Susceptibility of Cattle to First-passage Intracerebral Inoculation with Chronic Wasting Disease Agent from White-tailed Deer

Materials and Methods

Sample collection and tests

The affected animals were euthanatized when in extremis (Table 1) with pentobarbital sodium and subjected to a complete necropsy. Representative samples of lung, liver, kidney, spleen, salivary gland, thyroid gland, reticulum, rumen, omasum, abomasum, intestines (ileum, colon), adrenal gland, pancreas, urinary bladder, lymph nodes (retropharyngeal, prescapular, mesenteric, popliteal), tonsils (palatine, pharyngeal), striated muscle (heart, tongue, masseter, diaphragm), eye, sciatic nerve, trigeminal ganglion, pituitary gland and spinal cord (cervical, thoracic, lumbar) were immersion-fixed in 10% neutral-buffered formalin. The brain was cut sagittally; half was fixed in formalin and the remainder was frozen (−20° C). The formalin-fixed brain was cut into coronal sections, 2–4 mm wide. A minimum of 5 brain areas per animal were selected for examination by light microscopy. These were rostral cerebrum, hippocampus, superior colliculus, cerebellum, and medulla (at the level of obex). Two sections of spinal cord at each location (cervical, thoracic, and lumbar) were also selected for microscopic examinations. The tissues were processed for routine histopathologic examination, embedded in paraffin wax, and sectioned at 5 μm. All tissues collected at necropsy were stained with hematoxylin and eosin (HE) and examined by IHC for PrP^d, as described previously, ¹⁰ with a monoclonal antibody (F99/97.6.1). ¹³ This antibody recognizes PrP sequences conserved in most mammalian species in which natural TSEs have been reported.

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Table 1.

Findings in cattle intracerebrally inoculated with the agent of chronic wasting disease from white-tailed deer (CWD^wtd) and in uninoculated control cattle. ∗

Calf No.	Ear tag No.	Inoculum (CWDwtd)	Survival time (MPI)	CNS signs	IHC	WB
1	603	IC	18	+	+	+/++
2	600	IC	20	+	+	+
3	601	IC	20	+	+	+
4	610	IC	20	+	+	++
5	615	IC	21	+	+	+++
6	592	IC	22	+	+	++
7	604	IC	22	+	+	++
8	605	IC	22	−	+	++
9	606	IC	22	+	+	++
10	613	IC	22	+	+	++/+++
11	609	IC	23	+	+	++
12	611	IC	26	+	+	+++
13	612	IC	36	−	−	−
14	607	IC	36	−	−	−
15	591	Control	36	NA	−	−
16	A253	Control	36	NA	−	−

∗MPI = months postinoculation (mean = 21.5 MPI); CNS = central nervous system; IHC = immunohistochemistry for PrP^d; SE = spongiform encephalopathy; IC = intracerebral; NA = not applicable; + = lesions or antigen present; − = lesions or antigen absent; WB = Western blot for PrP^d; for WB reactivity − = no reactivity, + = weak, +/++ = weak to moderate; ++ = moderate, ++/+++ = moderate to strong; +++ = strong.

For detecting PrP^d in the brainstem by WB, a commercial kit (Bio-Rad, Marnes-La-Coquette, France) with antibody 6H4 (0.1 μg/ml of a mouse IgG1 monoclonal antibody antiserum raised against human residues 144–152 of the PrP peptide; Prionics AG, Switzerland) was used.

Results

Approximately 6 to 8 weeks before euthanasia, 12 of the 14 inoculated cattle gradually started to lose appetite and weight. Eleven of these showed intermittent abnormal neurologic signs (Table 1) that included various combinations of circling, bruxism, head pressing, depression, and nonresponsiveness to external stimuli. Between 18 and 26 MPI, 12 of the 14 calves became recumbent and were unable to rise without assistance; at this stage they were euthanatized. The 2 CWD-inoculated cattle without clinical signs and the 2 control calves were euthanatized 36 months after the study was initiated (Table 1).

Although the affected cattle had lost considerable weight, gross postmortem examination did not reveal obvious signs of emaciation, such as serous atrophy of fat.

