CASE REPORTS HlSTlOCYTlC ULCERATIVE COLITIS IN A BOXER DOG F. W. G. HILL’’’ and N.D. SULLIVAN1 SUMMARY: A 2-year-old male Boxer dog had passed loose faeces mixed with fresh blood and mucus for 8 months. Tenesmus after defaecation was a feature. Colitis was diagnosed from the proctoscopic appearance of the recto-colonic mucosa and confirmed from a biopsy. The disorder proved unresponsive to sulphasalazine therapy, but oral chloramphenicol. betamethasone and prednisolone enemas administered over a 6-week period produced a satisfactory clinical improvement, which persisted for a further 3 weeks without treatment. However, follow-up proctoscopy showed only a marginal improvement in the appearance of the mucosa and appeared to exacerbate further bloody diarrhoea, which persisted. The dog was destroyed and histiocytic ulcerative colitis confirmed at autopsy.
Introduction
History and Clinical Details
Histiocytic ulcerative colitis ( H U C ) of Boxer dogs was first described in the United States by Van Kruiningen, Montali, Strandberg and Kirk (1965). These authors chose to define the disease as a granulomatous colitis, characterised morphologically by a diffuse inflammation of the mucosa and submucosa of the descending colon and rectum. In the more advanced cases, the caecum, ascending colon and dependent lymph n o d e s may. b e m i l d l y a f f e c t e d . T h e histopathological feature is the infiltration and distortion of the lamina propria and stroma of the submucosa by niacrophages, whose cytoplasm stains strongly periodic acid - Schiff (P.A.S.) positive. Subsequently there have been reports of the disease from other locations in the United States (Kennedy and Cello 1966; Koch and Skelley 1967; Van Kruiningen 1967; Sander and Langham 1968; Lawson, Gomez, Stewart, Rambo a n d Margulis 1970; Russell, Gomez and Trowbridge 1971; Cockrell and Krehbiel 1972; Ewing and Gomez 1973) and from Holland (Gaag, Happe and Wolvekanip 1975). Confusion results from the different terminology used by authors to describe the disorder. Thus in Boxer dogs, colitis; granulomatous colitis; colitis resembling Whipple’s disease and the more descriptive histiocytic ulcerative colitis (Sanders and Langham 1968) are likely to be synonymous terms describing the same clinical entity. H U C of Boxer dogs has not previously been reported in Australia.
The subject, a 2-year-old male Boxer was referred to the Veterinary School Clinic on 3/3/77. For the previous eight months the dog had passed intermittently fresh blood, either mixed in loose faeces o r with mucus. The referring veterinarian’s treatment consisting of o r a l courses of trimethoprim a n d sulphadiazine, triple sulphonamide and astringents had controlled the signs initially. But, for the last two months, the dog passed persistently bloody diarrhoea in spite of the treatment. The owner reported that the dog was passing a motion, a t least 12 times daily with, at times, tenesmus after defaecation. Physical examination revealed n o obvious abnormality apart from bilateral cryptorchidism. The dog was well nourished (bodyweight 22.5 kg) and of an excitable, nervous disposition. The rectal temperature was 38.6”C and femoral pulse was 96/min. The red and white blood cell counts were within normal limits. A smear and routine culture of a rectal swab failed to demonstrate any significant bacteria. A routine faecal examination for parasites proved to be negative. In hospital the reported dysentery was confirmed. A proctoscopic (St. Mark’s Major proctoscope set, Adult sigmoidoscope tube) examinatipn of the mucosa of the rectum a n d distal colon, under acepromazine maleate ( 1 mg) and thiopentone sodium (2.5%) general anaesthesia, showed generalised thickening, loss of normal folds, and corrugation. There was hyperaemia with ecchymoses, shallow erosions a n d haemorrhages. The latter may have been a result of the procedure, since they were more aparent as the instrument was withdrawn from the lumen. The lesions were seen to extend beyond the 25 cm length of the proctoscope. A mucosal biopsy confirmed the 441
