Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy

Bone, 14, 59-67, (1993)

8756-3282/93 $6 .00 + .00 Copyright © 1993 Pergamon Press Ltd .

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Canine Distemper Virus Transcripts Detected in the Bone Cells of Dogs with Metaphyseal Osteopathy A . P . MEE,' M . T . GORDON,' C . MAY, 2 D . BENNETT, 2 D . C . ANDERSON; and P . T . SHARPE' 'Dow Disease Research Centre, Department of Cell and Structural Biology, Stopford Building, University of Manchester . Manchester M13 9PT, U.K . 2 Department of Veterinary Clinical Science, Small Animal Hospital, University of Liverpool, Liverpool L7 7EX, U .K . 3 Department of Medicine, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New territories, Hong Kong Address for correspondence and reprints : Dr . P. T . Sharpe, Department of Cell and Structural Biology, Stopford Building, University of

Manchester, Oxford Road, Manchester, M13 9PT, U .K. Abstract

further evidence to suggest that the disease may be the result of a slow-virus infection by one of the Paramyxoviridae . Those viruses implicated include respiratory syncytial virus (Mills et al . 1981 ; Singer & Mills 1983; Pringle et al . 1985), simian virus 5 (Basle et al . 1985), parainfluenza virus type 3 (Basle et al . 1985), measles virus (MV) (Rebel et al . 1980 ; Singer & Mills 1983 ; Basle et al . 1985, 1986), and canine distemper virus (CDV) (Gordon et al . 1991) . Further to our previous findings that CDV mRNA is present within bone cells of patients with Paget's disease, we have recently shown that this virus can infect bone cells of the natural host (Mee et al . 1992) . The virus was concentrated within the growth plate region of the long bones, and was especially prominent in osteoclasts . In contrast to our findings in Paget's disease, positive hybridisation was seen with both sense and antisense probes . The localisation of CDV in the metaphyses was particularly interesting, since the initial lesion of the canine bone disease metaphyseal osteopathy is found within this region . Metaphyseal osteopathy (MO) is an idiopathic canine bone disorder, particularly prevalent in young, rapidly growing dogs of the larger breeds . Affected dogs usually present with pyrexia and metaphyseal pain and swelling, which may be marked (Meier et al . 1957 ; Grondalen 1976 ; Woodard 1982) . These clinical signs may be preceded by respiratory or gastrointestinal disease . Dogs suffering from MO often recover spontaneously, but relapses can occur, and periosteal and extraperiosteal new bone may form around the metaphyses . Eventually, this bony proliferation may extend to involve the diaphyses (Meier et al . 1957 ; Grondalen 1976 ; Woodard 1982) (Fig . 1) . Dogs are sometimes euthanased because of the intense pain associated with the metaphyseal lesions, or the severe bony deformity seen in chronic cases, and unexplained deaths are also reported . The initial radiographic features, of radiolucent lines parallel and close to the growth plate, are similar to those seen in infantile scurvy (Meier et al . 1957) (Fig. 2) . It has been suggested that lack of vitamin C is the cause of MO, but the radiolucent lines correspond to areas of elongation and failure of ossification of the cartilaginous lattice, inflammatory infiltrate (neutrophils and lymphocytes), and trabecular necrosis (Grondalen 1976 ; Woodard 1982), which are not seen in infantile scurvy . Furthermore, reports show that treatment with vitamin C has been variably successful (Meier et al . 1957 ; Grondalen 1976 ; Rendano et al . 1977 ; Vaananen & Wikman 1979 ; Teare et al . 1979; Woodard 1982), and one group of authors suggested that vitamin C treat-

