spiroplasmosis and TSEs.

DISCLOSURE OF INVENTION

[0044] We have discovered a new method for detecting spiroplasmosis and transmissible spongiform encephalopathies, including Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy, scrapie, and other TSEs. We have discovered that Spiroplasma are present in substantial numbers in the vitreous humor, the aqueous humor, corneal endothelium, and other locations where Spiroplasma have not previously been reported. Samples taken from the vitreous humor, the aqueous humor, or tissues of the cornea can reveal the presence of Spiroplasma well before clinical symptoms arise, and can confirm a diagnosis once clinical symptoms are present. The novel method is only moderately invasive, and is readily suited for use in living human patients. The method may also be used in other animals susceptible to TSEs, such as cattle and sheep. In livestock the method may be used for diagnosis of live animals. After slaughter, the method may be used for the convenient and rapid identification of Spiroplasma or TSE in carcasses (either of individual animals, or pooled samples taken from many animals).

[0045] For example, fluid taken from the vitreous humor of eyes from

SMCA-infected deer and sheep has revealed the presence of extracellular Spiroplasma. Extracellular spiroplasma has also been seen in immunostained, vacuolated corneal endothelia from SMCA-infected animals. Indeed, Sp/rop/asma-infected corneal endothelia may be the explanation for previously-reported iatrogenic transmission of CJD via corneal transplants. Extracellular organisms within the vitreous humor may account for contamination of instruments secondary to ophthalmic surgical procedures involving the eye. We have also isolated Spiroplasma from vitreous humor and from cornea removed from scrapie-affected eyes, by direct isolation into M1 D media or in tissue cultures.

MODES FOR CARRYING OUT THE INVENTION

[0046] As used in the specification and claims, unless context clearly indicates otherwise, the term "transmissible spongiform encephalopathy" (or TSE) should be understood to include all forms of TSE recognized in the art (or later discovered). Creutzfeldt-Jakob disease (CJD) and new variant Creutzfeldt-Jakob disease (nvCJD) are the primary human TSEs. Other forms of human TSEs include kuru, fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome. TSEs in other species include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, transmissible mink encephalopathy (TME), and chronic wasting disease in deer and elk. Any mammal susceptible to spiroplasmosis or to a TSE may be diagnosed according to the method of the present invention, including for example humans, cattle, sheep, goats, deer, and elk. If familial (genetically-linked) TSEs also implicate Spiroplasma infection (e.g., vertical transmission of the pathogen, or genetically enhanced susceptibility to the pathogen), then the present invention may be used for detecting them; but if no Spiroplasma infection is implicated in familial forms, then the invention might find little or no applicability for those TSEs that are directly inherited.

[0047] Spiroplasma may be detected in samples taken from the eye, including for example the corneal endothelia, the aqueous humor, and the vitreous humor, by any of several methods known in the art (or to be discovered), including immunological detection of Spiroplasma antigens or antibodies (including, e.g., immune histochemistry by light microscopy or electron microscopy using enzyme-linked or particle-labeled antibodies or antigens), PCR, ELISA or other enzymatic methods, fluorescent microscopy, light microscopy including dark field microscopy, culture assay in medium or in tissue culture, Western blots, dot blots, and magnetic beads. Specular microscopy might be used to detect spiroplasmosis in the corneal endothelia. Samples may, for example, be inoculated in cell-free growth media; or into cell cultures that support Spiroplasma growth, such as rabbit lens cell cultures, neuronal cell cultures, or glial cell cultures; or injected into embryonated eggs. Vitreous or aqueous humor samples may also be studied directly by fluorescent microscopy of aliquots stained using DNA fluorescent dyes known to the art; however, the specificity of such an assay will be relatively low. A preferred method is PCR detection of Sp/rop/asma-specific 16S rDNA, as described for example in U.S. Patent No.6,033,858; and modified as reported in F. Bastian et al., "Linking chronic wasting disease to scrapie by comparison of Spiroplasma mirum ribosomal DNA sequences," Exper. Molec. Pathol., vol. 77, pp.49- 56 (2004).

[0048] In alternative embodiments, samples of saliva, tears, iris, ciliary body, lens, or cataract may also be tested for Spiroplasma; preliminary results suggest that organisms may be present in these locations in spiroplasmosis cases, although typically in smaller concentrations than in the vitreous humor. For example, cataracts may be tested in elderly patients following cataract surgery; and small amounts of iris are sometimes removed during glaucoma surgery.