presence of a colitis. without evidence of a specific in f i l trn t ion of h ys t ioc!,tes. A low residue diet, and an oral course of 500 nig sulphasalazine (salazopyrin, Pharmacia) tablets and 0.25 nig betarnethasone tablets both t.i.d. were prescribed for the dog. After a week the owner reported that there had been 110 improvement in the dog's condition. The animal was hospitalized and treatment continued for a further seven days. Loose bloody faeces were passed persistently and vornition of food became a daily feature. Treatment was changed to oral chloramphenicol 250 nig capsule b.i.d. Betamethasone therapy was continued and a 50 ml prednisolone enema (predsol enema, Glaxo) \vas given once daily. Over the next fourteen days there was a gradual improvement i n faecal consistency, and a reduction in the number of motions voided. Betamethasone tablet5 arid the enemas were stopped. A repeat proctoscopic examination showed there was n o appreciable improvement i n the appearance of the mucosa. Chloramphenicol was continued Tor a further tnenty-eight days and 60 rng phenobarbitone b.i.d. were given to reduce boisterousnes\ and oierexciternent. At the end of this period the dog pas\ed faeces of a firm consistency t\\ice daily. A small volume of fresh blood was always ebident on the faeces. Very occasionall!. blood and mucus \\ere passed alone. After eight weeks of continuous therapy all treatment was btopped. Over the next three weeks faecal consistency rernaincd firm but continued to be tinged u i t h fresh blood. Proctoscopic exarnination of the rnucosa revealed hyperaemia and thickening up to a distance of 15 cm from the anus, w i t h several raised haemorrhagic sites in the rnucosa. Elsewhere there appeared to be some improvement i n the appearance. Following on proctoscopy, loose bloody diarrhoea recurred and continued. The dog was destroyed, at the owner's request, 10 neeks after admission. The dog's pedigree was not a\,ailable for scrutiny.
tensity of the lesions in affected areas was subject to considerable variation. The inflammatory changes were most prominent in the subrnucosa which appeared distended and oedematous. The most striking feature of the inflammatory reaction \bas the accumulation of numerous large histiocytic cells whose abundant cytoplasm stained pink with Iiaeniatoxylin-eosin and had a red globular appearance with PAS (Figure I ) . These histiocytes were concentrated in the subrnucosa but could also be found in the glandular layer especially in its basal portions. In the submucosa there were only a few mononuclear cells admixed with the histiocytes but there was pronounced infiltration of the glandular lamina propria by plasma cells and lymphocytes. There were focal areas of usually shallow rnucosal ulceration which were associated ivith evidence of haemorrhage and neutrophilic infiltration of the underlying lamina propria. Some of the glandular crypts were dilated and in these there was regression of the epithelium towards a lo~v cuboidal type. I n the colonic rnesenteric lymph nodes there were sinusoidal oedenia. haemorrhage, erythrophagocytosis and haernosiderosis. Scattered throughout the nodes were light accumulations of the typical PASpositive his t i oc y t es .
Pathology
The dog was in reasonable body condition (21 kg bodyweight) and significant findings were confined to the alirnentar) tract. No helniinth parasites Here seen. The mucosae of the colon and rectum were thickened, corrugated and congested. There were four raised bleeding points in the rnucosa approximately 70 cm from the anus. Distal rnesenteric lymph nodes n e r e prominent. Selected tissue blocks of the colon, rectum and rnesenteric lyrnph nodes were fixed in forniolsaline, embedded in paraffin, sectioned at 6 um and stained w i t h haernatoxylin-eosin and PAS. The microhcopic changes in the colon \vere sornewhat patchy i n their occurrence and the in448
Figure 1 The colonic submucosa is distended with the characteristic histiocytic cells and there are minor accumulations of histlocytes adjacent to the basal lamina o f the glandular mucosa There is mononuclear cell infillration In the mucosa and some 01 the glands appear distended In this area there is only slight damage to the superficial epithelium PAS x 63
Au5tralian Vrrerinarj ./ourna/. Vol. 54, September, 1978
Discussion
The clinical signs of H U C may have features in common with, severe whipworm infestation, salmonellosis, colonic and rectal neoplasia, intussusception of the terminal ileum into colon and some forms of rectal impaction. Clinical features and investigative procedures indicated that none of these disorders were present in this case. The occurrence of persistent loose faeces tinged with fresh blood and mucus in young Boxer dogs should arouse suspicion of H U C . A greatly increased number of motions per day and tenesmus after defaecation are frequent features, otherwise affected dogs appear healthy. In the present case, a diagnosis of colitis was readily made, once the disorder had been suspected, from the gross appearance of the mucosa a n d the histological examination of biopsy specimens obtained at the proctoscopic examination. A problem for us, not mentioned in previous reports, was that characteristic histiocytes were absent from the mucosal biopsy specimens. These did not include lamina propria o r submucosa. Although we suspected H U C , a definitive diagnosis was only possible at autopsy, when