Microscopic examination of HE-stained sections of brain and spinal cord (cervical, thoracic, and lumbar) failed to reveal characteristic lesions of spongiform encephalopathy (SE). In one animal (No. 5) a focal area of vacuolation in the neuropil was seen in one section of the brain (rostral colliculus) and a few isolated neurons with single, clear vacuoles of variable size were seen in the red nucleus of one control and four inoculated animals. Nonneuronal lesions consisted of the presence of a few sarcocysts in striated muscles of 12 of 16 cattle.

Of the 14 inoculated cattle, 12 had evidence of PrP^d in CNS tissue, as determined by IHC (Table 1). However, the intensity of immunoreactivity varied between individual animals and at different anatomic locations in the brain and the spinal cord (Table 2). In 2 cattle (Nos. 3 and 9), the immunoreactivity was limited in both amount and distribution within the brain (primarily colliculi and hippocampus) and was absent in the obex of No. 3. In the other 10 cattle there was moderate to extensive labeling in almost all brain sections, except for the cerebellum, where the amount of labeling was usually minimal and in 5 calves was absent. In the 7 cattle that had IHC reactivity in cerebellum, labeled areas were present primarily in white matter, and only sparse foci appeared in the granular layer. Similarly, the IHC labeling in sections of hippocampus and cerebrum was predominantly in white matter. In spinal cord, almost all cervical and lumbar samples had reactivity (restricted to grey matter), but several thoracic samples were negative. It should be noted that in the 2 cattle that had limited labeling in brain sections (Nos. 3 and 9), all spinal cord sections were IHC negative.

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Table 2.

Immunohistochemical (PrP^d) findings in cattle intracerebrally inoculated with the chronic wasting disease from white-tailed deer (CWD^wtd) and in uninoculated control cattle.

		Brain					Spinal cord
Calf No.	Ear tag No.	Obex	Cerebellum	Colliculus	Hippocampus	Cerebrum	Cervical	Thoracic	Lumbar
1	603	++	−	+++	++∗	++∗	++	++	++
2	600	++	++∗	+++	++∗	+∗	+	−	+
3	601	−	−	+	+∗	−	−	−	−
4	610	++	−	++	+++∗	++	++	+	+
5	615	+++	++∗	++	+++∗	++	+	−	+
6	592	+++	++∗	++	+++∗	++∗	++	+	++
7	604	++	++∗	+++	++∗	+∗	++	−	−
8	605	++	−	++	+++∗	++	++	+	+
9	606	+	−	+∗	+∗	−	−	−	−
10	613	++	+∗	++	+++	++∗	++	−	+
11	609	+++	++∗	++	+++∗	++	++	+	+
12	611	++	++∗	+++	++∗	++∗	++	+	+
13	612	−	−	−	−	−	−	−	−
14	607	−	−	−	−	−	−	−	−
15	591∗∗	−	−	−	−	−	−	−	−
16	A253∗∗	−	−	−	−	−	−	−	−

∗ =  immunoreactivity primarily or exclusively in white matter; ∗∗ = control animals; + = minimal, ++ = moderate, +++ = extensive.

In most immunolabeled sections, the pattern of labeling was multifocal (Fig. 1), although in some areas of grey matter the reactivity was so extensive that it was essentially diffuse. Most of the labeling appeared as discrete particles or particulate, often concentrated in or around glial cells, but small aggregates and plaques were sometimes present. Notably, intraneuronal, perineuronal (Fig. 2), and perivascular immunolabeling were not observed. Other than brain and spinal cord, PrP^d was not found in any of the other tissues examined (including multiple lymphoid tissues, retina, and pituitary gland).

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Fig. 1.

Brain; brainstem (obex) of calf No. 1. Note the multifocally distributed concentrations of particulate labeling of PrP^d (red) in the section. Labeled for PrP^d by IHC. Bar = 130 μm.

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Fig. 2.

Brain; brainstem (obex) of calf No. 1. Higher magnification of a focal area of PrP^d labeling. Particulate labeling is scattered in the neuropil with very little labeling associated with neurons (arrows). Labeled for PrPd by IHC. Bar = 90 μm.

