Materials and Methods

Inoculum—Brains from 7 clinically affected 171 QQ sheep testing positive for scrapie were verified to have PrP^SC by IHC and western blot testing of brain obex. Whole brains were singly homogenized, combined, and diluted to a 20% solution with PBS solution. The combined scrapie-positive SBH was frozen and stored at −70°C. Samples of the single homogenates were later sequenced; 5 sheep were 136/154/171 AARRQQ, 1 was AVRRQQ, and 1 was VVR-RQQ.

Sheep and treatment—Twenty-seven Suffolk ewes, approximately 9 months old, with the genotype 171 QQ were purchased from 2 private owners in October 2001. Scrapie had never been observed in either flock and has not been observed since. In November 2001, ewes were exposed to a Suffolk ram with the genotype 136/154/171 AVRRQQ. In December 2001, sheep were given 5 mL of the thawed 20% SBH solution, which was deposited superficially on the back of the tongue via a dose syringe. Other than thawing, there was no special treatment of the inoculum. Thirty-three lambs were born during April 2002, and on the day of birth, each lamb was also given 5 mL of 20% SBH solution in the same manner.

Ewes and lambs were housed in an outside drylot pen with fence-line feeders and shelter from inclement weather. The study was approved by the University of Idaho Animal Care and Use Committee. Their base diet was alfalfa hay, and grain was fed during pregnancy and lactation. Heated, automatic waterers provided water ad libitum. A trace mineral salt was provided. Two technicians checked sheep twice daily and recorded any problems or abnormal behavior. Sick sheep with any illness were moved into a separate pen for specific care or supportive treatment as needed.

Biopsy protocol—A biopsy specimen was obtained from the lymphoid tissue of the third eyelid as described,¹² except xylazine (0.1 mg/kg) was given IV for sedation and anesthesia. For mandibular lymph node biopsies, the area beneath the base of the ear was prepared for surgery after xylazine injection. The lymph node was palpated just medial to the angle of the mandible, and an incision was made over the top of the lymph node with a disposable scalpel. After the lymph node was identified, it was grasped with a pair of forceps and removed with either scissors or scalpel. One or 2 sutures were placed in the skin, and sheep were permitted to recover. Instruments were soaked in 2.5N NaOH for at least 24 hours, rinsed, then sterilized in an autoclave for reuse.

Necropsy protocol—Ewes and lambs were euthanatized when they could no longer hold themselves in sternal recumbency, and necropsies were performed. Fresh samples of tissues in which gross abnormalities were detected on necropsy were taken for bacteriologic examination and placed into neutral-buffered 10% formalin for histologic examination. Several areas of the brain and spinal cord were also sectioned for H&E staining and microscopic evaluation. Samples of brain, palatine tonsils, retropharyngeal lymph nodes, mandibular lymph nodes, lymphoid tissue of the third eyelid (if still present), mesenteric lymph node, and ileum with Peyer's patches were obtained; 1 sample from each tissue was placed into neutral-buffered 10% formalin for IHC, and a second sample was frozen at −20°C for future experimental protocols.

IHC testing—Fixed, paraffin-embedded tissues were cut at 5 [.mu]m, and resulting slides were processed by use of a microprobe system.^a After tissues were deparaffinized and hydrated, the paired slides were placed in an autoclave at 121°C for 30 minutes in TB. The cooled slides were rinsed in TBGS used also to make a 1:1,000 dilution of the monoclonal antibody (cell line F89/160.1.5^b), which was applied to the slides for 12 hours at 2°C incubation.

Slides were rinsed with TBGS, and the secondary antibody (biotinylated anti-mouse IgG, H and L,^c heavy and light chains), diluted in a ratio of 1:200 in TBGS, was added for 30 minutes. After rinsing with TB, slides were loaded with avidin-alkaline phosphatase and incubated for 20 minutes. Slides were rinsed several times with 0.1M Tris HCl buffer (pH, 8.2) containing 0.25% Tween 20. Alkaline phosphatase substrate was prepared with 0.1M Tris HCl buffer (pH, 8.2) containing 0.25% Tween 20 and 0.1 mL of levamisole, then loaded onto slides. Slides were incubated for 5 minutes twice, then once for 10 minutes, rinsed with water containing 0.25% Tween 20, and counterstained with hematoxylin with TB used as a bluing reagent. Slides were dehydrated in graded alcohols and cleared, and a cover slip was placed on each slide.

For lymphoid tissue to be considered negative for scrapie, a minimum of 5 lymphoid follicles was required on the slide for evaluation. The presence of 4 negative follicles, for example, was considered a no test. This was only a problem in the case of eyelid tissue.

