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The case for mad pigs in the US
Response of Dr. Janice Miller
Dietary risk factors in CJD: pork, ham, hot dogs, roast lamb, pork chops, scrapple
Sundlof not aware of histopathology slides
Stephen F. Sundlof, D.V.M., Ph.D Center for Veterinary Medicine Food and Drug Administration 7500 Standish Place, Room 482, HFV1 RockvLIle, MD 20855
Dear Dr. Sundlof:
We are writing to you to submit information that has recently come to our attention which suggests that a TSElike disease (transmissible spongiform encephalopathy) might exist in pigs in the U.S. We believe this new informantion calls for intensive research and makes it urgent to ban the use of all mammalian proteins, including swine, in the feed of all food animals, until better answers are found.
The evidence for the potential PSE (porcine spongiform encephalopathy ) is as follows. In 1979, an FSQS veternarian, Dr. Masuo Doi, noticed some unusual central nervous system (CNS) symptoms in young (about 6 months old) hogs coming into a slaughter plant In Albany, New York. Since the plant received hogs from a wide variety of sources (New York, Canada, Indiana, Illinois, Ohio, and other Midwestern states) and was not a plant used to dealing with diseased animals, Dr. Doi thought that the problem might be affecting hogs slaughtered nationwide. So, he decided to conduct a detailed study on central nervous system (CNS) symptoms/disease in young hogs coming into that slaughter plant. The study ran for 15 months (January, 1979 to March, 1980) and consisted of extended observations of the behavior of animals with suspected CNS symptoms at the plant, followed by pathological, histopatholpgical, and microbiological work on tissues from various organs of particular animals after slaughter.
For his behavioral observational work, Dr. Doi extended the usual two day observation period to three to four days, during which he took careful notes on the animals' behavior and other vital signs. During the 15 month period of the study, some 106 animals exhibiting CNS symptoms were retained during antemortem inspection.
A 1980 paper that summarized Dr. Doi's findings on the clinical symptoms and incidence of the 'disease," contained descriptions of these symptoms that sound remarkably similar to the symptoms noted for bovine spongiform encephalopathy (BSE):
"Excitable or nervous temperament to external stimuli such as touch to the skin, handling and menacing approach to the animals is a common characteristic sign among swine affected with the disease.... In the advanced stage of the disease, manifestation of neurological signs are evidenced in the form of general ataxia . . . Many animals have been found to be "downers' at first observation; if the hindquarters of these downers are raised they may be able to walk one or two steps and then fall to the ground" (Doi et al., 1980: 2, 4).
Indeed, a table of symptoms includes, for the early stage: "excitability and nervousness (squealing, smacking of lips, grinding of teath, chewing, gnawing ant foaming at mouth); stiffness of limbs . . . 'tic'; weakness of hindquarters; focal tremors of skeletal muscles"; and for the advanced stage: depression; ataxia; crossing over of limbs . . . kneeling posture . . . crawling". In addition to his clinical observations, Dr. Doi also made an 8 mm film of thirteen of the affected animals; film of two of the pigs was shown at the MPI National Pathology Meeting in Seattle, Washington on flay 20, 1979.
Dr. Doi sent tissue samples from suspect cases to the USDA's Eastern Laboratory in Athens, GA for pathological, histopathogical and microbiological work. Known infectious diseases were ruled out. As Dr. Doi points out, "Histopathological studies of tissue collected from the brain and spinal cord of these animals in the early stage of the disease show congestion, hemorrhage and neuronal degeneration. All animals in the advanced stage of the disease have been confined to have Encephalitis or Meningitis by MPI laboratory" (Doi et al., 1980: 5). Eventually some 60 animals were confirmed by the MPI Laboratory to have encephalitis or meningitis, with no ldentifiable cause. As pointed out in a paper presented at the 1979 MPI National Pathology Meetings,
"Since January, a number of hogs in this establishment have been found, in antemortem, to show what appears to be CNS. Sets of tissue samples were sent to the laboratory for examination, various tests were done which include histological study (EH stain), fluorescence antibody technique, virus neutralization and viral and bacteriological isolation. Differential diagnosis was also done to exclude vitamin B deficiency, post vaccination reaction, chlorinated hydrocarbon, arthritis, and transport stress" (Doi et al., 1979).