all the large intestine and full thickness specimens of its wall were available for study. Other authors (Randell et a1 1971) have also noted the patchy distribution of the lesions in the descending colon a n d rectum and the vertical transition from acute to chronic inflammatory cell types within the mucosae, PASpositive staining histiocytes predominating in the deeper lamina propria and submucosa. Multiple biopsies of the colon a n d rectal wall t o include both the mucosa a n d submucosa may be necessary to obtain a diagnosis in life. Review of previously published cases indicates that once established H U C may be very refractory to treatment. The encouraging clinical response t o oral chloramphenicol therapy in the reported case is consistent with the finding of Van Kruiningen et a/ (1965). However this response may have been further favourably influenced by the simultaneous steroid therapy. The clinical improvement was not accompanied by a n overall improvement in either the gross o r histological appearance of the colonrectal mucosa and suggests the disease was only in remission and in a sub-clinical form. The recurrence of signs following stress, in this case a general anaesthetic and follow-up proctoscopic procedure, has been recorded previously (Lorenz 1975) and there a r e undocumented reports and beliefs amongst clinicians that the disease is influenced and exacerbated in affected o r susceptible dogs by stress o r trauma. In this respect H U C would appear to be very similar to human ulcerative colitis. At present the aetiology and epidemiology of Australian Veterinary Journal, Vol. 54, September, 1978
the disease is unclear. Van Kruiningen (1975) believes the likely infectious casual agent to be a lipid-, ribosome-rich, coccoid to coccobacillary organism. But a more recent ultrastructural study of the early mucosal lesion by Gomez et al(l977) failed to find convincing evidence for an infectious casual agent and any micro-organisms seen were considered to be opportunist secondary invaders. A genetic predisposition may underlie the disease process. The fact that H U C apparently occurs only in purebred Boxer dogs a breed that has been closely bred, suggests this possibility (Russell et a1 1971). Nothing comparable to the characteristic H U C macrophage infiltrate exists in chronic idiopathic ulcerative colitis of man, and although clinically there are many similarities, pathologically the two diseases are distinct (Kennedy a n d Cello 1966). Ulcerative colitis is recorded in other breeds of dogs (Ewing and Gomez 1973) and occurs sporadically in Afghans and other hound and working breeds (Hill, unpublished data). The proctoscopic appearance of the colon-rectal mucosa is similar to that of H U C but the disorders are histologically distinct. In these affected dogs there is a marked clinical response to sulphasalazine therapy combined with a low residue diet and to which is added flake bran. Acknowledgments We are grateful to Professor P.C. Kennedy, University of California and Dr D.F. Kelly, University of Bristol, for examining histological sections from this case and concurring with the diagnosis. References Cockrell, B . Y . and Krehbiel, J.D. (1972) - Am. 1. vet. Res. 33: 453. Ewing, G.O. and Comez, J.A. (1973) - J . Am. Anim. Hosp. Ass. 9: 395. Caag, 1. van Der., Happe, R.P. and Wolvekamp, W.T.C. ( 1975) - Proceedings Voorjaradagen Netherlands Small Animal Veterinary Association, P A 0 No. 6, 19-23. Comez, J .A ., Russell, S.W., Trowbridge, J.O. and Lee, J. (1977) - Am. J . dig. Dis. 22: 485. Kennedy, P.C. and Cello, R.M. (1966) - Gastroenterology. 51: 926. Koch, S.A. and Skelley, J.F. (1967) - J. Am. vet. med. Ass. 150: 22. Lawson, T.L., Gomez, J.A., Stewart, E.T., Rambo, O.N. and Margulis, A.R. (1970)- Am. J. Roent. 110: 377. Lorenz, M.D. (1975) - Textbook of Veterinary Internal Medicine Ed. S.J. Ettinger, W.B. Saunders Co., Philadelnhia. Vol. 2. on. 1203-1208. Russell, S.*W.,'Goniez,'j.A. and Trowbridge, J.O. (1971) Lab. Invest. 25, 509. Sander, C.H. and Langham, R.F. (1968) - Arch. Path. 85: 94. Van Kruiningen, H.J., Montali, R.J., Strandberg, J.D. and Kirk, R.W.(1965)- Path. V e t . 2 : 5 2 1 . Van Kruiningen, H.J. (1967) - Gastroenterology, 53: 114. Van Kruiningen, H.J. (1975) - Vet. Path. 12: 446. (Received forpublication 10 November 1977) 449
Introduction
History and Clinical Details