Results

Two lambs died at 0.7 and 1.4 months of age. In those lambs, the cause of death was determined to be starvation and pneumonia. Tissues were not retained for scrapie testing. Two other lambs died at 4.6 and 5.7 months of age of pneumonia, and results of IHC tests were negative. Five months after inoculation, 1 ewe died from a prolapsed uterus; this ewe was not tested for scrapie. One ewe died 9 months after inoculation during anesthesia for herniorraphy; PrP^SC was not detected in tissues via IHC at the time of death (Table 1).

In January of 2003, 12 and 9 months after inoculation of ewes and lambs, respectively, biopsy specimens of third eyelid lymphoid tissue were obtained from all inoculated sheep and tested for PrP^SC via IHC. Only specimens from 2 adults and 1 lamb tested positive. As of June 2005, the lamb and one of those adult sheep were alive 36 and 42 months after inoculation, respectively. The other adult had clinical signs compatible with scrapie¹³ for 3 weeks and was euthanatized 16 months after inoculation (in April 2003).

Twelve months after inoculation of adult sheep, 2 ewes, (ID No. 290 and 298) were ataxic and had difficulty maintaining their balance (Table 1). Five days later, one of those ewes (ID No. 290) was unable to maintain sternal recumbency and was euthanatized; all tissues, including the third eyelid, tested positive for PrP^SC (Table 2). The other ewe (ID No. 298) was euthanatized 3 weeks after showing clinical signs compatible with scrapie; all tissues, except the mandibular lymph node, tested positive for PrP^SC. One adult ewe (ID No. 283) in which tissue from the third eyelid tested positive was euthanatized 3 weeks after developing clinical signs of ataxia. Thirteen months after inoculation, 1 ewe (ID No. 293) became recumbent and was euthanatized. This ewe had appeared clinically normal 1 day earlier. All tissues, except for lymphoid tissue of the third eyelid, tested positive for PrP^SC via IHC.

These 4 sheep were in good body condition and did not have the wasting commonly associated with naturally occurring scrapie; 3 sheep did not have pruritus and 1 sheep had minimal pruritus, and they did not have visible tremors commonly seen in sheep with classic signs of scrapie. Clinical signs in these sheep progressed from lack of coordination to being unable to stand in a brief period of time: approximately 3 weeks, sometimes within 18 hours. The genotype of these 4 ewes was 136/154/171 AVRRQQ.

In the first 3 months of 2003, 12 lambs, 36.4% of those born in 2002 and inoculated at birth, died of scrapie. All had an incubation time <12 months, (mean, 10.2 months), and duration of clinical signs was < 2 weeks. In those lambs, brain and mesenteric lymph node tissue tested positive for PrP^SC via IHC. The genotype of those lambs was 136/154/171 AVRRQQ. Other tissue results from these lambs are described (Table 2).

The PrP^SC accumulation in tissues of SBH-inoculated AVRRQQ sheep was notably less as determined by IHC than that of sheep naturally infected, being fine granular deposits lightly sprinkled through affected cells and often difficult to see. Findings from several of the first cases of scrapie in sheep were shared with personnel in the USDA Agricultural Research Service^d to substantiate the diagnosis. Results of all bacterial cultures of brain and spinal cord were negative for Listeria spp, and other infectious bacteria and histologic examinations of sections obtained from obex, cerebrum, cerebellum, and spinal cord did not reveal classic scrapie lesions.

Sequencing the PRNP gene from brain tissue of 13 AVRRQQ sheep that died with a decreased incubation time was performed,^e starting at amino acid 54 through amino acid 254. The first 53 amino acids were not sequenced because there are no reported critical polymorphisms in these areas. There were 3 sheep that had an MT polymorphism at codon 112 (Table 3). This polymorphism has not been involved in resistance or susceptibility to scrapie. There were no additional polymorphisms or amino acid changes divergent from the known sheep PrP sequence.

The IHC results for third eyelid biopsy specimens from all dead lambs that were inoculated at birth were negative for PrP^SC, except for 1 lamb (ID No. 2146). The IHC results for third eyelid biopsy specimens in 1 other lamb (ID No. 2112) were positive also, but this lamb's genotype is 136/154/171 AARRQQ, and the lamb was still alive at the time of publication. The third eyelid tissue of 1 lamb (ID No. 2136) that originally tested negative for PrP^SC on biopsy tested positive for PrP^SC at the time of death.