The brains of the 60 animals were examined. The brain of one of these pigs, on histopathological analysis, exhibited signs reminiscent of a TSE. This histopathological work was performed by Dr. Karl Langheinrich, Pathologist-In-Charge at USDA's Eastern Laboratory in Athens, Georgia. According to the USDA FSQS laboratory report, dated early November, 1979, Dr. Langheinrich noted:
"Microscopic examination of the barrow tissues revealed a encephalopathy and diffuse gliosis characterized by vacuolated neurons, loss of neurons and gliosis in a confined region (nucleus) of the brain stem (anterior ventral midbrain). Only an empty sometimes divided vacuole was present instead of the normal morphology of a nerve cell. Occasionally a shriveled neuron was seen. According to . . . Pathology of Domestic Animals, . . . 'The degeneration of neurons, the reactivity of the glia .... are the classical hallmarks of viral infection of the central nervous system' .... Scrapie of sheep, and encephalopathy of mink, according to the literature, all produce focal vacuolation of the neurons similar to the kind as described for this pig. I was unable to locate any lead as to the cause of this interesting phenomenon in other species including swine'' (Langheinrich, 1979).
Indeed, Dr. Langheinrich's main diagnosis was, " Encephalopathy and diffuse gliosis of undetermined etiology." Portions of the brain were sent for microbiological testing to a neurologist at the University of Georgia, where they came up negative for pseudo-rabies. The brain was unique enough that USDA scientists, such as Dr. Langheinrich and Or. Dot, mentioned it to student and scientific colleagues over the years.
In 1979-1980, BSE was completely unknown. However, both the behavior of the pigs, as well as the histopathology on at least one pig, both showed sign consistent with a porcine TSE. This raises particular concern became the affected animal was only 6 months old; in an animal this young, one would rust expect to see any physical signs of TSE in the brain. Histopathology of TSEs can be very variable, so that spongiform appearance (i.e. vacuolated neurons) are not always present. Behavioral changes can be seen in TSE-infected animals before any changes in brain morphology are visible. Dr. Clarence Gibbs, in testimony before a Congressional hearing on the TSE issue on January 29, 1997 made just this point:
''In the mid-1960s, we demonstrated with our French and English collaborators that during the early incubation of the TSEs, when the virus titer in the brain was very low, there were already marked functional changes, even though no pathology was yet detectable, even ultrastructurally. A month or hero later, polynucleation of neurons appeared in spider monkeys, incubating kuru, and somewhat later, microvacuolation and membrane changes visible only by electron microscopy. This preceded the pest appearance of astrogliosis and spongiform change. It was only much later that the classical scrapieTSE pathology appeared with virus titers in brain of 10 -5 or higher" (Gibbs, 1997; pg. 4).
Given that TSEs can cause behavioral changes in infected animals before any physical changes in the brain can be seen, that the manifestation of TSE in the brain can be quite variable, and that changes in brain morphology are not usually seen in 6 month old animals, we are concerned that the brain of one pig actually showed physical evidence consistent with a TSE.
Following the announcement In March, 1996 of ten cases of new variant CJD (Creutzfeldt-Jakob Disease) in the United Kingdom and their possible connection to BSE, Drs. Doi, Langheinrich and others urged reinvestigation of this case.
In August, 1996, the USDA sent five slides, one of which was a histopathology slide, to Dr. Janice Miller of USDA's Agricultural Research Servicer . Dr. Miller stained four of the slides for prion protein (she didn't stain the H&E slide). Dr. Miller told Consumers Union that Dr. Patrick McCaskey, USDA/FSIS, in charge of the Research Center at Athens, GA, called her, told her that he had five slides that all showed "problems" and asked her to stain four of them. The H&E slide, which clearly show vacuoles in the neurons (one sign of TSE), wasn't stained because to stain for PrP entails removing the slide cover, baking the slide to destain it and then restaining it for PrP; they didn't want to risk destroying the H&E slide.