Using the technique of in situ hybridisation, we have recently extended our observations that canine distemper virus (CDV) is present in the bone cells of patients with Paget's disease, and have shown that CDV is also detectable in the bone cells of dogs that are naturally infected with the virus . Since hybridisation was localised to bone cells within the metaphyses of the affected dogs, we investigated the possibility that CDV might be involved in the canine metaphyseal bone disorder, meitaphyseal osteopathy . Bone samples from three cases of metaphyseal osteopathy were examined for the presence of the CDV nucleocapsid (CDV-N) gene and the measles virus nucleocapsid (MV-N) gene, using MS-labelled sense and antisense riboprobes . As with our previous findings in Paget's disease of bone, only the antisense probe was found to hybridise to the osteoblasts and osteoclasts within the affected metaphyses . No hybridisation was seen with the CDV-N sense and MV-N probes in any of the samples tested . Bone samples were also taken from one of the cases to check for the presence of the CDV-N gene using the polymerase chain reaction (PCR) . Our findings with in situ hybridisation were confirmed by PCR and subsequent Southern blotting and probing with a 32P-labelled cDNA probe . The detection of CDV RNA within the bone cells of dogs with metaphyseal osteopathy suggests that this virus may be a cause of the disease and provides further, indirect evidence that CDV might be responsible for the bony abnormalities seen in Pag et's disease of bone . Key Words : Bone-Dog-Canine distemper virusMetaphyseal osteopathy-Paget's disease .

Introduction Paget's disease is a chronic skeletal disorder, characterised by excessive remodelling and deformity of bone (Kanis 1991) . In the U .K . it affects approximately 5% of the elderly population, with a disproportionate prevalence (in some towns up to 8%) in the North West region of the country (Barker et al . 1977, 1980) . Following the discovery of virus-like inclusions in osteoclasts from pagetic bone : (Rebel et al . 1974 ; Mills & Singer 1976 ; Gheradi et al . 1980; Howatson & Fornasier 1982), there has been 59

60

A . P . Mee et al . : Distemper and metaphyseal osteopathy in dogs

(a)

(b)

Fig. 1 . Metaphyseal osteopathy (chronic) . (a) Distal forelimb of a seven-month-old Great Dane showing marked swelling and deformity of the antebrachium . (b) Post-mortem specimen of the same limb . Note the extensive new bone formation extending along the length of the diaphyses (small arrows) and the calcification and swelling of the soft tissues (open arrows) . ment is contraindicated (Teare et al . 1979) . Other reports have suggested that overnutrition and excess vitamin and mineral intake might contribute to the disease, perhaps by causing an abnormal "stress" in rapidly growing bone (Hedhammar et al . 1974 ; Bennett 1976) . In view of our findings in distemper-infected dogs, we have examined for the presence of CDV, within the bone cells of three dogs suffering from MO, using sense and antisense radioactively labelled riboprobes to the CDV-N gene, and also, in one of the dogs, using the PCR technique with primers to the CDV-N gene . Materials and Methods Case details Metaphyseal osteopathy was diagnosed in all three cases, based on the clinical findings of pyrexia and pain localised to swollen metaphyses, and on the radiographic findings of irregular radio-

lucent lines running parallel to the growth plates in several of the long bones (Fig . 2)- Subsequent histology confirmed the diagnosis in each case . The details of each case are summarised in Table 1 . Tissue samples and preparation The samples taken from each case are summarised in Table II . Metaphyseal tissue was obtained, under sterile conditions, either at autopsy (within 30 minutes of euthanasia), or at surgery . A bone trephine was used to produce cores of tissue approximately 7 mm in diameter . Some of the samples were fixed in buffered formalin for 24 hours and then decalcified in 10% EDTA before being processed for routine paraffin-wax embedded histology . Sections (7 p.m) were mounted onto organosilanated slides (Rentrop et al, 1986) for in situ hybridisation . Other samples, from the metaphyses of case 1, were placed into liquid nitrogen and stored at -70° C for subsequent RNA extraction .



A . P . Mee et al . : Distemper and mctaphyscal osteopathy in dogs

61 Table 11. Samples and results Results Case

Samples taken

1

Left proximal tibia Right proximal tibia Left distal tibia Right distal radius Right distal ulna Right distal radius Right distal ulna Spicen Bladder Left distal radius

2

3

In

Situ

+ + + + + +

PCR + + nd nd nd nd nd nd nd nd

+ : Positive . - : Negative . nd: not done .

background . Control slides were treated with 100 .g/ml p RNase for 30 minutes at 37° C to render them RNA free . Hybridisation was carried out overnight at 50° C in 50% formamide and 0 .3 M NaCl, in a sealed, humidified container . Approximately 1 .0 x 105 counts of riboprobe was added to each section . Posthybridisation procedures included washing at 50° C and treatment with 20 lsgIml RNase A . The slides were then dipped in Ilford K5 emulsion diluted 1 :1 with distilled water, exposed for seven days, and developed . After counterstaining with haematoxylin and eosin, the sections were viewed by light and dark field microscopy .

