Abstract

This report describes ten farmed elk (Cervus elaphus) calves (four male, five female, one unknown sex) with cerebellar abiotrophy from western Canada. Calves ranged in age from six weeks to six months. The time from onset of disease to euthanasia of animals ranged between a few days to several weeks. Multiple calves within affected herds showed similar lesions. Two animals from one affected herd were related through a grandsire. There was significant reduction in the number of Purkinje cells, the width of the molecular cell layer, and the density of neurons in the granular layer of the cerebellum was also reduced in all cases examined. Diagnostic testing for potential causes such as nutritional deficiencies, toxicities or infectious agents was inconsistent among cases. No etiology for this disease was determined but a genetic cause is suspected.
 

Introduction

WR Gowers first used the term ‘abiotrophy’ in a lecture in 1902 (1) to describe premature degeneration or aging of tissues, especially within the nervous system, which was not due to external factors. Many of the diseases called abiotrophies have since been proven to be due to metabolic derangements within cells, and the majority of these are known or suspected to be genetically transmitted (2).

Cerebellar abiotrophy in domestic animals has been reviewed (2,3) but has not been reported in elk (Cervus elaphus). In recent years a number of farmed elk calves with clinical signs of cerebellar dysfunction and histological lesions typical of an abiotrophy have been presented to diagnostic laboratories in western Canada. This paper examines various clinical and pathological aspects of this disease presentation, and will compare this syndrome with cerebellar abiotrophies described in other species.
 

Materials and Methods

Between 1993 and 1999, ten farmed elk calves with a progressive neurologic disorder suspected to be a cerebellar dysfunction were presented to three laboratories in western Canada. Four males, five females and one calf of unknown sex ranging from six weeks to six months (approximate ages) were included in this study. All had similar clinical histories of being normal at birth followed by an acute onset of clinical signs referable to cerebellar disease. Clinical signs progressed over days to weeks until the calves were unable to function normally or were recumbent. All calves were euthanized.

Necropsy records and formalin-fixed, paraffin-embedded sections of brain and spinal cord from elk calves were obtained from three diagnostic laboratories in western Canada (Department of Veterinary Pathology, Western College of Veterinary Medicine (WCVM), Saskatoon SK; Prairie Diagnostic Services, Saskatoon, SK and Regina SK; Animal Health Laboratories Branch, Alberta Agriculture, Fairview AB). Tissues of nine of these calves were examined histologically. The cerebellar sections from calf #5 were not available for examination, and descriptions of histologic abnormalities were taken from the necropsy report. This calf was not included in statistical analysis. Between one and four slides that contained cerebellar tissue were available from the other calves. Hematoxylin and eosin stains were used on all tissues. Luxol Fast Blue (for myelin) and Holmes stains (for axons) were used on selected sections of cerebellar tissue. Purkinje cells were counted where the Purkinje cell layer formed a straight line across a field of view using a 20X objective lens (4). Granular cell and molecular layer thicknesses were measured using computer image analysis (Northern Eclipse 5.0, Empix Imaging Inc.). Twenty fields per glass slide of cerebellar sections were examined. Means of each variable were calculated for each animal and these values were compared with control animals using a signed rank test. A p-value of ≤0.05 was accepted as indicating significance.

Other diagnostic tests, used on individual animals, included: Fluorescent Antibody Test (FAT) for Rabies virus (1 calf); virus isolation, FAT and/or immunohistochemistry for Bovine Viral Diarrhea (BVD) virus (3 calves); liver copper status (3 calves); bacterial culture (4 calves); and fecal examination for parasites (1 calf).

Five neurologically normal farmed elk calves (three male, two female) ranging in age from 1-12 weeks that were presented to the Department of Veterinary Pathology, WCVM for necropsy were used as controls. The cause of death in four of these animals was determined to enteritis/enteropathy and one calf died due to malnutrition. Liver copper was evaluated in two of the control elk calves. Brain weights and weights of the cerebellums (after formalin fixation) of all of the control elk were recorded, as well as from seven other neurologically normal elk less than four weeks old whose brains were not examined histologically.
 

Results

Clinical History: All elk were ataxic and six were reported to have had a head tremor. This tremor was noted to become significantly worse when the calves were trying to eat or drink. The calves were alert, normal strength was maintained, and all were able to eat and drink, although three needed assistance to do so. Six of the calves were reported to be circling or falling to one side and five had sustained head injuries after stumbling and falling.