Dr. Doi had kept frozen samples of the brain and spinal chord of the suspect PSE pig in case the Eastern lab wanted more material for analysis. Unfortunately, these samples were discarded when the packing plant in Albany, NY closed in 1991. It appears that the brain material sent to the Univcrsity of Georgia may have been discarded. [pers com.. Dr. Doi 3/13/97]
Dr. Miller found that the PrP stained in the four pig slides was found only on the inside of neurons, while a positive control slide from a scrapie sheep showed massive amounts of extraneuronal staining. In a letter summarizing her results (copy attached), she concludes that the PrP stained in this pig was normal: "In the pig sections you will see a small particulate type of staining that is confined to neurons and as I indicated on the phone, I would interpret as normal PrP. It is in marked contrast to the massive amount of extraneuronal staining seen in the scrapie section" (Miller, 1996).
Unfortunately, Dr. Miller's finding toes not conclusively rule out a TSE. We are concerned that while British BSE and serapie create a massive amount of extraneuronal staining, there are TSEs where this isn't the case. Three experiments were done in He U.S. -- in Mission, TX (APHIS work), Pullman, Washington (ARS work), and Ames, Iowa (ARS work) -- to see whether sheep scrapie can possibly infect cows. In all the experiments, cattle were inoculated with tissue from scrapie-infected sheep primarily by intra-cranial injection, but in the case of the Texas and Iowa studies also by oral feeding -- to see if cattle were susceptible to scrapie at all. In all three experiments, the majority of cows injected in the brain with scrapie-infected sheep material (usually brains) also developed a fatal spongiform encephalopathy.
However, in all three examples, the symptoms of the spongifonn encephalopathy differed from "mad cow" disease ~ England, as did the appearances of slides from their brains. The brain lesions seen in ail these animals were more variable than those seen in England. When Dr. Miller did similar staining for PrP from these brains (what she called "bovine scrapie") she only found PrP stains on the inside of the neurons, not the massive extraneuronal staining seen in BSE (Miller, pers. comm., March 7, 1997). Thus, Dr. Miller's finding of PrP stains only inside the neurons in the suspect pigs is not particularly reassuring.
In November 1996, USDA sent the single histopathology slide to Dr. William Hadlow, one of the foremost spongiform encephalopathy pathologists in the world. (For unknown reasons, Dr. Hadlow was only sent the one slide; he was not told of the existence of the other slides, nor of Dr. Miller's findings, nor was he told or given the behavioral report from Dr. Doi or the morphology work by Dr. Langheinrich, or shown film of the affected pigs [Dr. Hadlow, pers. com., 3/13/97] From this single slide, Dr. Hadlow found some evidence consistent with TSEs but not enough for a conclusive diagnosis. He noted that the slide contained vacuoles inside neurons, one of the signs of a TSE (Dr. Langheinrich had noted this as well).
However, since such vacuoles occasionally occur normally in pigs, he thought that was not something special: "About twelve (12) neurons in the parasympathetic nucleus have unilocular optically empty vacuoles in the perikaryon. This is the site where such vacuolated neurons have been seen in the swine (as well as in cats and sheep) as an incidental finding. So I do not think such cells have any significance in this pig" (Hadlow, 1996). However, he did see evidence, Including changes in astrocytes, that suggested a TSE, but without examining other parts of the brain to look for other evidence of TSE, he couldn't be sure:
"I am impressed, though, with what seems to be an increase in the number of astrocytes in the section. Some astrocytes are in clusters, some are enlarged and vesicular. Where they are most numerous, a few rod cells (activated microglia) are seen. These findings suggest some perturbation of the nervous tissue. Although such a global response occurs in the transmissible spongifonn encephalopathies, I do no! know its significance in this case without examining other parts of the brain for changes characteristic of these diseases. Thus, from looking; at this one (1) section of brain, I cannot conclude that the pig was affected with a scrapie-like spongiform encephalopathy" (Hadlow, 1996).
In sum, Dr. Hadlow~s letter does not rule out the possibility of a TSE. He says that there is suggestive evidence, but that he would need to look at other slides/sections of the brain, to make a conclusive diagnosis.