Fig. 2. Metaphyseal osteopathy (acute) . Radiograph of the distal radius and ulna of the Japanese Akita in this study . Note the irregular radiolucent lines running parallel to the growth plates (arrows) .

Polymerase Chain Reaction In Situ Hybridisalion All molecular biology reagents and enzymes were obtained from Promega UK, or Boehringer Mannheim . To prevent contamination, all reagents were aliquoted and discarded following single usage (Innis et al . 1990) .

Riboprobes

Using the appropriate SP6 or T7 RNA polymerise, sense and antisense riboprob .s were prepared, using 73 S-UTP, by transcription of sequences to the CDV and MV nucleocapsid genes, which had been subcloned into the polylinker of the vector pSP72 (Gordon et al . 1991) . A probe to a-actin was used as a positive control, and a probe transcribed from a random SP6 vector sequence served as a further negative control . Probes were reduced to approximately 250 base pairs by limited alkaline hydrolysis (Angerer et al . 1987) .

Extraction of total RNA from bone

After fragmentation in a liquid nitrogen cooled "bomb," total RNA was isolated from the metaphyseal bone samples according to the method used by Chomczynski and Sacchi (1987) . Briefly, the bone particles were denatured in a solution of 4 .0 M guanidinium thyocyanate, 0 .1 M (i-mercaptoethanol, 25 mM sodium citrate, and 0 .5% sarcosine . They were then homogenised for 15 seconds in a polytron . Following isolation in an acidified phenoll-chlorofornd-isoamyl alcohol mix, the RNA was precipitated at -20° C in an equal volume of isopropanol and then resuspended in RNase free water and ethanol .

Hybridisation

The sections were hybridised according to the method described in detail by Gordon et al . (1991) . Briefly, sections were permeabilised with 0 .2 M HCI and 10 pgfml proteinase K . Treatment with 0 .25% acetic anhydride was used to reduce non-specific Table 1 . Case details Breed

Age

1

Japanese Akita

4 months

2

Boxer

3

Border Collie

Case

13 weeks 5 monthsb

Sex

Vaccination history'

Clinical outcome

male

4 weeks prior to MO

female

I week prior to MO

male

I

Initial improvement, relapse, euthanasia Neospora eaninum diagnosed, euthanasia Recovery after treatment

week after MO

Time of vaccination with polyvalent vaccine (including live CDV) in relation to first clinical signs of MO . 5First clinical siens of MO at nine weeks . but not seen at the University of Liverpool until five months old .



62

A . P . Mm et al . : Distemper and metaphyseal osteopathy in dogs

fig . 3. In situ hybridisation results . Low power, dark field views of the metaphyseal area, showing (a) positive hybridisation with the CDV-N antisense probe, and (b) no hybridisation (background level of silver grains) with the MV-N probe . Growth place (GP) . Magnification x60 . High power (c) light and (d) dark field views of the metaphyseal area, showing positive hybridisation with the CDV-N antisense probe . Osteoclast (OC) . Magnification X500 .



63

A . P . Mee et at . : Distemper and metaphyseal osteopathy in dogs

,, 016 .

.0

~

f.

;

4k

4

04" 0 i

Fig. 3. (contd) . No hybridisation with the CDV-N antisense probe in the bladder of case 2 viewed by (e) light and (f) dark field microscopy . Magnification X90 . No hybridisation with the CDV-N antisense probe in the spleen of case 2 viewed by (g) light and (h) dark field microscopy . Magnification x90 .