[0049] Although detection by actual isolation of a pathogen is usually considered the gold standard for diagnosis, the detection of antigens or the detection of specific sequences of Spiroplasma DNA, for example by PCR₁ will often be faster, less expensive, and more convenient. Alternatively, a sample may be inoculated directly into special culture media of high osmolality (e.g., M1 D or Sp-4). Spiroplasma growth in the media will result in increased acidity; and spiral organisms may be seen by dark-field microscopy after a -2-3 week incubation. Similarly, inoculation of tissue cultures (e.g., corneal endothelia, astrocytic, or neuroblastoma tissues cultures) results in growth of Spiroplasma both within the cells and in the culture media. It is interesting that Spiroplasma will not grow in MEM culture media alone, but that they readily proliferate in MEM media that has been exposed to tissue culture cells. See F. Megraud, "Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture," Infect. Immun., vol. 42(3), pp. 1168-1175 (1983).

[0050] Detection of spiroplasma in the eye of a ruminant would suggest the presence of TSE, and would lead to culling of the affected animals from the flock or herd. Post-mortem detection of spiroplasma in eyes from meat packing plants would be a simple means for mass-screening for TSE cases before meat is distributed.

[0051] In humans, screening of eyes (especially corneal endothelia, vitreous humor, or aqueous humor) for Spiroplasma will be an important diagnostic for CJD, and should be effective either before or after clinical symptoms of CJD have developed. A preferred diagnostic technique, one that may be used either before or after the appearance of clinical symptoms, is to draw a sample from the aqueous humor, and to test it for the presence of Sp/rap/as/na-specific antigen or antibody, by any of the various immunological detection techniques that are known in the art or that may later be developed. It is noteworthy that antibody levels in the aqueous humor in other types of bacterial eye infections have been observed to be roughly double that observed in serum from the same patient.

[0052] Because the vitreous and aqueous humors are located in a

"privileged" location, a positive test for Spiroplasma in the vitreous or aqueous humor, by any method, should have a low rate of false positives with a low background level of other bacteria; whereas serology could be more susceptible to false positives, resulting from prior exposure - in the gut or otherwise. Confirmatory tests, where desired, may always be performed with an alternative assay, such as a Western blot or PCR.

[0053] In experimentally-induced spiroplasmosis, Spiroplasma are difficult to detect in the CNS. However, we have discovered that Spiroplasma migrate from brain tissue, presumably via the optic nerve to the retina, and thence to the vitreous humor, where they aggregate in large numbers. We initially detected the presence of Spiroplasma in the vitreous humor, aqueous humor, and corneal endothelia by immunohistochemistry of fixed, embedded and sectioned eyes from experimentally infected animals using hyperimmune rabbit sera specific for S. mirum. For isolation studies we have shown that vitreous or aqueous humor can be removed from the eye ex vivo using a large-gauge needle (e.g., a 16 gauge) and then assayed for presence of Spiroplasma. For in vivo evaluations, a smaller needle (e.g., 25 gauge) is preferred. A small quantity of aqueous humor may be aspirated in vivo by techniques known in the art, e.g., by inserting the needle into the anterior chamber of the eye near the limbus.

EXAMPLE 1 Discovery of a Spiroplasma-L\ke Organelle" in Infected Brain Samples (Work Previously Reported)

[0054] We first became interested in exploring a more conventional microbial etiology for CJD upon discovering a peculiar spiral-shaped "organelle" in neuronal cell processes in a CJD brain biopsy. These structures have not been found in normal brain tissue samples. This observation was later confirmed, both by us and by others. The morphology of the organelle was reminiscent of Spiroplasma. Spiroplasma lies within the apparent size range of the transmissible agent (>35 nm by filtration studies). Small tubular forms of Spiroplasma <40 nm have been seen by transmission electron microscopy in rat brains infected with GT-48 spiroplasma and in culture pellets of S. mirum at all stages of the log phase growth cycle. These small forms may represent the more resistant elements of Spiroplasma cultures, analogous to spores in other bacteria. Identical inclusions have been reported in scrapie-infected mouse brains, and in brains from animals and humans affected with TSEs. We believe that these inclusions are pleomorphic structures associated with Spiroplasma.

EXAMPLE 2 Spiroplasma Causes CJD-Like Pathology in Rats (Work Previously Reported)

[0055] Initial tests of our Sp/rop/asma-degenerative brain disorder hypothesis used a rat model inoculated with the Spiroplasma mirum strain GT-48. We found that ~30 to ~300 organisms injected intracranial^ into suckling rats multiplied to a cell density of ~10⁶ CFUs per gram of tissue within two weeks of infection. Although the incubation period for experimental Spiroplasma infection was substantially shorter than that for experimental TSE infection, the microcytic changes in the rat brain closely resembled the spongiform alterations seen in TSE. Electron micrographs of the Spiroplasma-\ nfected brain tissues showed pathology essentially identical to that seen in CJD. Vacuolization without inflammation was seen in long-standing disease (8 weeks in this study). We found that Spiroplasma localized to neurons and gray matter, as determined by immunocytochemistry. Spiroplasma were clearly evident as spiral- shaped organisms in the diseased tissue at two weeks, but had become less apparent at eight weeks. However, even at eight weeks we were able to cultivate GT-48 Spiroplasma from the brains of the infected animals.