WB analyses of brainstem samples from these animals revealed that all cattle inoculated with CWD^wtd and showing clinical signs of TSE disease (Table 1) were positive for the presence of PrP^d as detected by monoclonal antibody 6H4 (Fig. 3). In addition, calf No. 8 did not exhibit CNS signs (Table 1) and was also positive for PrP^d. In contrast, Nos. 13 and 14, which were inoculated with CWD^wtd and sacrificed 36 MPI (Table 1) as well as Nos. 15 and 16, the noninoculated control animals, were negative for PrP^d (Fig. 3). Both of the positive control samples, from a bovine infected with CWD^md and a white-tailed deer infected with CWD^wtd, had a strong positive WB reaction (Fig. 3).

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Fig. 3.

Western blot analysis of brain homogenates of cattle infected with CWD^wtd. M = protein weight marker; calf Nos. 2, 3, 1, 7, 8, 9: brain homogenates from cattle infected with CWD^wtd. No. 16 = brain homogenate from control cattle; Cattle MD CWD = brain homogenate from cattle infected with CWD^md; WTD CWD =  brain homogenate from white-tailed deer infected with CWD^wtd. Positive samples show a clear reaction with the 3 protease resistant polypeptide isoforms (di-, mono-, and unglycosylated) of PrP^d; no reaction is found with the negative sample No. 16. Monoclonal antibody 6H4 (Prionics AG, Switzerland) was used for immunodetection of PrP^d.

Discussion

This study used the intracerebral route of inoculation to provide the most efficient means of testing the absolute susceptibility of cattle to the agent of CWD from white-tailed deer and to provide a comparison with similarly designed previous studies. ^{2– 5, 7} Such studies do not inform on susceptibility by natural routes of transmission but, if negative, can provide evidence supporting the existence of a considerable species barrier. In instances of successful transmission they also provide an understanding of the descriptive pathology that might characterize the natural disease in the recipient species.

CWD, like all other TSEs, is characterized by a long incubation period, which in deer is seldom less than 18 months. ^{15, 16} In an experimental study of cattle inoculated intracerebrally with CWD from mule deer (first passage; CWD^md), PrP^d was demonstrated in only 5 of 13 cattle (38%), after incubation periods that ranged from 23 to 63 MPI. ^{4, 5} In contrast, 12 of 14 inoculated cattle in the present study were positive for PrP^d (86% attack rate) within 26 MPI (21.5 ± 0.5 MPI). The increased incidence and shorter incubation periods indicate that the CWD^wtd inoculum was more pathogenic in cattle than the CWD^md inoculum on first passage. ^{4, 5} However, there may be several reasons for these differences, such as genetic variability of the recipient hosts, the differences in PrP genotype of pooled brain material in the inoculum, and the infectivity titer of the inocula used in the CWD transmission studies of cattle. ^{4, 5} Regarding the latter, in a separate study of intracerebral CWD inoculation of white-tailed deer, we found both sources of CWD (from mule deer and white-tailed deer) successfully transmitted to each of two groups of white-tailed deer within two years, and there was no significant difference in incubation times and lesions between the groups (Kunkle et al., unpublished data).

In cervids, clinical CWD is characterized by emaciation, changes in behavior, and excessive salivation. ^{15, 16} Although the latter was not observed in the CWD^wtd-inoculated cattle, 12 of 14 inoculated calves showed anorexia and considerable weight loss, and the majority of these cattle also showed intermittent abnormal CNS signs. However, despite the advanced clinical signs in the affected cattle, none of the animals had histopathologic changes of SE, but all were positive for PrP^d by IHC and WB. The absence of morphologic lesions in cattle with cross-species transmitted TSEs has been documented previously with cattle inoculated intracerebrally with the sheep scrapie agent. ³ In that study, 100% of cattle succumbed to the disease between 14 and 18 months after inoculation with a US strain of the scrapie agent derived from sheep. None of the cattle showed microscopic lesions, and all were positive for PrP^d. A similar lack of morphologic changes was observed in cattle inoculated with CWD^md, even after the second passage. ^{4, 5, 8}

The predominant characteristics of IHC reactivity reported previously for cattle inoculated with mule deer CWD ^{4, 5, 7} were also observed in the diseased cattle that died after inoculation with CWD from white tailed-deer. The PrP^d accumulations were primarily multifocal and presumably glial cell–associated, with no evidence of intraneuronal or perineuronal labeling. The only difference noted between calves inoculated with CWD^md and CWD^wtd was that the latter seemed to show more extensive labeling, both in amount and anatomic distribution within the CNS. In addition, labeling of white matter was more prominent in the CWD^wtd-inoculated calves.