64

A . P . Mee et al . : Distemper and metaphyseal osteopathy in dogs a. a) Y i A

(I)

E

0 U

Ct D 0 a

(L

-CDV Fig . 5. Southern blot results . Positive hybridisation with the eDNA probe to CDV-N, showing as a dark band on the X-ray film (Probe), in the expected position (arrows) . Marker-pSP72 cut with Hpa 11 . Primers used were to the CDV-N gene (CDV) . carried out in laboratory premises distant from those used for the bone samples and with a different set of reagents . Reverse transcription

Fig. 3. (contd) . Low power dark field view (i) showing no hybridisation (background level of silver grains) with the CDV-N antisense probe in the bone of an uninfected dog (as previously reported (Mee et al . 1992)) . Growth Plate (GP) . Magnification X60 .

Single-stranded cDNA was synthesised by incubating approximately 20 .pg of the extracted total RNA (from either bone or veto cells) with 10 u of avian reverse transcriptase enzyme, I pg random hexamers, and 10 mM each of dATP, dCTP, dGTP, and dTTP, in a final volume of 20 id, at 42 ° C for one hour, A further 10 u of enzyme was then added and the incubation continued for another hour . To terminate the reaction, the mixture was then heated to 95 ° C and cooled on ice .

RNA extraction from vero cells PCR primers Viral RNA was also extracted from veto cells infected with the Onderstepoort strain of CDV (2 X 10 5 pfu/ml), or with the Edmonton strain of MV (2 x 105 pfu/ml) (Gordon et al . 1992) . Control cells were grown without virus . These experiments were

a m Y

m

2

M O

tRNA C

C

O > U 2

u

Q

Specific primers were manufactured to encompass the region of the CDV-N gene from position 1231 to 1464 bp, and also to incorporate restriction endonuclease sites ; hence the expected size of the fragment was 249 bp . Primers were also made for M V (position 601 to 1620) and (as a positive control) for exons 4 and 5 of the 3-actin gene . These gave fragments of expected size 1019 and 434 bp, respectively .

0 U

PCR reaction

2

Q

Fig. 4. PCR results . Photograph of an agamse gel viewed by ultraviolet light transillumination . Note bands of the expected size fm CDV-N (249 bp) (arrows) and for 0-actin (434 bpi using the cDNA from the dog with metaphyseal osteopathy (MO) . No band is seen with the primers to MV-N . No bands are seen with any of the primers using the eDNA tram reverse transcribed tRNA. Marker-pSP72 cut with Hpa II . Primers used were to the CDV-N gene (CDV), the MV-N gene (MV), and exons 4 and 5 of the (3-actin gene (Actin) .

Aliquots (5 µl) from the reverse transcription reaction were amplified in a solution of 20 mM Tris-HCI pH 8 .0, 1 .5 mM MgCl,, 100 mM each of dATP, dCTP, dGTP, and dTTP, 10 pmol each of the specific primers, and 2 .5 u of Taq DNA polymerise . The reaction volume was made up to 100 µl with nuclease free water, and two drops of mineral oil were added to each tube to prevent evaporation during the amplification reactions . The denaturation (94 ° C for 50s), annealing (55° C(CDV and (3-actin) or 65° C(MV) for I min), and extension (72 ° C for I min) reactions were repeated 40 times in a Techne cycler . To ensure complete strand synthesis, a final extension step at 72° C for 10 min was carried out . As a negative control, reverse transcribed tRNA was also used with each set of primers . After the final extension reaction, 30 0 aliquots from each reaction were run in a 2 .5% agarose gel in I X Tris-borateEDTA buffer . The DNA was then visualised with an ultraviolet light transilluminator .

65

A . P . Mee et at. : Distemper and metapbyseal osteopathy in dogs Southern blotting To confirm the identity of the bands of expected size, the DNA was transferred from the gel onto Gene Screen Plus membrane (NEN ® Research Products) and Southern blotted according to standard procedures (Ausubel et al . 1987) . The eDNA probe to CDV, obtained from Dr . S . L . Cosby in Belfast, was labelled with "P-dCTP, andd then hybridisation and subsequent washes were carried out at high stringency (1% SDS, 0.2 M SSC at 65° C) .

With the cDNA obtained from the MO tissue from case 1, bands of the expected size for the CDV-N gene (249 bp) and a-actin (434 bp) were visualised by ultraviolet illumination of the agarose gel (Fig . 4) . Subsequent Southern blotting and probing confirmed the identity of the 249 by fragment as CDV (Fig . 5) . With the MO eDNA, no band was seen with the primers to MV (Fig . 4) . No bands were seen with any of the primers using the cDNA from the reverse transcribed tRNA (Fig . 4) .