EXAMPLE 3 Scrapie-Associated Fibrils are Morphologically Identical to Spiroplasma Fibril Proteins (Work Previously Reported)

[0056] A characteristic, diagnostic feature of both CJD and scrapie is the presence of fibrils in homogenized/protease-treated brain tissue. These fibrils are composed of two or four, helically-wound, 4 nm subfilaments, with straight segments 200 nm long, and a 10 nm periodicity. These structures, called scrapie-associated fibrils (SAF), are distinctive from other twisted amyloid fibrils. Even though the fibrils are protease-resistant, they are not composed of PrP. The term SAF has sometimes been inappropriately used as if it were a synonym for prion rods. Because SAF accumulates in tissues in proportion to infectivity, it is a candidate for the infectious agent, or at least a candidate for being a part of the infectious agent. Several investigators, including us, have identified fibrils in Spiroplasma that appear morphologically identical to SAF. We have demonstrated that Spiroplasma fibrils are protease-resistant. Protease-resistant proteins have also been reported from other Mycoplasmas.

EXAMPLE 4 Protease-Resistant Spiroplasma Proteins Cross-React With Anti-Scrapie Antibody (Work Previously Reported)

[0057] Additional evidence connecting Spiroplasma to prion diseases came from the use of anti-scrapie antibody to probe Spiroplasma mirum proteins by Western blot analysis. Polyclonal antiserum was raised in rabbits against protease- resistant proteins (including prion proteins) from the brains of mice that had been inoculated with scrapie strain ME7(courtesy of Richard Rubenstein). The antiserum was tested against a protease-resistant extract of Spiroplasma mirum that had been cultured in vitro. Four Spiroplasma protein bands reacted with the antiserum. An explanation that is consistent with these results is that Spiroplasma antigens are present in scrapie tissues.

EXAMPLE 5 CJD Brain Extracts Contain Spiroplasma mirum 16S Ribosomal RNA Gene Sequences (Work Previously Reported)

[0058] A recent and more compelling piece of evidence supporting a

Spiroplasma etiology for CJD was the discovery, through PCR and Southern blot analyses, of S. mirum 16S ribosomal RNA gene sequences in TSE-affected brain samples from humans (CJD), sheep (scrapie), and deer (CWD). For this study we designed oligonucleotides suitable for PCR analysis from conserved sequences present in Spiroplasma 16S rRNA genes. These primers amplify a 270 bp product from Spiroplasma DNA preparations. See generally U.S. Patent No.6,033,858; and Bastian et al. (2004), supra.

EXAMPLE 6 Sp/rop/asma-induced Astrogliosis in Ruminants

[0059] In ruminants with experimentally-induced spiroplasmosis, we have demonstrated astrogliosis and retinopathy. Both are hallmarks of neuropathology in naturally-occurring TSEs. The topography of the astrogliosis and neuronal lesions in the Spiroplasma-\ nfected ruminant brains was identical to that in scrapie-affected sheep and mouse brains. Spiroplasma infection in astrocytic tissue cultures induced glial fiber formation, a finding that indicated that astrogliosis in the ruminant model was a direct result of the bacterial infection.

[0060] We focused initially on a triad of lesions that, collectively, are specific for TSEs: spongiform degeneration of neuropils, neuronal loss, and astrogliosis. Haematoxylin and eosin staining of Sp/rop/asma-infected ruminant brains revealed neuronal degeneration and spongiform changes in the neuropil associated with neurons, most prominently in the brain stem in the region of the obex, the thalamus, the hippocampus, and the cerebellum. Glial fibrillary acidic protein (GFAP) immuno-staining of the Sp/rop/asma-infected neural tissues showed prominent astrogliosis in the same topographical distribution as has been reported in both naturally-occurring and experimental TSEs. Hypertrophic astrocytes were also seen in the infected brains. By contrast, similar lesions were not seen in controls. Astrogliosis was seen in infected deer 1.5 months after inoculation, relatively early and well before the onset of clinical symptoms. Early onset of astroglial proliferation is typical of both naturally-occurring and experimentally-induced TSEs, occurring before prion deposits are detectable. The astrogliosis that we have seen in both the spiroplasmosis model and in experimental scrapie in mice is so striking that it raises the possibility that astrogliosis might be a toxic host response to the infection.