In this and in the earlier study of CWD^md in cattle, ^{4, 5} IHC labeling differed from that seen in cattle with bovine spongiform encephalopathy (BSE) or experimental transmissible mink encephalopathy (TME). ⁶ In both (BSE and TME) the IHC labeling was similar and was characterized by widespread diffuse (usually perineuronal) labeling of grey matter neuropil, with labeled particles that were not cell-associated except occasionally at neuronal cell membranes. ^{5, 14} The IHC pattern in bovine CWD also contrasts markedly with that seen in scrapie-inoculated cattle, in which intracytoplasmic labeling of neurons was a prominent feature. ^{2, 3}

Compared with experimental TME in cattle, ⁷ the experimental bovine CWD in this study was associated with less extensive IHC labeling in neural tissues other than brain and spinal cord. Whereas the retina was positive in all cattle inoculated with TME, none of the CWD-infected cattle in this experiment had any retinal labeling. Similarly, in the present study there was no labeling in the pituitary gland, a tissue that in some TME-infected cattle is positive. ⁷ The different affinities of tissues may reflect different abilities of these agents to amplify in these tissues. However, given sample sizes and uncontrolled experimental variables, other features, including host genetics, titers of inocula, and stage of disease cannot be excluded.

IHC labeling for PrP^d was not observed in striated muscles (heart, tongue, masseter, diaphragm) of the experimental calves. This observation is in accordance with our previous findings ⁹ in which striated muscle from 20 animals (cattle, sheep, elk, and raccoons) was examined for PrP^d by IHC. In these animals, all of which had developed a TSE after experimental inoculation, PrP^d was found by IHC examination in the CNS, but not in striated muscle. However, recent investigations with an enriched WB technique ¹² have enabled us to detect PrP^d in the tongues of some sheep and elk that were experimentally inoculated with scrapie and CWD, respectively (Bessen et al., unpublished data).

When brainstems of CWD-infected cattle were analyzed by WB for the presence of PrP^d, 12 of 14 samples were found to be positive for PrP^d (Table 1; Fig. 3), showing a clear reaction with the three protease resistant polypeptide isoforms (di-, mono-, and unglycosylated). Interestingly, No. 3, which was found by IHC to be negative in the obex area, but positive in midbrain region (Table 2), was found positive by WB (Fig. 3). This might indicate that the WB technique used for these studies could be more sensitive than the IHC method applied. However, this difference could also be attributable to the sampling of different portions of obex used for IHC and WB analyses. The WB molecular mass was similar in all cattle inoculated with CWD^wtd and similar to that of cattle-passaged CWD^md (Fig. 3). However, PrP^d isoforms from deer infected with CWD^wtd had a higher molecular mass (Fig. 3).

This study indicates a high susceptibility of cattle to the CWD^wtd agent by the most direct route of inoculation of the CNS, but it remains to be shown if natural routes of exposure would result in infection and disease and whether differences in attack rates and incubation periods between cattle inoculated with CWD^md and CWD^wtd agents reflect true differences in susceptibility or are attributable to differences in experimental factors.

Although susceptibility of cattle to intracerebral inoculation of CWD^wtd was demonstrated, it should be noted that this is an unnatural route, and it suggests only a potential for cattle to become infected under natural conditions of exposure. It is likely that transmission of CWD to cattle by a more natural route, such as per os, would require a much larger dose of inoculum and might even be difficult to accomplish within the normal life span of the animal. Because the present study resulted in a higher attack rate and shorter incubation periods than similar inoculation with CWD^md, the CWD^wtd agent would be the initial choice for experimental oral inoculation of cattle.

Further transmission studies of CWD isolates (e.g., CWD of elk or moose in cattle) are required to explore possible differences in clinicopathologic features that might indicate different disease phenotypes, which may in turn, reflect different CWD agent strains.

Finally, the findings of this study suggest that diagnostic techniques presently used for confirming BSE (IHC and WB) would also detect CWD^md and CWD^wtd in cattle should it occur naturally.