Discussion Results Histology The histological changes varied in severity between cases and, indeed, between different bones from the same case . Pathology was restricted to the metaphyseal areas of bone . The most prominent feature in all cases was an inflammatory infiltrate between the bony trabeculac, immediately adjacent to the physes . The infiltrate included both polymorphonuclear and mononuclear cells . There was also increased vascularity within the metaphyses and occasional thrombus formation ; haemorrhage and fibrin deposits were also sometimes seen . Many bony trabeculae were irregular, with loss of their normal orientation and, in some sections, this area of disruption appeared as a discrete band within the metaphyses, running parallel to the physis . Some trabeculae contained necrotic bone cells, and an increased number of osteoclasts was a common feature . Occasionally, the adjacent intertrabecular spaces, on the diaphyseal side of this disrupted area, were filled with a fibrocellular tissue which had replaced the marrow elements . In some sections, periosteal woven bone was evident, sometimes associated with trabecular organisation at its inner aspect . In situ hybridisation The specificity of the CDV and MV probes has been shown previously on both human and canine tissues (Gordon et al . 1991 ; Mee et al . 1992) . The results are summarised in Table II . In all three cases, positive hybridisation was seen in the bone samples with the CDV-N antisense probe, but not with the CDV-N sense or the MV-N probes (Fig. 3a-d) . Hybridisation was found in osteoclasts and osteoblasts in the diseased areas, but not in adjacent normal bone, in chondrocytes of the growth plate, or in epiphyseal bone . No hybridisation was seen with the CDV or MV probes in the bladder and spleen samples from case 2 (Fig . 3e-h) . As previously reported (Mee et al . 1992), there was no hybridisation with t h! CDV-N antisense (or sense) probe to bone cells of dogs that were not infected with distemper (Fig . 3i). Accessibility to hybridisation was confirmed in all samples using the p-actin probe, and no hybridisation was seen with the random SP6 vector sequence probe, or with any of the probes following RNase pre-treatment (not shown) .

Polymerase chain reaction The specificity and efficiency of the primers were confirmed using CDV, MV, or mock infected vero cells (Gordon et al . 1992) (results not shown here) . With the CDV-infected cells, a product of 249 by was seen with the CDV primers, and the primers to MV did not amplify . The converse was true for the MV-infected cells, where a product of 1019 by was seen with the MV primers . All primers were negative against uninfected vero cells .

Canine distemper is a highly contagious disease of dogs and other carnivores, which presents initially with respiratory and gastrointestinal signs . Neurological signs develop later in some cases (Appel 1987) . Using the technique of in situ hybridisation, we have previously shown that CDV infects metaphyseal bone cells in dogs with naturally acquired distemper (Mee et al . 1992) . In the present study, bone obtained from three young dogs with metaphyseal osteopathy (MO) was examined by PCR (one case) and in situ hybridisation (all three cases) for the presence of CDV RNA . A band of the expected size for the CDV-N gene was detected using PCR, and this was confirmed as CDV by subsequent Southern blotting and probing . The probe used has only approximately 40% homology with MV (Rozenblatt et al . 1985), and the levels of stringency used were sufficient to prevent any cross reaction with MV . There is always a risk of contamination with PCR, hence the use of in situ hybridisation as a further test for the presence of CDV . Using in situ hybridisation, positive hybridisation was seen within osteoblasts and osteoclasts using a probe to the CDV-N gene, again confirming the presence of the virus within the affected areas . The negative findings in the bladder and spleen samples from case 2 suggest that the specific localisation of CDV within the metaphyses was not an incidental finding consequent of viraemia. In contrast to our findings in the distemper-infected dogs, and in keeping with our findings in Paget's disease (Gordon et al . 1991), only the antisense probe (hybridising with mRNA) was positive for the bone cells . This suggests t hat . as in Paget's disease, viral genome is not present in large amounts in MO . This might be due to mutations within the genome that, although favouring transcription of the virus genome within the bone, might preclude active replication of the virus, Metaphyseal osteopathy has been reported in both vaccinated and unvaccinated dogs (Meier et al . 1957 ; Grondalen 1976 ; Rendano et al . 1977 ; Vaananen & Wikman 1979 ; Woodud 1982) . All of the dogs in this study were vaccinated with a polyvalent commercial vaccine (which included live attenuated CDV) . Two of the dogs were vaccinated prior to the onset of clinical signs of MO, one four weeks before and another one week before . However, it is not certain that the CDV RNA that we detected was from the vaccine, since the dogs could have had concurrent wild strain infections . If the vaccine strain does infect bone cells, it is possible that attenuation of the virus during manufacture of the vaccine causes mutations, or acts in some other way to prevent replication of the virus within the bone cells . In subacute sclerosing panencephalitis, a neurological disease caused by measles virus, mutations of the viral RNA are seen (Cattaneo et al . 1988), and we have recently detected similar mutations in CDV transcripts obtained from pagetic bone (Gordon et al . 1992) . It is possible that the failure of in situ hybridisation to detect genomic RNA of CDV in bone from both MO and Paget's disease patients is a result of these mutations . If this is the case, it may be that vaccination protects against the natural infection by preventing active replication of the virus, but allows transcription into mRNA . The third dog was vaccinated one week after the initial signs of MO . If CDV is responsible for