[0061 ] We observed astrogliosis in the deer brains inoculated with SMCA by GFAP-immunostaining. Astrogliosis was absent from controls. The hippocampi and cerebella of Sp/rop/asma-infected deer showed marked astrocyte proliferation compared to controls. The greatest proliferation of astrocytes was seen in the thalamus of Sp/rop/asma-infected deer brain at 1.5 months. Scattered hypertrophic astrocytes were also present at other sites. The well-developed astrogliosis seen in the deer at 1.5 months indicated the early onset of this process in the spiroplasmosis ruminant model. Much of the glial staining in the cerebellum was in the Purkinje cell layer, which likely represents excitation of Bergmann astrocytes. Glial fiber proliferation in the brain stem and obex were seen most prominently immediately beneath the ependymal surface of the 4th ventricle. Obex from Sp/rop/asma-infected deer at 3.5 months showed subependymal vacuolization, with organisms attached both to the ependymal surface and within vacuoles. The distribution and extent of astrogliosis in the Sp/rop/asma-infected deer brains were remarkably similar to that seen in naturally occurring or experimental scrapie in ruminants. EXAMPLE 7 Spiroplasma Infection of Astrocytes and Glial Fiber Proliferation

[0062] We then investigated whether astrogliosis in the TSE model was due to the bacterium itself, and whether Spiroplasma could infect astrocytes directly. We inoculated a log phase culture of SMCA onto in vitro monolayers of astrocytes (ATCC number CRL-2541 : astrocyte type I clone from mouse cerebellum). At seven days post-inoculation, cytopathology was seen: cell granularity, multinucleation, and enlarged nuclei with minimal vacuolization of the cytoplasm, lmmunohistochemistry staining for GFAP showed glial fibers seven days post-inoculation, suggesting that Spiroplasma itself stimulated glial fiber proliferation.

EXAMPLE 8 lmmunohistochemical Staining of Sp/rop/asma-inoculated Ruminant Brains

[0063] Hyperimmune rabbit serum specific for the SMCA strain of S. mirum was the kind gift of Maureen Morrison (Purdue University). We used the hyperimmune serum for immunohistochemical localization of SMCA in the inoculated ruminant brains. We observed Spiroplasma widely distributed within neurons of the infected brains, with particular prominence in the brain stem in the region of the obex beneath the ependyma of the 4th ventricle. Spiroplasma were seen in the Purkinje cell layer of the cerebellum, and along dendritic processes in the overlying layer. Spiroplasma were also seen scattered through the granular and pyramidal neurons of the hippocampus, and were sparsely present throughout the cerebral cortex. No inflammation was seen in the Sp/rop/asma-infected brain tissues. Spiroplasma were documented by electron microscopy in the obex region beneath the floor of the fourth ventricle of a Spiroplasma-\^' nfected deer showing clinical signs of CWD. EXAMPLE 9 Sp/rop/asma-induced Retinopathy in Ruminants

[0064] We also studied eyes from the ruminant Spiroplasma model. The eyes from deer and sheep that had been inoculated with SMCA displayed retinopathy that was consistent with that seen in both natural and experimentally-induced TSE. Degeneration occurred in the inner retinal layers. The neuronal cell layer was depleted of neurons, and the surviving neurons were vacuolated. The inner nuclear layer of neurons was vacuolated. The outer plexiform layer was depleted, and had merged into the outer nuclear layers of neurons. The layer of photoreceptors was disorganized and atrophied. There was increased glial staining in the neuronal cell layer, with extension into the inner nuclear layer and outer plexiform layer, indicating astrogliosis. No inflammatory response was evident in the retinal tissues. Controls showed no evidence of any retinopathy.

[0065] lmmunohistochemistry showed the presence of Spiroplasma in the neuronal layers of the retina, especially in the neuronal cell layer and inner nuclear layer. We were surprised to observe large numbers of extracellular Spiroplasma organisms in the vitreous humor. Apparently the organisms had escaped the retina into a growth medium (the vitreous humor) rich in sterols and polysaccharides, where they grew extracellularly - almost as if in a culture. Spiroplasma were also seen, although less frequently, in the aqueous humor - presumably because most of the aqueous humor tends to wash out during processing for tissue sections. Tissue sectioning is not the preferred method to assay the aqueous humor. The presence of organisms free in the aqueous humor suggests that there is a substantial concentration of Spiroplasma organisms, antigen, and perhaps also Sp/rop/asma-specific antibodies there. These Spiroplasma organisms, antigens, and antibodies will be detected in fluid taken from the aqueous humor by immunological techniques otherwise known in the art. The endothelia of the cornea were vacuolated, with Spiroplasma present intracellular^ and attached to the surface. Spiroplasma were not seen in peripheral nerve elements in the limbic border of the cornea and sclera, nor within the epithelia or stroma of the cornea. Notwithstanding the massive infiltration of Spiroplasma into the eye, no inflammation was seen in the eye tissues. This unusual distribution of the organisms was most surprising, and does not correlate with reported distributions of prion protein. Deposits of prion protein have been observed only in the retinal layers, and not in other parts of the eye. In particular, prion protein has not been reported in significant amounts in the vitreous and aqueous humors, nor in the cornea. Despite the absence of prion protein, it has nevertheless been observed that corneal tissues are infectious in both naturally-occurring and experimentally-induced TSEs.