Histiocytic ulcerative colitis ( H U C ) of Boxer dogs was first described in the United States by Van Kruiningen, Montali, Strandberg and Kirk (1965). These authors chose to define the disease as a granulomatous colitis, characterised morphologically by a diffuse inflammation of the mucosa and submucosa of the descending colon and rectum. In the more advanced cases, the caecum, ascending colon and dependent lymph n o d e s may. b e m i l d l y a f f e c t e d . T h e histopathological feature is the infiltration and distortion of the lamina propria and stroma of the submucosa by niacrophages, whose cytoplasm stains strongly periodic acid - Schiff (P.A.S.) positive. Subsequently there have been reports of the disease from other locations in the United States (Kennedy and Cello 1966; Koch and Skelley 1967; Van Kruiningen 1967; Sander and Langham 1968; Lawson, Gomez, Stewart, Rambo a n d Margulis 1970; Russell, Gomez and Trowbridge 1971; Cockrell and Krehbiel 1972; Ewing and Gomez 1973) and from Holland (Gaag, Happe and Wolvekanip 1975). Confusion results from the different terminology used by authors to describe the disorder. Thus in Boxer dogs, colitis; granulomatous colitis; colitis resembling Whipple’s disease and the more descriptive histiocytic ulcerative colitis (Sanders and Langham 1968) are likely to be synonymous terms describing the same clinical entity. H U C of Boxer dogs has not previously been reported in Australia.
The subject, a 2-year-old male Boxer was referred to the Veterinary School Clinic on 3/3/77. For the previous eight months the dog had passed intermittently fresh blood, either mixed in loose faeces o r with mucus. The referring veterinarian’s treatment consisting of o r a l courses of trimethoprim a n d sulphadiazine, triple sulphonamide and astringents had controlled the signs initially. But, for the last two months, the dog passed persistently bloody diarrhoea in spite of the treatment. The owner reported that the dog was passing a motion, a t least 12 times daily with, at times, tenesmus after defaecation. Physical examination revealed n o obvious abnormality apart from bilateral cryptorchidism. The dog was well nourished (bodyweight 22.5 kg) and of an excitable, nervous disposition. The rectal temperature was 38.6”C and femoral pulse was 96/min. The red and white blood cell counts were within normal limits. A smear and routine culture of a rectal swab failed to demonstrate any significant bacteria. A routine faecal examination for parasites proved to be negative. In hospital the reported dysentery was confirmed. A proctoscopic (St. Mark’s Major proctoscope set, Adult sigmoidoscope tube) examinatipn of the mucosa of the rectum a n d distal colon, under acepromazine maleate ( 1 mg) and thiopentone sodium (2.5%) general anaesthesia, showed generalised thickening, loss of normal folds, and corrugation. There was hyperaemia with ecchymoses, shallow erosions a n d haemorrhages. The latter may have been a result of the procedure, since they were more aparent as the instrument was withdrawn from the lumen. The lesions were seen to extend beyond the 25 cm length of the proctoscope. A mucosal biopsy confirmed the 441
presence of a colitis. without evidence of a specific in f i l trn t ion of h ys t ioc!,tes. A low residue diet, and an oral course of 500 nig sulphasalazine (salazopyrin, Pharmacia) tablets and 0.25 nig betarnethasone tablets both t.i.d. were prescribed for the dog. After a week the owner reported that there had been 110 improvement in the dog's condition. The animal was hospitalized and treatment continued for a further seven days. Loose bloody faeces were passed persistently and vornition of food became a daily feature. Treatment was changed to oral chloramphenicol 250 nig capsule b.i.d. Betamethasone therapy was continued and a 50 ml prednisolone enema (predsol enema, Glaxo) \vas given once daily. Over the next fourteen days there was a gradual improvement i n faecal consistency, and a reduction in the number of motions voided. Betamethasone tablet5 arid the enemas were stopped. A repeat proctoscopic examination showed there was n o appreciable improvement i n the appearance of the mucosa. Chloramphenicol was continued Tor a further tnenty-eight days and 60 rng phenobarbitone b.i.d. were given to reduce boisterousnes\ and oierexciternent. At the end of this period the dog pas\ed faeces of a firm consistency t\\ice daily. A small volume of fresh blood was always ebident on the faeces. Very occasionall!. blood and mucus \\ere passed alone. After eight weeks of continuous therapy all treatment was btopped. Over the next three weeks faecal consistency rernaincd firm but continued to be tinged u i t h fresh blood. Proctoscopic exarnination of the rnucosa revealed hyperaemia and thickening up to a distance of 15 cm from the anus, w i t h several raised haemorrhagic sites in the rnucosa. Elsewhere there appeared to be some improvement i n the appearance. Following on proctoscopy, loose bloody diarrhoea recurred and continued. The dog was destroyed, at the owner's request, 10 neeks after admission. The dog's pedigree was not a\,ailable for scrutiny.