6

the disease, it is reasonable to assume that the positive finding in this case was due to wild type strain (although it is possible that the vaccine strain was also present) . The lack of CDV in some of

the bone samples from case I could be due to differing stages of the disease in each bone, or it may be that the levels of CDV in these samples were too low to be detected by in situ hybridisation . Our failure to detect genomic RNA of CDV could also be due to one or more virus or host factors, which include differences in infectivity of different strains of CDV (Appel 1987) . It may even be that we are detecting a previously unknown Morbillivirus that has high homology with CDV . Previous attempts to isolate an organism from bone affected with MO and to transmit the disease to other dogs have proved unsuccessful (Grdndalen 1979 ; Woodard 1982) . However, Grondalen reported that, of seven dogs given blood from dogs with MO, three developed symptoms of distemper . Reports in the literature provide further, circumstantial evidence that CDV might be involved . The initial clinical signs of pyrexia, pain, and swelling and the histological evidence of inflammation all support the theory that some infective agent is involved in the disease . Several authors report that the clinical manifestations of MO are often accompanied by, or preceded a few days earlier by respiratory or gastrointestinal symptoms (Meier et al . 1957 ; Gr9ndalen 1976 ; Rendano et al . 1977 ; Vaananen & Wikman 1979; Woodard 1982), both of which are seen with CDV infection . Meier et al . (1957) even described "distemper-like symptoms" in their study . Tooth enamel hypoplasia occurs as a sequel to CDV infection (Appel 1987), and has also been reported as a post mortem finding in three littermates that died with MO (Woodard 1982) . Bacterial osteomyelitis in young children occurs most commonly in the metaphyses, due to the hairpin loops of the metaphyseal vessels "trapping" bacterial emboli (Rhinelander & Wilson 1982), and it is possible that CDV could also localise in these areas . There are no previous clinical reports of viral osteomyelitis in the dog, although Boyce et al . (1983) reported metaphyseal lesions associated with CDV infection . The disease is also rare in humans, although several viruses, including rubella, variola, vaccinia, and cytomegalovirus, have been shown to produce metaphyseal bone lesions (Silverman 1976) . Experimental herpes virus infection in cats was also found to produce necrosis of metaphyseal bone (Hoover & Griesemer 1971) . The similarities between the lesions reported in human andd feline viral osteomyelitis and those seen in MO could support a viral aetiology for this canine disease . Our findings from both in situ hybridisation and PCR, that CDV RNA was present in bone cells from three cases of MO, support the hypothesis that CDV is involved in the pathogenesis of this disease . However, these results are only preliminary, and further cases will need to be evaluated to confirm the presence, or absence, of the virus . If our initial observations are confirmed and expanded, we believe that MO might prove valuable in understanding the pathogenesis of Paget's disease .