In our view, the implications of this data are extremely serious. Experiments in the United Kingdom have shown that pigs are susceptible to BSE. Pigs inoculated with BSE develop a TSE (Dawson et al., 1990). Feeding experiments are underway in the UK to see if BSE can be orally transmitted to pigs; as of March, 1997, some 6 years after the start of the experiment, none of the pigs fed BSE brain have come down with a TSE. Unfortunately the design of this experiment severely limits what we will learn from it, and will most likely not tell us conclusively if pigs can get BSE from feed. It turns out that the pigs were not fed BSE brain continuously. Rather, the pigs were only fed BSE brain material on three days, over a three week period (i.e.. one day each week). Following these three doses, the pigs were never fed contaminated material again. The total amount of infective material given to the pigs was therefore quite small. Thus, a negative finding would be hard to interpret and would not mean that BSE is not orally active in pigs.
We believe that as a top priority USDA should conduct follow-up studies to look for potential CNS/PSE cases in pigs (we plan to communicate about this to USDA separately). In brief, we feel that the following kinds of studies need to be done:
i) TSE pathology experts should examine all the slides from the suspect pig (2709). To our knowledge, at least 12 separate slides exist.
ii) Determine if any brain material from the suspect pig (2709) still exists at the Unlverslty of Georgia. If so, this material should be retrieved and used for transmission studies. In particular, suckling pigs should be inoculated with the material and then permitted to live unto they die of a disease or old age, at which point their brains should be examined for physical signs of a TSE as well as for immunchistochemical evidence (i.e. staining looking for the abnormal PrP).
iii) Increase antemortem inspection for CNS symptoms at hog facilities. Inspectors should be trained to detect the subtle CNS symptoms seen in the Doi et al. study. At a select number of slaughter facilities, animals exhibiting CNS symptoms should be removed and held for observation until they die, at which time their brains should be examined for evidence of a TSE.
iv) Research on CNS symptoms among Me 6,000 or so breeding sows which are permitted to live for 3+ years. Sows exhibiting CNS symptoms should be removed and held for observation until they die, at which time then brains should be exernined for evidence of a TSE.
While such work is underway, given the above inforrnabon, we believe that as a precutionary measure the FDA must expand the proposed ruminant plus mink-to-ruminnant feed ban to prevent protein from any material, including hogs, being fed to any food animal.
Michael Hansen, Ph.D Research Associate
Jean Halloran Director
Dawson, M., Wells, G.A.H., Parker, B.N;J. and A.C Scott. 1990. Primary parental transmission of bovine spongiform encephalopathy to the pig. Veternary Record, pg. 338.
Doi, M., Matzner, N.D. and C. Rothaug. 1979. Observation of CNS disease in market hogs at Est. 893 Tobin Packing Co., Inc. Albany, New York. United States Department of Agriculture, Food Safety and Quality.Service, Meat and Poultry Inspection Service. 7pp.
Doi, M, Langheinrich, K. and F. Rellosa. 1980. Observations of CNS signs in hogs at Est. 893 Tobin Packing C:o., Inc. Presented by Dr. Lngheinrich at the MPI National Pathology Meeting in Seattle, Washington on July 20, 1979.
Gibbs, C. 1997. Statement to the Committee on Governnent Reform and Oversight, Subcommittee on Human Resources and Intergovernmental Relations, U.S. House of Representatives. January 29,1997.
Hadlow, WJ. 1996. Letter to Patrick McCaskey, USDA/FSIS/Eastem Lab, dated November 13, 1996.
Langheinrich, KA. 1979. USDA/FSQS Laboratory report on specimen 2709. Dated November 8, 1979
Miller, J. 1996. Letter to Patrick McCaskey, USDA/ESIS/Eastern Lab, dated September 6, 1996.
My involvement in the "pig incident" (I refuse to say "mad pig disease" since no such disease has been recognized):
I was asked by Dr. Al Jenny at the National Veterinary Services Laboratory if I had ever done immunohistochemistry on slides that had already been stained by hematoxylin and eosin, the standard stain used for histopathology. I had done it on a few scrapie cases so he asked if I would do the procedure on some pig brain slides that he had received from Dr. Pat McCaskey, an FSIS pathologist in Athens, GA.