tensity of the lesions in affected areas was subject to considerable variation. The inflammatory changes were most prominent in the subrnucosa which appeared distended and oedematous. The most striking feature of the inflammatory reaction \bas the accumulation of numerous large histiocytic cells whose abundant cytoplasm stained pink with Iiaeniatoxylin-eosin and had a red globular appearance with PAS (Figure I ) . These histiocytes were concentrated in the subrnucosa but could also be found in the glandular layer especially in its basal portions. In the submucosa there were only a few mononuclear cells admixed with the histiocytes but there was pronounced infiltration of the glandular lamina propria by plasma cells and lymphocytes. There were focal areas of usually shallow rnucosal ulceration which were associated ivith evidence of haemorrhage and neutrophilic infiltration of the underlying lamina propria. Some of the glandular crypts were dilated and in these there was regression of the epithelium towards a lo~v cuboidal type. I n the colonic rnesenteric lymph nodes there were sinusoidal oedenia. haemorrhage, erythrophagocytosis and haernosiderosis. Scattered throughout the nodes were light accumulations of the typical PASpositive his t i oc y t es .
Pathology
The dog was in reasonable body condition (21 kg bodyweight) and significant findings were confined to the alirnentar) tract. No helniinth parasites Here seen. The mucosae of the colon and rectum were thickened, corrugated and congested. There were four raised bleeding points in the rnucosa approximately 70 cm from the anus. Distal rnesenteric lymph nodes n e r e prominent. Selected tissue blocks of the colon, rectum and rnesenteric lyrnph nodes were fixed in forniolsaline, embedded in paraffin, sectioned at 6 um and stained w i t h haernatoxylin-eosin and PAS. The microhcopic changes in the colon \vere sornewhat patchy i n their occurrence and the in448
Figure 1 The colonic submucosa is distended with the characteristic histiocytic cells and there are minor accumulations of histlocytes adjacent to the basal lamina o f the glandular mucosa There is mononuclear cell infillration In the mucosa and some 01 the glands appear distended In this area there is only slight damage to the superficial epithelium PAS x 63
Au5tralian Vrrerinarj ./ourna/. Vol. 54, September, 1978
Discussion
The clinical signs of H U C may have features in common with, severe whipworm infestation, salmonellosis, colonic and rectal neoplasia, intussusception of the terminal ileum into colon and some forms of rectal impaction. Clinical features and investigative procedures indicated that none of these disorders were present in this case. The occurrence of persistent loose faeces tinged with fresh blood and mucus in young Boxer dogs should arouse suspicion of H U C . A greatly increased number of motions per day and tenesmus after defaecation are frequent features, otherwise affected dogs appear healthy. In the present case, a diagnosis of colitis was readily made, once the disorder had been suspected, from the gross appearance of the mucosa a n d the histological examination of biopsy specimens obtained at the proctoscopic examination. A problem for us, not mentioned in previous reports, was that characteristic histiocytes were absent from the mucosal biopsy specimens. These did not include lamina propria o r submucosa. Although we suspected H U C , a definitive diagnosis was only possible at autopsy, when all the large intestine and full thickness specimens of its wall were available for study. Other authors (Randell et a1 1971) have also noted the patchy distribution of the lesions in the descending colon a n d rectum and the vertical transition from acute to chronic inflammatory cell types within the mucosae, PASpositive staining histiocytes predominating in the deeper lamina propria and submucosa. Multiple biopsies of the