[0066] The distribution of Spiroplasma seen in ourTSE model may explain the mechanism underlying iatrogenic CJD infections following ophthalmic surgery. The transmission of CJD via cadaver corneal transplants has previously been presumed to result from peripheral nerve elements, since the epithelia of the anterior cornea were not shown to be infectious. However, based on our observations, we suggest that corneal infection is related to bacterial infiltration of the corneal endothelia. The posterior portions of the eyes are, nevertheless, substantially more infectious than the anterior portions, including the cornea. The large numbers of Spiroplasma we observed in the vitreous humor in the experimental Spiroplasma TSE ruminant model could explain this phenomenon; we hypothesize that surgical instruments would pass through a Spiroplasma "culture" in the vitreous humor during surgery on CJD patients. Thus our observations of the effect of Spiroplasma on the eye logically explain the iatrogenic spread of CJD, either by corneal transplants or by contamination of surgical instruments.

[0067] These observations provide a novel means to detect spiroplasmosis and TSEs, both clinically and pre-clinically. Samples may be taken from the aqueous humor, vitreous humor, or corneal endothelium and assayed for the presence of Spiroplasma. Neither the presence of Spiroplasma in these locations, nor the testing of samples from these locations to diagnose spiroplasmosis and TSEs, has previously been suggested. EXAMPLE 10

[0068] Neonatal rats were experimentally infected with the GT-48 strain of S. mirum. These rats later exhibited spongiform encephalopathy remarkably similar to the neuropathology of TSE. Clinical signs of disease were seen in the rats in a dose-dependent fashion; and the Spiroplasma organism was recovered in cell-free media from infected rat brains.

EXAMPLE 11

[0069] Intracranial inoculation of 5-day-old deer with Suckling mouse cataract agent (SMCA), a laboratory strain of S. mirum, maintained in cell-free media through several passages, induced clinical symptoms identical to those of CWD in neonatal deer (3/4). Spongiform encephalopathy was seen on necropsy at 4.5 months in all deer inoculated with SMCA (4/4). No evidence of disease or pathology was seen in non-inoculated twins of these animals (0/4).

EXAMPLE 12-13

[0070] Retinopathy characteristic of that for natural or experimentally- induced scrapie was seen in both deer and sheep inoculated with SMCA. Neonatal sheep and goats were inoculated with either SMCA or Spiroplasma spp. isolates, derived from ruminant brains affected with either scrapie or CWD. These animals showed no clinical signs 12 to 16 months post-inoculation; but at necropsy their brains showed widespread pathology. All infected ruminants displayed a triad of neuropathic lesions consistent with TSE: spongiform degeneration of the neuropil, neuronal loss, and marked astrogliosis. EXAMPLE 14

[0071] Using ELISA we have detected circulating antibodies against

Spiroplasma HspβO protein in peripheral blood from more than 95% of Creutzfeldt-Jakob disease patients (30 human patients), with positives in fewer than 5% of age-matched controls (40 human patients).

EXAMPLE 15

[0072] Using ELISA we have detected circulating antibodies against

Spiroplasma Hsp60 in scrapie-affected sheep (8/8), but not in normal sheep (0/40).

EXAMPLES 16-18

[0073] Spiroplasma spp. have been recovered via passage in embryonated eggs from 100% of all forms of TSE we have tested to date: scrapie in sheep (2/2); chronic wasting disease (CWD) in deer (1/1 ); CJD in humans (4/4). The organism was not recovered from normal sheep (0/3), deer (0/2), nor age-matched humans (0/3).

[0074] Examples 19-20 Additional studies in deer and sheep models of spiroplasmosis

Neuropathological studies were carried out on all brain samples from the deer and sheep inoculated with SMCA in Examples 12-13. The deertissues, including sham- inoculated controls from a subsequent study, were examined at 4.5 months. The SMCA-inoculated sheep and sham-inoculated sheep samples were obtained 16 to 19 months after inoculation. In addition, formalin-fixed eyes from these ruminants were sectioned horizontally to include retina, optic disc, fovea, vitreous humor, and the anterior components of the eye, including cornea and lens. All sections were stained with haematoxylin and eosin (H&E). All brain and eye tissues were further examined by immunohistochemistry using antibodies directed against GFAP to determine the presence of astrogliosis. Adjacent tissue sections were also examined by immunohistochemistry using hyperimmune rabbit sera directed against SMCA (courtesy of Dr. M. Morrison, Purdue University), to show the distribution of Spiroplasma in the brain and eye tissues in relation to neuropathological brain lesions and retinopathy.