Acknowledgments: We would like to thank Dr. S . L . Cosby for the CDV

probe, P . Lockey and J . Brownie for help with preparing the tissue sections, and L. Lackey and 1 . Denton for assistance with the photographs . This study was funded by the National Association for the Relief of Paget's Disease (APM), the Sir Jules Thom Trust (MTG), the Wellcome Trust, and the Salford Paget's Disease Appeal.

References Angerer, L. M . ; States, M . H . ; Angerer, R . C . In situ hybridisation with RNA probes: An annotated recipe . Valentine, K. L. ; Eberweine, J . H. ; Harachas,

A . P . MM et al . : Distemper and metaphyseal osteopathy in dogs 1 . D ., eds . In situ hybridisation: Applications to neurobiology. New York, Oxford: Oxford University Press ; 1987 ; 42-70 . Appel, M . J . Canine distemper virus . Appel, M . J ., ed . Virus infections of venebrares . Amsterdam: Eksevier ; 1987 ; 133-159 . Ausubel, F . M . ; Brent, R . : Kingston, R . E. ; Moore, D . D . ; Seidman, J . G . ; Smith, 1 . A. ; Stmhl, K ., eds. Current protocols in molecular biology. New York : Wiley lnterscience; 1987. Barker, D . J . P . ; Clough, P . W . L . ; Guyer, P . B . ; Gardner. M. L Paget's disease of bone in 14 British towns . Br. Med. J . 1 :1181- 1183 ; 1977 . Barker, D . J . P . ; Chamberlain, A . T. ; Guyer, P . B . ; Gardner. M . J . Paget's disease of bone : The Lancashire focus. Br . Med . J . 280:1105-1107 ; 1980 . Basle, M . F . ; Russell, W . C . ; Goswami, K . K . A . ; Rebel, A . ; Girauden, P . ; Wild . F . ; Filmon, R. Paramyxovirus antigens in osteoclasts from Paget's bone tissue detected by monoclonal antibodies . J . Gen . Viral . 66 :2103-2110 ; 1985. Basic, M . F .'. Fournier, J . G. : Rozenblatt, S . ; Rebel, A . ; Bouteille, M . Measles virus RNA detected in Pager's disease bone tissue by in situ hybridisation . J. Gen. Viral. 67 :907-913 ; 1986. Bennett, D . Nutrition and bone disease in the dog and cat . Vet . Rec . 98:313--320 ; 1976 . Boyce, R . W . ; Axthelm, M. K . ; Krakowka, S . ; Weisbrode, 5 . E . Metaphyseal bone lesions associated with canine distemper virus infection . Proceedings of the Annual Meeting of the American College of Veterinary Pathologists, San Antonio, Texas, p . 126:1983 . Cattaneo, R . ; Schmid, A . ; Fschle, D . ; Baczko, K . ; Meulen, V . ; Billeter, M . A . Biased hypermutations and other changes in defective measles viruses in brain infection . Cell 55 :253-265 ; 1988 . Chomczynski, P. ; Sacchi . N. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction . Analytical Biochem . 162:156-159 ; 1987 . Gheradi, G .; La Cascio, V. ; Bonucci, E . I use structure of nuclei and cytoplasm of osteoclasls in Paget's disease of hone . Histopah . 4 :63-74 ; 1980 . Gordon, M . T . ; Anderson, D . C . ; Sharpe, P . T . Canine distemper virus localised in bone cells of patients with Paget's disease . Bone 12 :195-201 ; 1991 . Gordon, M . T . ; Men, A . P . ; Anderson, D . C . ; Sharpe, P . T . Canine distemper virus transcripts sequenced from pagetic bone . Bone Min . 19 :159-174 . Grendalen, J . Metaphyseal osteopathy thypertrophic osteodystrophy) in growing dogs . A clinical study. L Small Anim . Pena. 17 :721-735 ; 1976 . Gmndalen, 1 . Letter to the editor. J . Small Anim . Preer . 20 :t24; 1979 . Hedhammar, A. ; Wu, F . M. : Krouk, L . ; Schryver, H . F. ; de Lahunta, A . ; Whales, J . ;Kallfelz, F . A . ; Nunez, E . A . ; Hints, H . F. ; Shelly, B . 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Dare Received : February 28, 1992 . Date Revised: September 8, 1992 Dare Accepted: September 17, 1992