At the time I didn't know the history of the situation but Dr. Jenny said I should call Dr. McCaskey and discuss it with him before proceeding. Only then did I learn a little about the history of the case. We decided that I wouldn't try to stain all of the slides because I was afraid the procedure required to remove the cover slips might damage the sections and Dr. McCaskey was concerned about preserving the sections for other pathology consultations, if necessary.
We agreed that I would stain 4 of the 5 slides, leaving the slide with the best lesions untouched. I was also concerned that I didn't know whether the antiserum we use would stain pig PrP but decided it was worth a try. When I completed the staining procedure the only positive material I observed was a small amount of particulate staining within the cell body of some neurons. We have occasionally observed that kind of staining in brains from control cattle and sheep in our experiments and interpret it to be normal PrP. (A similar finding was reported by Dr. Haritani, who first described the technique for BSE).
That observation was reported in our 1994 paper and we stated that consequently we could not interpret intraneuronal staining as indicative of scrapie (although it may be present, the bulk of staining is in neuropil, around vessels and neurons, etc). In that study I think our interpretation was somewhat validated by the very close correlation we had between immunohistochemistry and western blot results. At any rate, I told Dr. McCaskey that my interpretation on the slides was that the only staining present was consistent with normal PrP. The good news was that the antiserum did in fact stain something and that it was in the correct location for normal PrP, indicating that the antiserum would have detected abnormal PrP, had it been present. Subsequently, I called Dr. Richard Rubenstein, who provided the antibody we use, and asked if he knew whether it would react with pig PrP and he said he didn't know. However, he said it reacted with almost all mammalian species, except ferret and mink, that he had tried so he would be surprised if it wasn't reactive with pig PrP.
So, having all of this information at hand, people can decide whether the immunohistochemical test means anything or not. The lack of a positive control pig tissue (positive sheep tissue is included in every test) may be viewed by some people as diminishing the value of a negative result, but feel we did the best we could under the circumstances.
The above recitation describes my experience with the case in question. I did not photograph the slides and returned all 5 to Dr. McCaskey. It was later that he had Dr. Hadlow look at the case for histopathologic interpretation. I did not examine the slides for that purpose because I do not have experience in scrapie diagnosis and would not consider my observations meaningful.
I appreciated the additional information about the original study done by Dr. Doi. Although I've heard bits and pieces of the story from different people, this was the first time I had heard that 60 of the pigs were diagnosed as having encephalitis or meningitis. I think that fact, plus the fact that the pigs were only about 6 months old, should certainly indicate that it's highly unlikey that a spongiform encephalopathy epidemic was causing the CNS signs observed. Whether the 1 pig with the questionable encephalopathy lesions was a TSE could be debated, I suppose. The age would seem to argue against it and the immunohistochemistry result would also (at least that's my opinion).
We disagree about the implications of age regarding the liklihood of TSE in a 6-month old pig. Certainly dose has an effect on incubation period in experimental transmissions and probably also in the "natural" acquired transmissions. However, regardless of the manner of transmission, I don't know of any first passage experimental interspecies transmission where the incubation period was as short as 6 months.
Early onset in mice were achieved only after adaptation through at least 1 intraspecies transfer. I believe the same is true for development of the hamster models. With regard to acquired transmissions, Linda Detwiler's review on scrapie cites research that indicated infectivity was found in CNS tissues of lambs as early as 4 months of age: however, they were not showing clinical signs. From what I can find in the literature, a clinical case of scrapie under 2 years of age would be exceptional, but with the amount of material published on that disease I wouldn't want to say it hasn't happened.
With TME the shortest incubation I've seen reported was 9 months. Elizabeth Williams has indicated that the youngest case of CWD observed in their wildlife facilities was 18 months old. You stated that in England calves were getting BSE by one year. In the experimental BSE transmissions cattle didn't develop clinical signs until the second year of observation and the earlest sign we observed in cattle inoculated with sheep scrapie was 14 months.