[0075] Example 21. Further study of neuropathology of spiroplasmosis in deer

The Suckling Mouse Cataract Agent (SMCA) strain of S. mirum, (courtesy of Dr. M. Morrison, Purdue University), was used for the inocula. This strain had undergone multiple passages in M1 D broth. SMCA was grown to log phase (1x10⁹), aliquoted into

1 ml portions, and frozen at -80⁰C until used. After thawing, the number of organisms was inferred to be 1x10⁸AnI, based on work with previous, similarly-prepared cultures. Four pen-raised twin pairs of deer were used in the study; for each pair, one twin was used as a control and one was inoculated. Four 4-month-old white-tailed deer (Odocoileus virginianus) were inoculated intracranial^ (IC) with the SMCA strain of S. mirum through the bregmatic fontanelle into the left cerebral hemisphere with 2 ml of log phase SMCA culture in M1 D media. The twin control group was inoculated IC with

2 ml of M1 D media and housed separately from the Sp/rap/asma-infected group. The deer were followed for development of neurological signs, at which time the animals were anesthetized by overdose of Xylazine (100 mg/ml) administered intravenously at a dose of 1 mg/lb, exsanguinated, and necropsied. All studies were conducted according to protocols approved by the Louisiana State University animal use committee. At necropsy, brains were removed and split saggitally: the right half was frozen at -20⁰C, and the left half was fixed in 20% buffered formalin for two weeks. The fixed hemisphere was sectioned at the juncture of brain stem and cerebrum, and the cerebellum was separated from the brain stem. Standard sections were taken from each hemisphere, including cerebral cortex, hippocampus, basal ganglia, upper brain stem, lower brain stem including obex, cerebellum, and spinal cord. Eyes were removed from inoculated and control animals: the right eye frozen at -20⁰C, and the left eye was fixed in 20% buffered formalin. The fixed eye was sectioned horizontally and the central portion was submitted for histology. All paraffin sections were stained with H&E. Immunohistochemistry was conducted using standard procedures on all brain and eye sections using monoclonal anti-GFAP. Adjacent serial sections were immuno- stained using primary antibody against SMCA (courtesy of Dr. Gail Gasparich, Towson University, Baltimore MD) that had been generated in rabbits by direct inoculation of the organism. (Spiroplasma does not induce disease in adult rabbits.) [0076] In the inoculated animals, but not in controls, we observed the triad of brain lesions that is characteristic of experimental and naturally-occurring scrapie or CWD: neuronal degeneration, vacuolization of the neuropil, and marked astrogliosis, in the same topographic distribution as seen in naturally occurring TSE.

[0077] Example 22. Retinopathy and other changes in the eyes.

The eyes of the Sp/rop/asma-infected deer and sheep showed a retinopathy remarkably similarto that seen in naturally-occurring scrapie. The pathological changes appeared to progress from the inner portions of the retina toward the photoreceptor regions. The depletion and vacuolization of cells in the neuronal cell layers gave the appearance of an overall atrophy to the retina. The retinas from deer 6 months after SMCA inoculation showed marked thinning and degeneration compared to those from controls. The neuronal cell layer was depleted of neurons, and those neurons that remained were vacuolated. The inner plexiform layer had thinned, and the inner granular cell neuronal layer was markedly thinned and vacuolated. The outer plexiform layer was severely atrophied, almost obliterated in some areas. The outer granular nuclear layer, representing the photoreceptor nuclei, was markedly depleted of neurons with some vacuolation. The photoreceptor layer was disorganized and atrophic. GFAP immune staining of the retina revealed astrogliosis in the inner regions of the retina. The remarkable similarity of the retinopathy of spiroplasmosis to the retinopathy of TSE in the same ruminant host species, in combination with the presence of the same triad of neuropathological lesions strongly supports the use of experimental spiroplasmosis in ruminants as a model for naturally-occurring TSE.

[0078] Example 23. Distribution of Spiroplasma in the brain and eyes.

We also compared the lesion triad and retinopathy in the spiroplasmosis model to the distribution of Spiroplasma antigen. Brain or eye tissue sections were immuno- stained for Spiroplasma using rabbit hyperimmune sera against S. mirum. The bacteria were found to be widespread, though sparse, throughout the ruminant brain tissues, but became plentiful in proximity to the vacuolated neurons, and near the vacuolated ependyma. The most surprising findings were in the eye, where large numbers of the bacteria were concentrated. Immune staining revealed sparse numbers of Spiroplasma in the retina, but those that were present appeared in close association with the vacuolated neuronal layers. Spiroplasma antigen was abundant just beneath the inner limiting Bruch's membrane of the retina, and extended beyond the retina into the vitreous humor, where large numbers of extracellular Spiroplasma were seen, embedded in mucoid material. Smaller numbers of Spiroplasma were also seen in the aqueous chamber. The most prominent collections of immuno-stained organisms were seen in the cytoplasm of vacuolated endothelial cells of the cornea. Spiroplasma were not seen in corneal stroma, nor corneal epithelia, nor in peripheral nerve elements in sclera at the limbus margin. Occasional organisms were seen in epithelial cells lining the ciliary body and lens. Despite the presence of Spiroplasma throughout the eye, no inflammatory reaction was seen in any of the eye tissues. The Spiroplasma spread into the non-neural tissues of the eye, concentrating particularly in the vitreous humor, the aqueous humor, and the corneal endothelia. Spiroplasma were not seen in any of these locations in samples from control animals. Indeed, the concentration of Spiroplasma in the corneal endothelia could account for the cloudiness of the cornea that we sometimes saw in SMCA-infected deer suggesting corneal edema.