All sheep (20 adults and 11 lambs) that were alive as of July 2004 (26 to 31 months after inoculation) were genotype 136/154/171 AARRQQ. The first scrapie-positive AAQQ sheep died 32 months (August 2004) postinoculation after having classic clinical signs of scrapie for several months. Fourteen of 31 AAQQ inoculated sheep and lambs were still alive 37 to 42 months, respectively (as of June 1, 2005), after inoculation.

Discussion

The study reported here was initially undertaken to provide additional scrapie-positive tissues and blood for research. At that time, codon 136/154 genetics were not considered particularly important in the United States, and only after inoculated sheep began to die at an early age was genotyping for the 136/154 codons performed. Because the 136 AV genotype was believed to be rare, the fact that many sheep in our study died from scrapie within 15 months and that they were all 136 AV was completely unexpected.

Six of 30 (20%) sheep (5 ewes and 1 ram) chosen from 2 private flocks on the basis of 171 QQ genotype were 136 AV, suggesting that the 136 AV genotype in some sheep flocks in the United States is not as rare as reported. The 136 V allele was also detected in our naturally infected research flock at a prevalence of 18.8%. In this flock, we found that the genotype also affected the incubation time, which was in agreement with results reported in sheep of all breeds in Great Britain.¹⁴

Typically, clinical signs in sheep with scrapie include wasting, incoordination and weakness of the hind limbs, pruritus, CNS signs, and behavioral changes that last 3 months or more.¹³ In our study, duration of clinical signs in inoculated AVRRQQ adult sheep and lambs from the first observed clinical sign to complete inability to stand continued for as little as 1 day to less than a month. Wasting was not detected and pruritus was rare. A producer or veterinarian would not have diagnosed scrapie in sheep with these clinical signs. In most diagnostic laboratories, samples from young adult sheep that die suddenly and yield no diagnosis are never considered for IHC testing unless perhaps sheep were from a flock known to have scrapie.

Other remarkable disparities included differences in the accumulation of PrP^SC in lymphoid tissue. In our naturally affected sheep, PrP^SC has been detected in the lymphoid tissue of the third eyelids of 57% of sheep testing positive for PrP^SC,¹⁵ whereas in this study, third eyelid lymphoid tissue from only 5 (31.2%) inoculated 136/154/171 AARRQQ sheep tested positive for PrP^SC at death. Additionally, in dead sheep inoculated at birth, palatine tonsils and mandibular and retropharyngeal lymph nodes had lower rates of IHC positive diagnoses (59%, 50%, and 75%, respectively) than mesenteric lymph nodes and brains (100%), whereas in naturally infected sheep, the mesenteric lymph nodes had the lowest percentage of IHC positive diagnoses (66%).¹⁵ Perhaps the long incubation time permitted a longer duration for absorbed PrP^SC to migrate, replicate, and accumulate in the CNS and lymph nodes. Concentration of PrP^SC IHC staining in brains (obex) of all inoculated sheep was much less than in naturally infected sheep, requiring that additional time be spent searching slides for cells containing PrP^SC. Furthermore, no histologic lesions were detected in obex, cerebrum, cerebellum, and medulla oblongata; therefore, the cause of the rapid and severe clinical signs is not known. The amount of PrP^SC has been measured in scrapie-infected sheep brains by the use of a new conformation-dependent immunoassay¹⁶ and supports our finding that amounts of PrP^SC will differ depending on genotype. However, the brains of 136/154/171 VVR-RQQ sheep experimentally inoculated with the SSBP/1 scrapie strain had larger amounts of PrP^SC than that measured in the brains of AVRRQQ and AVRRQR sheep at the time of death. Unfortunately, that does not substantiate our observation (using IHC) of less PrP^SC deposition in tissues of AVRRQQ inoculates than AARRQQ naturally exposed sheep with the longer incubation times. This may be a scrapie strain difference.

In the past, the observed comparison between accumulation of PrP^SC, brain lesions, and clinical signs had been taken as strong evidence for a direct neurotoxic effect of PrP^SC.^17,18 Certainly, when microscopic changes and neuronal loss are seen during histologic examination and large accumulations of PrP^SC are identified in neurons by IHC staining, the theory is easy to accept. However, wild-type mice can accumulate high concentrations of PrP^SC and have little if any brain changes, suggesting that the molecule may not be the neurotoxic factor.¹⁷ On the other hand, results of the IHC test for detection of PrP^SC in tissue have been used to diagnose numerous cases of scrapie in sheep that had clinical signs but did not have the typical brain lesions that had formerly been required for a positive diagnosis.¹⁹ Indeed, bovine spongiform encephalopathy infectivity has been transmitted in mice despite the absence of detectable abnormal prion protein.¹⁷ Other cases of lightly scattered deposits of PrP^SC with few brain lesions have also been detected in sheep with severe clinical signs.²⁰ Therefore, clinical signs and death do not correspond well with brain lesions or the concentration of PrP^SC. Thus, whether PrP^SC is a cause or an effect is not known. The observation that shorter incubation time and duration of clinical signs equate to more lightly scattered PrP^SC supports the idea that PrP^SC may actually be an effect of the disease, and time is required for abnormal prion to accumulate. This is not a novel idea; several hypotheses have been put forth that bacteria, viruses, or other factors, not abnormal prions, are actually the causative agents of prion diseases.^21–25