Pedigree/Herd History: Partial family histories were obtained for three of the ten calves. One (calf #2) was not related to any other calves in this study, but the owner reported the 3 other cases of cerebellar disease had been previously diagnosed on this ranch (Ranch B). All three previous suspected cases were female and shared the same sire. The sire in question was the grandsire to calf #2. Calves #5 and #6 were from Ranch D and were related through a grandsire (Fig. 3). Further history from this ranch indicated a similar clinical case (sex not reported) suspected to be abiotrophy in the early 1990's but no record of post mortem examination was found. This earlier suspected case had two ancestors in common with both calves #5 and #6. Calves #1 and #3 originated from the same ranch (Ranch A), calves #7 and #8 originated from Ranch E and calves #9 and #10 were from Ranch F but pedigree information was not available from this animals. Calf #4 originated from Ranch D and pedigree information was also not available from this animal.

Pathology: All calves were reported to be in fair to poor nutritional condition. Seven whole carcasses were submitted, fresh and formalin fixed portions were submitted from two calves, and the head and cervical spine were submitted from one calf. Five calves had recent traumatic injuries to the head including avulsion of the lower lip. The brains appeared grossly normal in five of the calves. In calves #1,2, 3, 4, and 10 the cerebellum appeared small and the folia were interpreted as thin by the examining pathologists. A small portion of the dorsal vermis of calf #2 was reported to lack grey matter.

Histological abnormalities in the cerebellum were seen or described (calf #5) in all affected calves and none of the control calves. The cerebellar folia were markedly thinned in all sections examined(Fig 1). There was a diffuse, moderate to severe depletion of Purkinje cells (Fig. 2). Purkinje cells present were often shrunken and hypereosinophilic, and interpreted to be necrotic. Large neurons resembling Purkinje cells were seen within the granular cell layer. The granular cell layer was irregular in thickness and appeared depleted of cells. Swollen axons were occasionally seen in the granular cell layer and clear spaces bordered by axons (‘empty baskets’) were seen at the external surface of the granular cell layer. In three of the affected animals the boundary between the granular cell layer and the Purkinje cell layer appeared vacuolated (Fig. 2). The molecular layer was also mildly to moderately thinned. In three calves (#4,8,10) occasional neurons in deep cerebellar nuclei were shrunken and hypereosinophilic. White matter tracts of the folia in calf #2 appeared pale and these areas stained poorly with Luxol Fast Blue. Swollen axons were frequently seen in the white matter of the folia. In calf #4 swollen myelin sheaths were noted in the ventral and lateral white matter tracts of the cervical spinal cord. No histologic abnormalities were noted in any of the control elk brains.

Quantitative analysis of the cerebellums is summarized in Table 1. Average counts of Purkinje cells in the clinical cases ranged from 1.28-5.1 cells/20X objective. This was significantly fewer (p=0.001) than that seen in the control animals (10.35-11.17). A significant difference was also seen in the thickness of the molecular layer (p=0.001). The molecular layer in control animals ranged in thickness between 0.293-0.307 mm, and in affected calves this layer ranged from 0.212-0.256 mm. No significant difference was seen in the thickness of the granular cell layer (p=0.383).

Other Diagnostic Tests: Bacterial culture was performed on tissues from calves #1, 4, 7, and 8. No significant pathogens were isolated. FAT for rabies virus was negative on calf #1. Liver copper was evaluated for calves #2 (19ppm- interpreted as deficient), #5 (116ppm-normal) and #8 (95ppm-normal). Virus isolation and/or immunohistochemistry for BVD virus were performed and were negative on calves #5, 6, and 7. Fecal examination for parasites was performed and was negative on calf #6.

Cerebellar weight as a percentage of total brain weight was evaluated on 12 neurologically normal farmed elk calves. The mean of these measurements was 10.41%. This is approximately the same as that reported for cattle of this age (5). Brain weights were not obtained from the affected elk so no comparison could be made. Liver copper values were obtained from two control animals. These values were 150ppm and 97ppm, respectively.
 

Discussion

The clinical histories and histological findings in these cases are consistent with cerebellar abiotrophy as it is described in other domestic species (2). Typically, affected animals are normal at birth followed within weeks to months by an acute onset of signs suggestive of diffuse cerebellar disease. Clinical signs are progressive and most affected animals are euthanized. Histologically, there is evidence of ongoing loss of Purkinje cells characterized by a lack of mature neurons as well as acute degenerative changes. Empty spaces on the external surface of the granular cell layer secondary to Purkinje cell loss and thinning of the molecular layer are also noted in this disease (6). Swollen axons are commonly seen in the granular cell layer. Secondary degeneration and subsequent loss of cells in the granular layer is a frequent finding, as granule cells are dependent on connections with the dendritic processes of mature Purkinje cells for their survival (3).