[0079] Example 24. Clinical Issues

Although 10% of CJD patients present with blindness, this clinical sign has typically been ignored as contributing to the work up of the patient. Furthermore, ~50% of CJD patients develop ophthalmic symptoms during the course of the illness. Blindness in CJD can be due to cortical lesions or retinal degeneration. In fact, retinal degeneration can be diagnosed long before the onset of other signs of CJD by ERG. Opacity of the cornea and blindness are common clinical signs of disease in animals, but blindness is difficult to detect in ruminants since the animals tend to follow a leader even following significant visual impairment. It is well known that surgery in the posterior eye in a CJD patient can contaminate surgical instruments, and that CJD has been transmitted by corneal transplant. It has generally been accepted that corneal transplants are infectious due to the infectivity of peripheral nerve elements in the cornea or sclera; this assumption is challenged by our findings. There is also a major problem arising from delay in diagnosis of CJD in the corneal donor. The lack of a rapid, reliable test poses a substantial problem for eye banks in avoiding transmission of CJD. Where blindness or other ophthalmic symptoms suddenly appear in a demented patient, the possibility of CJD should be investigated.

[0080] Example 25. Methods of Diagnosis

TSE may be diagnosed by detecting Spiroplasma organisms, antigen, or antibody. Diagnosis may be conducted on samples taken from the eye, particular the aqueous humor, the vitreous humor, and corneal endothelia. Sampling from the aqueous humor is preferred for in vivo diagnosis, to minimize the invasiveness of the procedure. While we have not yet conducted experiments to confirm the presence of anW-Spiroplasma antibodies, we expect to find such antibodies, particularly in the aqueous humor, as antibodies are typically present in the aqueous humor relating to eye infections. Establishing cultures with samples taken from the eye is another method that may be used for diagnosis. These detection procedures are simple and straightforward to implement. PCR, Western blots, and other immunological techniques may also be used for diagnosis.

[0081] Example 26. Spiroplasma from Eyes of Scrapie Research

Sheep Flock

We investigate whether Spiroplasma can be isolated directly from the eyes of infected sheep, for example the scrapie research flock of sheep maintained at the University of Idaho. Spiroplasma are identified in eyes sampled from scrapie-affected sheep, for example, by dark-field microscopy, electron microscopy, or IFA of inoculated tissue cultures or in broth media (e.g. M1 D), to increase the number of organisms. Eyes and corresponding sera are obtained from several sheep with scrapie, all of which are positive for prion protein at necropsy. Eyes and sera are also obtained from sheep experimentally inoculated IC with SMCA. Sheep eyes unaffected by scrapie are used as controls. Some eyes are stored at 4°C until used, others are frozen at -70⁰C until used, and others are fixed in formalin. [0082] SMCA cultures are grown and are maintained in another physically separate laboratory. Thawed samples are cultured in M1 D media. Standard Western blots are conducted using available sera, and using electrophoresed, boiled SMCA as the antigen.

[0083] Fixed sheep eyes are examined for Spiroplasma, for example by dark-field microscopy, immunofluorescent staining with SMCA-hyperimmune rabbit sera, or scanning electron microscopy. Direct observations of Spiroplasma in the vitreous humor and the cornea from scrapie-affected sheep eyes, and in culture will support our hypothesis that Spiroplasma are involved in the pathogenesis of scrapie. [0084] We also directly culture the organism from the eyes of affected animals. Samples are removed aseptically from scrapie-affected sheep eyes and control eyes, and placed in M1 D culture fluid. The portions of eye to be sampled include the optic nerve, retina, vitreous humor, aqueous humor, and corneal endothelia. After 14 days incubation at 37°C, the cultures with Spiroplasma show the clear, acidic fluid broth that is characteristic of Spiroplasma growth in M1 D medium. [0085] Samples of optic nerve, retina, vitreous humor, aqueous humor, and corneal endothelia from scrapie-affected eyes and from control eyes are overlaid onto mouse neuroblastoma cell cultures, or onto bovine corneal endothelial cell cultures, both of which are commercially available. Following incubation, the cultures are examined for the presence of Spiroplasma, for example by dark field microscopy, immunofluorescent staining, or PCR. Alternatively, such samples may be cultured in embryonated eggs.

[0086] We will also confirm the presence or absence of Spiroplasma from the various samples by PCR using primers specific for Spiroplasma mirum 16S rDNA, or by immunological detection of antigen or antibody.