The oral route was chosen because it is the natural route of infection, although reportedly the most inefficient experimental route for PrP^SC neuroinvasion,²⁶ making the short incubation time in these sheep even more unexpected. In our study, sheep merely had the inoculum placed superficially on the back of the tongue; however, hamsters inoculated into the tongue had the shortest incubation period for transmissible mink encephalopathy ever reported for that type of hamster.²⁷ This suggests that inoculation into peripheral tissues innervated by cranial nerves originating from the brainstem will result in a more rapid direct prion neuroinvasion of the brain. In our study, older sheep may have had small abrasions around teeth, tongue, or cheeks, but it is unlikely that neonates had such lesions. On the other hand, in lambs, large protein molecules move easily through the intestinal wall after birth for approximately 24 hours, facilitating the passive uptake of maternally derived antibody. This may have facilitated absorption of PrP^SC into the intestinal lymph tissue (Peyer's patches) and mesenteric lymph nodes and may explain why the incubation time in lambs was 3 months shorter than that in the inoculated ewes. Mesenteric lymph nodes in all of the inoculated 136 AV sheep tested positive for PrP^SC, supporting results of earlier studies^28,29 indicating that PrP^SC enters the body via the intestines as evidenced by the fact that accumulation of PrP^SC is first detected in lymphoreticular tissue of the intestines and associated lymphoid tissue in the naturally occurring disease.

The dose of 5 mL of SBH was purely arbitrary. When the first sheep inoculated with SBH died quickly, we believed that the sheep had been given a large overdose. However, because the inoculated AARRQQ sheep were dying of scrapie within the usual time frame with typical clinical signs, dose likely was not a factor.

The new Uniform Methods and Rules for Scrapie Control¹¹ recognizes that sheep that are carriers for codon 136 V are at a high risk for scrapie infection even when they also carry codon 171 R. The definition of genetically susceptible sheep now includes AVQR ewes epidemiologically associated with 136 VV or AV-positive sheep.¹¹ Veterinarians from the USDA Animal and Plant Health Inspection Service indicate that in flocks in which scrapie-positive 171 QR sheep have been identified, all or nearly all sheep testing positive for scrapie have a 136 AV or VV codon. Epidemiologists from the USDA suggest that these data strongly support the premise of a second scrapie type in the United States in which codon 136 V is the primary determinant of susceptibility,^f most likely similar to the SSBP/1 strain reported in Cheviot sheep at the NPU in Edinburgh, Ireland.³⁰ Neuropathogenesis unit Cheviot sheep infected with the SSBP/1 scrapie strain reportedly have a short (2 to 3 years) incubation gene (later determined to be VV on codon 136) for scrapie manifestation and a long (4 to 6 years) incubation genotype in sheep with codon 136 AV.³¹ The incubation period in sheep homozygous for the 136 AA genotype is believed to be longer than the sheep's lifetime or to be totally resistant.^30,31 However, the 136/171 AAQQ NPU Cheviots died of scrapie when infected with another strain of scrapie (CH1641), whereas the 136/171 AARR genotype conferred resistance to that strain.³²

It was hypothesized that sheep used in our study may have had another nucleotide variation of the PRNP not heretofore described, which might have affected the incubation time of the traditional scrapie strain found in the United States. Thus, brain tissue of 13 of these sheep was sent to USDA for DNA sequencing of the PRNP gene. There were no notable abnormal sequences to explain the reduced incubation time. It would seem, then, that our scrapie inoculant had a V-dependent strain or a strain similar to the SSBP/1 strain, which may have originated from the two 136 V sheep brains in the inoculum.

Therefore, there are several possible explanations for the rapid incubation time seen in sheep in the study reported here: the strain may have been the traditional strain of scrapie detected in the United States, and the incubation time may have been influenced by only the 136 genotype of the sheep; this may be a V-dependent strain of scrapie similar to the SSBP/1 strain that can also affect 136 AA sheep (ie, a new, unreported strain); or the inoculum may have contained both a traditional strain detected in the United States and a V-dependent strain of scrapie.