With respect to swine, the only model we have is the experimental transmission of BSE. The animal first developed signs about 17 months after inoculation. I think that it would be highly unlikely for a 6 month old pig to be showing CLINICAL signs of a TSE (the claim in this particular situation). One can never say never but it seems reasonable to at least examine what is known and make an educated estimate about what is likely.
Am J Epidemiol 122 (3): 443-451 (1985) Davanipour Z, Alter M, Sobel E, Asher DM, Gajdusek DCThe mode of natural transmission of Creutzfeldt-Jakob disease remains unknown. In a case-control study conducted in 1981-1983 to evaluate possible dietary and other sources of the disease, 26 cases were ascertained in the mid-Atlantic region of the United States, 23 of which were obtained from accumulated records of the Laboratory of Central Nervous System Studies of the National Institutes of Health. Controls included 18 family members and 22 hospital-matched individuals (total sample size, 66).
An increased consumption among patients was found for roast pork, ham, hot dogs (p less than 0.05), roast lamb, pork chops, smoked pork, and scrapple (p less than 0.1). An excess consumption of rare meat (p less than 0.01) and raw oysters/clams (p less than 0.1) was also reported among the patients. Liver consumption, among organ foods, was greater (p less than 0.1) among the cases. If Creutzfeldt-Jakob disease is acquired through ingestion of foods containing the agent, then the food items identified may be among those which need to be evaluated more intensively. Larger case-control studies with more focused dietary questions are warranted.
From: Dr. Stephen Sundlof D.V.M., Ph.D. Director, Center for Veterinary Medicine Food and Drug Administration:
At the present time FDA is in the process of developing a final rule which will regulate the feeding of certain animal-derived protein to other animals. In addition to studying the scientific literature pertaining to TSE's, we have received 700 comments relating the proposed rule that was published in the Federal Register on January 3, 1977.
The information provided by Dr. Hansen and others will be considered in developing the final rule along with all of the other information and comments that have been officially submitted to FDA. Until the final rule is published, FDA is prohibited from commenting on information that might impact the final rule. Therefore, I am unable to respond to the documents in Dr. Hansen's letter.
I do not have access to the photomicrographs of the histopathology slides, and I was unaware of their existance until Dr. Hansen brought the issue to my attention. Furthermore, I do not have addresses or telephone numbers for Drs. Doi. Langheinrich, or Hadlow. Someone from the USDA would have this information but I am not sure who that would be.
Webmaster had written: " Do photomicrographs of any of the 12 slides exist? If you have any of them, I would like to scan a few of these and post them at high resolution on the internet so that pathologists world-wide could view and comment on them.
Veterinary Record 1990 127 13 338 Dawson, M.; Wells, G. A. H.; Parker, B. N. J.; Scott, A. C.Ten, weaned one- to two-week old piglets from a specific pathogen free breeding herd were inoculated under halothane anaesthesia by simultaneous injections intracerebrally (0.5 ml) intravenously (1 to 2 ml) and intraperitoneally (8 to 9 ml) with an inoculum consisting of 10% saline suspension of pooled homogenised brainstem from 4 natural bovine spongiform encephalopathy cases. Control piglets were similarly inoculated with saline. After 69 weeks one challenged pig showed mild aggressive behaviour towards the animal attendants. Intermittent inappetence and depression were also noted.
Within one week the behavioural changes included aimless biting activity and there was mild symmetrical ataxia. The ataxia progressed and 5 weeks after onset of signs the gait ataxia was generalised with hypermetria and wide-based stance. At this time the pig was killed. Histopathological examination of the brain revealed spongiosis of grey matter neuropil with greatest intensity in the medial geniculate body, superior colliculus and corpus striatum. There was sparse vacuolation of neuronal perikarya in the dorsal nucleus of the vagina nerve and widespread astrocytic reaction. Characteristic fibrils associated with transmissible spongiform encephalopathies were detected by electron microscopy.
One good question is what _pooled_ medical products do they make from pigs. The key issues for spread of this disease are the amplification cycle and distribution pooling. That is, one rotten apple by itself is less of a problem than if it is in a barrel. -- webmaster