[0087] Preliminary studies have been inconclusive as to whether prion protein is deposited in the brain tissues of the experimental spiroplasmosis TSE ruminant model. The inconclusive finding may be related to the degree to which a particular strain of Spiroplasma has adapted to a particular animal species; we expect that more prion protein will be seen following a greater number of passages in the same species. We note that prion deposits have not always been seen following inoculation of scrapie brain homogenates into rodents. [0088] Example 27. Differentiation of CJD from other diseases.

The clinical diagnosis of CJD can be problematic, in that other degenerative brain processes may present with similar clinical histories or physical findings. Lewy body dementia is an example of an unrelated degenerative brain disease that is often confused with CJD. CJD often presents with psychiatric manifestations suggestive of psychotic mental illness; and such patients are often interred in a psychiatric institution until manifestations of organic brain disease are recognized. Brain tumors such as gliomas or lymphomas may present a similar diagnostic problem, especially when they are not readily diagnosed from brain scans. Also, physicians can be reluctant to order a brain biopsy or necropsy on such patients because of fear of the unknown nature of the transmissible agent of TSE. The present invention may be used to diagnose CJD, and to differentiate CJD from other causes of dementia.

Miscellaneous

[0089] As used in the specification and claims, unless context clearly indicates otherwise, the term "Spiroplasma" is defined to mean one of the group of microorganisms presently classified as bacteria of serological group V within genus Spiroplasma, class Mollicutes, and characterized by having a spiral morphology, being motile, lacking a cell wall, and being an obligate or facultative pathogen of one or more species of mammals. (We note that other Spiroplasma are known in the art, organisms that do not satisfy the conditions of this definition. However, these other organisms are not of direct relevance here. For simplicity of nomenclature only those organisms satisfying this definition shall be considered to be "Spiroplasma.") [0090] The complete disclosures of all references cited in this specification are hereby incorporated by reference. Furthermore, the entire disclosures of U.S. Patent No. 6,033,858; of published international application WO/2005/115483; and of priority application 61/035,946 are specifically incorporated by reference herein. In the event of an otherwise irreconcilable conflict, however, the present specification shall control.

Claims

What is claimed:

1. A method for detecting a disease in a mammalian subject; wherein the disease is selected from the group consisting of spiroplasmosis and the transmissible spongiform encephalopathies; said method comprising the steps of:

(a) taking a sample from the mammalian subject, wherein the sample is selected from the group consisting of aqueous humor, vitreous humor, corneal endothelium, iris, ciliary body, tears, and saliva; and

(b) assaying the sample for the presence of Spiroplasma;

wherein a positive indication for the presence of Spiroplasma is indicative of a disease selected from the group consisting of spiroplasmosis and the transmissible spongiform encephalopathies; and wherein a negative indication for the presence of Spiroplasma is indicative of the absence of such a disease.

2. The method of Claim 1 , wherein the transmissible spongiform encephalopathy is Creutzfeldt-Jakob disease, and wherein the subject is a human.

3. The method of Claim 1 , wherein the transmissible spongiform encephalopathy is bovine spongiform encephalopathy, and wherein the subject is a bovine.

4. The method of Claim 1 , wherein the transmissible spongiform encephalopathy is scrapie, and wherein the subject is a sheep.

5. The method of Claim 1 , wherein the transmissible spongiform encephalopathy is chronic wasting disease, and wherein the subject is a cervid.

6. The method of Claim 1 , wherein the sample comprises aqueous humor drawn in vivo.

7. The method of Claim 1 , additionally comprising the step, after said sample-taking step and before said assaying step, of culturing at least part of the sample in vitro under conditions conducive to the growth of Spiroplasma.

8. The method of Claim 7, wherein said culturing step is conducted in the presence of mammalian cells that support the growth of Spiroplasma.

9. The method of Claim 7, wherein said culturing step is conducted in the presence of neuroblastoma cells.

10. The method of Claim 7, wherein said culturing step is conducted in the presence of corneal endothelial cells.

11. The method of Claim 7, wherein said culturing step is conducted in the presence of astroglial cells.

12. The method of Claim 1 , wherein said assaying step includes observing by dark-field microscopy.

13. The method of Claim 1 , wherein said assaying step includes immunological testing for Spiroplasma antigen.

14. The method of Claim 1 , wherein said assaying step includes immunological testing for Spiroplasma antibody.

15. The method of Claim 1 , wherein the subject is human, and wherein said method is used as part of a differential diagnosis to distinguish between Creutzfeldt-Jakob disease and one or more other types of dementia.

16. The method of Claim 1 , wherein the disease is spiroplasmosis.

17. The method of Claim 1 , wherein the disease is a transmissible spongiform encephalopathy.

18. The method of Claim 1 , wherein the disease is a transmissible form of a prion- associated disease.