Cerebellar abiotrophy in Kerry Blue Terriers has been well described (7). These dogs are clinically normal at birth and a sudden onset of progressive cerebellar disease is seen in juvenile animals. Lesions begin in the Purkinje cell layer and are similar to those described in elk calves in this study. Retrograde degeneration secondary to Purkinje cell loss was seen in the olivary nuclei, caudate nuclei, and in the substantia nigra. Ultrastructural studies of affected puppies suggests that the damage to neurons may be mediated by the excitatory neurotransmitter, glutamate (8). A number of other breeds of dogs are reported to be affected by cerebellar abiotrophy (2,3,9-13) and lesions are generally restricted to the cerebellum.

Many cattle breeds are reported to be affected by this disorder (4,14,15) and it has also been reported in crossbred animals (16,17). A similar clinical history and histologic lesions as described in canines is seen. Diagnostic tests have failed to reveal infectious or toxic causes of this lesion and a familial pattern of disease occurrence is often noted. Cerebellar abiotrophy has also been diagnosed in numerous other species including sheep (18,19), swine (2), horses (20) and a number of laboratory animals (3).

Cerebellar hypoplasia can cause a clinical disease similar to cerebellar abiotrophy and must be considered as a differential diagnosis. However, lesions of cerebellar hypoplasia are distinct from those seen in cases of abiotrophy (21). Hypoplastic cerebellums are consistently reduced in size and their anatomical structure is abnormal. Although Purkinje cells are reduced in number and occasionally secondary degeneration of the granular cell layer is seen, evidence of orderly differentiation of Purkinje cells is lacking and ongoing destruction of neurons is not a prominent feature of this disease. The most common cause of cerebellar hypoplasia in cattle is BVD virus. Clinical disease produced by in-utero infection at approximately 150 days of gestation is generally present at birth and is nonprogressive. Tests for BVD virus in three affected elk calves were negative.

Inflammatory or infectious disease may also produce a clinical disease similar to abiotrophy. No inflammatory lesions were found in any of the affected animals, and no significant bacteria were isolated.

Other causes of cerebellar degeneration which were considered as possible etiologies include toxicities e.g. mercury, and deficiencies e.g. copper. These cannot be entirely ruled out, as in many of the clinical cases no toxicology was done. Liver copper was normal in two cases and low in another compared to values previously reported in adult farmed elk (22). A single report of cerebellar abiotrophy in an adult moose suspected to be due to copper deficiency is found in the literature (23). Clinical histories may be useful in ruling out these types of conditions as only single animals seemed to be affected within a herd in any given year. Detailed nutritional information and/or exposure to toxic substances is unknown in most of these cases, but if a toxin was the cause it is likely that multiple animals would have been affected and possibly from different age groups.

Lesions in all ten calves examined are consistent with cerebellar abiotrophy as it is described in other species. Several problems were encountered in this study which made assessment of potential etiologies difficult. Cerebellar abiotrophy is a progressive disease and a certain degree of variability in the severity of lesions is to be expected depending on the duration of the clinical disease. Time between recognition of clinical disease and euthanasia of the calves ranged from a few days to a few weeks and this variation may explain the difference in severity of the histologic lesions. In some species where cerebellar abiotrophy has been described, neuronal necrosis has been noted in areas other than the cerebellum. However, such animals were followed clinically for several weeks to months prior to euthanasia. It is possible that the elk calves in this study were not alive long enough for the development of extracerebellar lesions.

A moderate degree of irregularity in the distribution of Purkinje cells and variability in the thickness of both the granular and molecular layers was noted in the brains of the control animals. This, combined with the relative sensitivity of the cerebellum to artifactual loss of neurons post mortem, may have influenced the results of objective measurements.

Specific diagnostic tests varied among cases. This inconsistency makes determination of an etiology impossible at this time. Testing for BVD virus and bacterial culture were negative in the cases evaluated. Analysis of liver copper levels was inconclusive. Pedigree and herd history suggested a familial association.

Clinical histories and histologic lesions are consistent with a diagnosis of cerebellar abiotrophy in all cases examined and a genetic cause is suspected. Further studies are warranted including additional toxicologic analysis, further evaluation for BVD virus, and possible test breeding of suspected carriers.
 

Table 1
Quantitative analysis of cerebellums. Data are means of multiple measurements (20 fields/slide) from each animal.

N/E= not evaluated

* mean value of 5 control elk
 
Case Number     Purkinje Cells/20X field     Granular Layer width (mm)     Molecular Layer width (mm)
        1                                 3.0                                         0.218                                             0.221
        2                                 2.4                                         0.206                                             0.210
        3                                 1.79                                       0.204                                             0.221
        4                                 1.5                                         0.212                                             0.256
        5                                 N/E                                         N/E                                                 N/E
        6                                 2.0                                         0.194                                             0.212
        7                                 1.43                                       0.203                                             0.228
        8                                 1.28                                       0.141                                             0.219
        9                                 5.1                                         0.201                                             0.231
        10                               3.2                                         0.208                                             0.221
Controls*                          10.67                                     0.198                                             0.286

 

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