colon a n d rectal wall t o include both the mucosa a n d submucosa may be necessary to obtain a diagnosis in life. Review of previously published cases indicates that once established H U C may be very refractory to treatment. The encouraging clinical response t o oral chloramphenicol therapy in the reported case is consistent with the finding of Van Kruiningen et a/ (1965). However this response may have been further favourably influenced by the simultaneous steroid therapy. The clinical improvement was not accompanied by a n overall improvement in either the gross o r histological appearance of the colonrectal mucosa and suggests the disease was only in remission and in a sub-clinical form. The recurrence of signs following stress, in this case a general anaesthetic and follow-up proctoscopic procedure, has been recorded previously (Lorenz 1975) and there a r e undocumented reports and beliefs amongst clinicians that the disease is influenced and exacerbated in affected o r susceptible dogs by stress o r trauma. In this respect H U C would appear to be very similar to human ulcerative colitis. At present the aetiology and epidemiology of Australian Veterinary Journal, Vol. 54, September, 1978
the disease is unclear. Van Kruiningen (1975) believes the likely infectious casual agent to be a lipid-, ribosome-rich, coccoid to coccobacillary organism. But a more recent ultrastructural study of the early mucosal lesion by Gomez et al(l977) failed to find convincing evidence for an infectious casual agent and any micro-organisms seen were considered to be opportunist secondary invaders. A genetic predisposition may underlie the disease process. The fact that H U C apparently occurs only in purebred Boxer dogs a breed that has been closely bred, suggests this possibility (Russell et a1 1971). Nothing comparable to the characteristic H U C macrophage infiltrate exists in chronic idiopathic ulcerative colitis of man, and although clinically there are many similarities, pathologically the two diseases are distinct (Kennedy a n d Cello 1966). Ulcerative colitis is recorded in other breeds of dogs (Ewing and Gomez 1973) and occurs sporadically in Afghans and other hound and working breeds (Hill, unpublished data). The proctoscopic appearance of the colon-rectal mucosa is similar to that of H U C but the disorders are histologically distinct. In these affected dogs there is a marked clinical response to sulphasalazine therapy combined with a low residue diet and to which is added flake bran. Acknowledgments We are grateful to Professor P.C. Kennedy, University of California and Dr D.F. Kelly, University of Bristol, for examining histological sections from this case and concurring with the diagnosis. References Cockrell, B . Y . and Krehbiel, J.D. (1972) - Am. 1. vet. Res. 33: 453. Ewing, G.O. and Comez, J.A. (1973) - J . Am. Anim. Hosp. Ass. 9: 395. Caag, 1. van Der., Happe, R.P. and Wolvekamp, W.T.C. ( 1975) - Proceedings Voorjaradagen Netherlands Small Animal Veterinary Association, P A 0 No. 6, 19-23. Comez, J .A ., Russell, S.W., Trowbridge, J.O. and Lee, J. (1977) - Am. J . dig. Dis. 22: 485. Kennedy, P.C. and Cello, R.M. (1966) - Gastroenterology. 51: 926. Koch, S.A. and Skelley, J.F. (1967) - J. Am. vet. med. Ass. 150: 22. Lawson, T.L., Gomez, J.A., Stewart, E.T., Rambo, O.N. and Margulis, A.R. (1970)- Am. J. Roent. 110: 377. Lorenz, M.D. (1975) - Textbook of Veterinary Internal Medicine Ed. S.J. Ettinger, W.B. Saunders Co., Philadelnhia. Vol. 2. on. 1203-1208. Russell, S.*W.,'Goniez,'j.A. and Trowbridge, J.O. (1971) Lab. Invest. 25, 509. Sander, C.H. and Langham, R.F. (1968) - Arch. Path. 85: 94. Van Kruiningen, H.J., Montali, R.J., Strandberg, J.D. and Kirk, R.W.(1965)- Path. V e t . 2 : 5 2 1 . Van Kruiningen, H.J. (1967) - Gastroenterology, 53: 114. Van Kruiningen, H.J. (1975) - Vet. Path. 12: 446. (Received forpublication 10 November 1977) 449
